In contrast, migraineurs and healthy controls displayed no and minimal differences in response to positive and neutral emotional stimuli, respectively. These findings support the notion that migraine may feature more generalized altered cerebral processing of aversive/negative stimuli, rather than exclusively to sensory stimuli. A generalized hypersensitivity to aversive stimuli may be an inherent feature of migraine, or a consequential alteration developed over the duration of the disease. This proposed cortical-limbic hypersensitivity may form an important part of the migraine pathophysiology, including psychological comorbidity, and may represent an innate sensitivity to aversive stimuli that underpins attack triggers, attack persistence and  gradual headache chronification.Pain is both an unpleasant sensory and emotional experience. This is highly relevant in migraine where cortical hyperexcitability in response to sensory stimuli  has been extensively reported. However, migraine may feature a more general enhanced response to aversive stimuli rather than being sensory-specific. To this end we used functional magnetic resonance imaging to assess neural activation in migraineurs interictaly in response to emotional visual stimuli from the International Affective Picture System. Migraineurs, compared to healthy controls, demonstrated increased neural activity in response to negative emotional stimuli. Most notably in regions overlapping in their involvement in both nociceptive and emotional processing including the posterior cingulate, caudate, amygdala, and thalamus (cluster corrected,  Pain, by its definition, is comprised of both “an unpleasant sensory and emotional experience” . This isThe relationship between pain and emotion is also highlighted by the overlap in their central representation in the brain. Pain  and emotIn support of this view a limited number of studies have shown altered processing of emotional stimuli in migraine, specifically in regards to negative or aversive emotion. The purpose of the present study was to assess the brain correlates of emotional processing in patients with episodic migraine during the interictal period using functional Magnetic Resonance Imaging (fMRI). We hypothesized we would observe enhanced neural activation in migraineurs in areas involved in emotional and pain processing  in response to viewing negative pictures. In contrast, we expected no differences between migraineurs and controls in neuronal response to positive and neutral emotional stimuli.n = 23 migraine patients and n = 23 healthy control subjects) who underwent an imaging session at McLean Hospital were included in this study. All migraine patients fulfilled the International Classification for Headache II criteria for episodic migraine (confirmed by a neurologist) and, as part of the inclusion criteria, suffered from migraines for more than 3 years. Clinical characteristics (including medication usage) for individual migraine subjects are given in Supplementary Table S1. Migraine patients were scanned during their interictal (headache-free) period, defined as migraine free at least 48 h before and 24 h after the imaging session. Healthy control subjects had no significant history of migraine, or other headache condition. All subjects were screened for depression . Mean (and standard error of the mean) ratings for migraine and control groups are shown in Figure 2.Migraine and control subjects did not differ in their ratings of valence or arousal in any emotional category [Positive valence: Supplementary Figure S1).Separately, both migraine and control subjects showed similar networks of activated brain regions in response to emotional stimuli .Positive IAPS stimuli: during positive emotional stimuli migraineurs displayed no significant different in neural response, in comparison to controls . This increased activation was observed in visual areas, notably the intracalcarine cortex and lingual gyrus.Neutral IAPS stimuli: during neutral emotional stimuli migraineurs displayed only a single region of increased neural response, in comparison to controls . These included increased activation in the superior and middle frontal gyrus, the frontal medial cortex, the frontal pole, posterior cingulate gyrus, precuneus and cuneal cortex, caudate, thalamus, left amygdala and right hippocampus, brainstem (midbrain and pons) and the cerebellum.Negative IAPS stimuli: during negative emotional stimuli migraineurs displayed a range of regions with increased neural response, in comparison to controls  compared in this study were induced using IAPS , a standFigure 4).By definition, pain includes both a sensory and affective component . NocicepHowever, the region of increased activation in the cingulate region was ascribed to the posterior cingulate cortex (PCC), which is thought to have little or no involvement in pain processing . Rather the posterior cingulate has extensive connections with the parietal lobe  and is proposed to be involved in emotional evaluation including assessment of the self-relevance of emotional stimuli . While tThe amygdala has long been known for its important role in processing the emotional dimension of pain . Among iOur understanding of migraine pathophysiology has dramatically evolved over the last half-century in increasing acknowledgment that this complex neurological disorder affects multiple cortical, subcortical and brainstem regions that regulate sensory, autonomic, cognitive and affective functions . The majAs episodic migraine progresses to its chronic form, psychological dysfunction often increases concurrently . PatientWhile this is the first study to demonstrate increased brain activity in response to general negative emotional stimuli in migraineurs, there are some caveats of our study that should be acknowledged. Though we have interpreted the observed increased activity in migraineurs in response to negative emotional stimuli as evidence of cortico-limbic hyperexcitability we are unable to determine if this represents increased facilitation or dishabituation of emotional processing networks . AdditioMigraine is a multifactorial disorder encompassing not only pain and sensory disturbances but also a complex psychological/affective component active both during attacks and interictaly. A complete model of migraine pathophysiology must integrate all of these diverse components that give rise to the condition . Our finSW contributed to the analysis and interpretation of data, drafting of manuscript and critical revision; RV and SS contributed to the acquisition of data; JL contributed to the analysis of data; DH contributed to critical revision; RB contributed to study conception and design; LB contributed to study conception and design, analysis of data and critical revision and DB contributed to study conception and design and critical revision.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.


Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review. Conventionally closely related species are easier to breed than inter species/genus due to compatibility issues, and this is considered a major limitation. Transgenic technologies have allowed gene transfer to completely unrelated organisms. All these advances have increased the global transgenic plant cultivation to 181 million hectares . TransgeTable 1).A major prerequisite for plant expressing a transgene is stability and segregation. Several reports have documented a deviation from the Mendelian segregation ratios in transgenic plants . This reThe British developmental biologist Conrad H. Waddington coined the term “epigenetics”. Epigenetics deals with studies related to interactions of genes and their products, which determine the phenotype of a system . During Two main classes of transgene-silencing phenomena have been reported to date. The first concerns position effects, in which the expression of a foreign gene is negatively regulated by flanking host DNA or chromosomal location . The expFigure 1A).The second class of silencing phenomena is based on epigenetic regulation and is a type of inactivation mechanism that can arise when multiple copies of the same or homologous sequence are introduced in a genome. Since interactions between homologous nucleic acid sequences are responsible for these silencing, it is also called homology-dependent gene silencing (HDGS) . Over thA major breakthrough in understanding epigenetic silencing in transgenic plants was first identified in transgenic tobacco, where interaction between two homologous promoters led to DNA methylation and silencing . Two typDe novo DNA methylation can be highly sequence-specific for a specific transgene .Transgene-induced viral resistance, recovery from infection and proteins encoded by viruses that counteract PTGS suggested it as a potential defense response to check viral infections . It is scis-inactivation is observed when foreign genes like β-Glucuronidase, neomycin phosphotransferase, etc., were driven under strong 35S promoter .A hallmark of PTGS in plants is that it systemically transmitted in a sequence-specific manner known as systemic acquired silencing (SAS). Remarkable and recurrent features in silencing patterns during developmental stages revealed propagation of a silencing message across the plant . Co-supplled SAS . Transget of SAS . A SAS Ptroduced . The siltroduced . The recde novo DNA methylation at homologous regions. Until then, DNA/RNA hybrids were believed to play a role in generating a target for de novo methylation. chsA co-suppression studies in Petunia led to the identification of mobile RNAs as potential candidates responsible for the induction of co-suppression  revealed transcript-dependent silencing of miRNA target. Thus, a crucial regulatory role of miRNAs might be conserved in other plants also, which are under investigation. siRNAs are another class of small RNAs that are involved in epigenetic modification  are produced from hairpin-like precursor RNA, which is essential for biogenesis of trans-acting siRNAs (ta-siRNAs). miRNAs are involved in regulation of gene expression by base-pairing with target RNAs further leading to their cleavage in plants. fication . EndogenSilencing cannot be considered as a mechanism that evolved to regulate transgene expression; it is a part of natural plant processes. TGS and PTGS can be considered as host defense responses against ‘foreign invading’ viruses. Hence, transgenes or their products can be equated to cellular invaders triggering defensive reactions leading to silencing of “trans”gene. PTGS recruits cellular components acting against foreign DNA that replicates extra-chromosomally in the nucleus, or RNA in the cytoplasm. A clear connecting link between PTGS and viral resistance was established after the discovery and characterization of various viral proteins that suppress PTGS . TGS may(1)Selection of transgenic lines with single T-DNA insert(2)Organelle targeting/transformation(3)Selection of favorable/unique integration sites(4)Reactivation of silent transgenes(5)Use of Scaffold Matrix Attachment Regions in silencing mutant host system to prevent silencing.The last three decades have seen immense progress and better understanding of epigenetic effects and silencing mechanisms; transgenic technologies have played a pivotal role for these achievements. Common phenomena behind different types of silencing and recent finding of involvement of siRNAs/miRNA continue to inspire efforts of scientific community to formulate comprehensive models, which also explain the silencing mechanism from an evolutionary view point. Our understanding of the influence of various factors on stability of transgene expression is improving rapidly. We cannot control or predict integration of gene into a recipient genome, nor predict the number of copies or integrity of a transgene. Hence, a comprehensive knowledge of underlying mechanisms in integration process and the influence of chromatin remodeling leading to transgene regulation are crucial. Finally, it might be useful to keep in mind that epigenetic silencing was an unexpected phenomenon; it is still hard to foresee overcoming epigenetic related silencing in transgenic system. Nevertheless, transgenic research will continue as a platform to discover new aspects of epigenetic silencing.SR and PA prepared the manuscript and RS revised it.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.


Microbes numerically dominate aquatic ecosystems and play key roles in the biogeochemistry and the health of these environments. Due to their short generations times and high diversity, microbial communities are among the first responders to environmental changes, including natural and anthropogenic disturbances such as storms, pollutant releases, and upwelling. These disturbances affect members of the microbial communities both directly and indirectly through interactions with impacted community members. Thus, interactions can influence disturbance propagation through the microbial community by either expanding the range of organisms affected or buffering the influence of disturbance. For example, interactions may expand the number of disturbance-affected taxa by favoring a competitor or buffer the impacts of disturbance when a potentially disturbance-responsive clade’s growth is limited by an essential microbial partner. Here, we discuss the potential to use inferred ecological association networks to examine how disturbances propagate through microbial communities focusing on a case study of a coastal community’s response to a storm. This approach will offer greater insight into how disturbances can produce community-wide impacts on aquatic environments following transient changes in environmental parameters. Most people and development reside near water bodies, so human activities profoundly affect both freshwater and marine ecosystems . In thesnon-disturbed state following a disturbance. This definition of resilience requires understanding the disturbance-independent temporal dynamics of microbial communities. Although marine microbial communities exhibit regular seasonal patterns at monthly timescales , and diversification  are known for their streamlined genomes, small cell sizes, and efficient use of resources , they occur frequently enough to allow comparison across different storms, environments, and microbial communities . Here weTo investigate this concept further, we will follow the progression of storm-mediated impacts on a simplified microbial community association network where an alga serves as a keystone microbe and a network hub. In our model system Figure , the majHere, we have presented a cartoon storm as a pulse of nutrients, in reality storms and other ecological disturbances are complex. In addition to nutrients, storms introduce human pollutants into aquatic ecosystems, including pesticides, oil, untreated human waste, etc., that will have direct and interaction-mediated effects on the microbial community. Unlike our simple example in Figure Here, we discuss the potential for association networks to track the propagation and persistence of disturbance in a microbial community. We have identified two major opportunities afforded by this approach: (1) to quantify the importance of interactions in a microbial community’s response to disturbance and (2) to generate biological hypotheses about the network’s inferred interactions. However, a major challenge of this approach is that to characterize a microbial community’s resistance and resilience we first need to understand disturbance-independent microbial community dynamics , suggestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The transmission of genes and culture between human populations has major implications for understanding potential correlations between history, biological, and cultural variation. Understanding such dynamics in 19th century, post-contact Native Americans on the western Great Plains is especially challenging given passage of time, complexity of known dynamics, and difficulties of determining genetic patterns in historical populations for whom, even today, genetic data for their descendants are rare. Here, biometric data collected under the direction of Franz Boas from communities penecontemporaneous with the classic bison-hunting societies, were used as a proxy for genetic variation and analyzed together with cultural data. We show that both gene flow and “culture flow” among populations on the High Plains were mediated by geography, fitting a model of isolation-by-distance. Moreover, demographic and cultural exchange among these communities largely overrode the visible signal of the prior millennia of cultural and genetic histories of these populations. It is evident that historical (long term) patterns of divergence and descent can be created in both the genetic and cultural systems of human communities given that both are based on systems of inheritance, which facilitates some degree of continuity1356Bison bison)13Native American communities that occupied the western portion of the Great Plains during the post-contact  period have come to be globally famous12These combined push and pull effects ensured that by the 1800s, communities from at least five different language families were occupying the western Great Plains , pursuin1016“linguistic diversity was much greater than the diversity of other aspects of their cultures and must be a retention from a time when they had less contact with each other and were culturally more distinct … The general picture that is suggested is of diverse peoples retaining their distinct ancestral languages while adopting new and to a large extent shared lifeways after coming into contact with each other.”Examining potential congruence between cultural diversity and genetic patterns and their pathways of transmission under such dynamic and historically contingent conditions obviously presents a major challenge. Moreover, in this specific case, additional analytical problems are present. Most obviously, there is the passage of time between the late 19th century and the present day, meaning we are dealing with historical rather than directly contemporary populations. Furthermore, there is currently an absence of contemporary genetic data for the living descendants of the equestrian bison-hunting populations of the pastcirca 1888–1903 under the direction of Franz Boas232425Here, we sought to overcome these challenges and simultaneously examine both biological and cultural pathways of transmission in nine ethno-linguistic tribes who occupied the western Great Plains during the 19th century . More spIn a situation such as the historical Great Plains, geographic distance between communities provides a primary hypothesis for predicting both cultural and biological similarities and differences between those groups. This is because under such circumstances, geographic distance is likely to mediate likelihood and intensity of between-group interactions, whereupon similarities and differences in cultural and/or genetic patterns are, most parsimoniously, simply a function of mutation (copying and transmission errors), drift (stochastic trait loss), migration (between group movements of people), and/or diffusion . In other words, under these conditions, what has long been referred to as “isolation by distance” in population genetics parlance28No statistical evidence was found for a correlation between the geographic distances among tribes and linguistic distances among them . This result thus accords with the known historical situation, in that many groups were recent migrants to the region and hence their geographic position, and the geographic relationships between tribes, had nothing to do with deeper historical factors as measured by linguistic affinity.With respect to tests of our central hypotheses, both the biological and cultural data sets independently showed a statistical fit to the isolation-by-distance model . That isWe also tested whether among-tribe cultural patterns of variation correlated with among-tribe biometric variation directly. We found that among-tribe cultural and biometric patterns of variation were not significantly correlated with each other .Here, we have examined the extent to which geographic distances among Native American communities occupying the western Great Plains during the 19th century mediated patterns of transmission in both their genetic and cultural inheritance systems. Our central results identify that among-tribe patterns of cultural and biometric variation correlate with the geographic distances among these tribes. In other words, our results identify that both gene flow and “culture flow” among these communities was mediated partly by the geographic distances between pairs of tribes in any given instance.Our results also show that deeper, historical patterns of cultural and biometric differentiation among these tribes, as measured by correspondence with their ancestral languages, were largely overridden by their more recent geographically mediated interactions, which led to genetic admixture and exchange of cultural ideas. Admittedly, use of direct genetic data for these historical populations rather than necessary use of proxy biometric data, may have revealed stronger evidence of deeper historical signal in terms of biological patterns of differentiation. Nevertheless, our results indicate that recent historical events of population exchange among these groups led to empirically recognizable effects in the available data in a relatively short time span. Indeed, there are two ways in which “culture flow” may occur: either via demographic exchange between populations or by transferal of ideas in the absence of demographic exchange31Linguistic differences among the various groups appear to have provided little barrier to either gene flow or culture flow among groups, at least such that these led to a deviation from patterns that can be predicted on the basis of geographic distances among tribes alone. Such a pattern is explicable, however, when it is considered the many of the cultural variants we examined here (moccasin decorative features and parfleche characteristics in particular) were the product of female craftswomen. Given that girls and women were taken as captives by tribes hostile toward one another, as well as married into allied tribes31and cultural patterns . In this sense, our analytical framework shares much in common with those who have previously advocated such an approach on the basis of identifying broad and deep historical correlations between genetic, linguistic, and/or other cultural dimensions of human variability  measurements for several thousand individuals from North American tribes, which were recorded on living individuals between 1888 and 1903353637383923242542n = 82 traits), moccasin decorative features (n = 10 traits), parfleche design features (n = 23 traits), and a single trait associated with tipi design (3 pole or 4 pole base). Traits were typically recorded on a presence/absence basis, although three trait variants relating to parfleche design were treated as categorical variables. An among-tribe cultural distance matrix  and alliance as they stood during the latter decades of the 19th century are reasonably well documented31Correlations between distance matrices were examined for statistical correlation. Since matrices violate the assumptions of traditional correlation statistics, matrices were analyzed using Mantel tests58How to cite this article: Lycett, S. J. and von Cramon-Taubadel, N. Transmission of biology and culture among post-contact Native Americans on the western Great Plains. Sci. Rep.6, 25695; doi: 10.1038/srep25695 (2016).


Recent advances in the modeling of microbial growth and metabolism have shown that growth rate critically depends upon the optimal allocation of finite proteomic resources among different cellular functions and that modeling growth rates becomes more realistic with the explicit accounting for the costs of macromolecular synthesis, most importantly, protein expression. The “proteomic constraint” is considered together with its application to understanding photosynthetic microbial growth. The central hypothesis is that physical limits of cellular space (and corresponding solvation capacity) in conjunction with cell surface-to-volume ratios represent the underlying constraints on the maximal rate of autotrophic microbial growth. The limitation of cellular space thus constrains the size the total complement of macromolecules, dissolved ions, and metabolites. To a first approximation, the upper limit in the cellular amount of the total proteome is bounded this space limit. This predicts that adaptation to osmotic stress will result in lower maximal growth rates due to decreased cellular concentrations of core metabolic proteins necessary for cell growth owing the accumulation of compatible osmolytes, as surmised previously. The finite capacity of membrane and cytoplasmic space also leads to the hypothesis that the species-specific differences in maximal growth rates likely reflect differences in the allocation of space to niche-specific proteins with the corresponding diminution of space devoted to other functions including proteins of core autotrophic metabolism, which drive cell reproduction. An optimization model for autotrophic microbial growth, the autotrophic replicator model, was developed based upon previous work investigating heterotrophic growth. The present model describes autotrophic growth in terms of the allocation protein resources among core functional groups including the photosynthetic electron transport chain, light-harvesting antennae, and the ribosome groups. However, assuming it is the burden of synthesizing amino acids, then the question arises whether this is because of the energetic cost of making amino acids, such as ATP consumed per amino acid and “opportunity costs” of not using ATP for other cell functions that contribute to cell reproduction. A bioinformatics analysis using codon bias as an indicator for expression rates found that less expensive amino acids are preferably utilized for highly expressed protein . Of the possible computational approaches, constraint-based FBA has already proven useful and appropriate for the predictive analysis of the production of engineered bioproducts , which uses pulse-labeling of cells with substrate atoms tagged using stable isotopes. This approach has been applied in cyanobacteria to analyze metabolic fluxes under autotrophic conditions  with the consequent wasteful release of energy-rich compounds such as ethanol, acetate, and lactate despite the fact that environmental and physiological conditions would allow for more energy-efficient utilization of the carbon source. For example, the well-known Pasteur effect, which involves metabolic switching between efficient and less efficient pathways. Here, yeast cells are observed to switch between less energy-efficient fermentation and more efficient aerobic metabolism by depending upon the availability of oxygen. However, what is less discussed are the circumstances where the Pasteur mechanism is over-ridden and fermentative metabolism occurs even under aerobic conditions – i.e., overflow metabolism occurs. This situation, known as the Crabtree effect in yeasts   of cyanobacteria. The CCM is a metabolic process that not only satisfies the requirement of the inorganic carbon supply by energetically acquiring bicarbonate, but it also dissipates excess energy under high light conditions by spilling of large amounts of acquired bicarbonate by release into the environment  pathways leads to maximal growth rates under conditions of nutrient excess and accounts for the efficacy of overflow metabolism in maximizing cell growth rates. On the other hand, expression of metabolically more efficient (non-overflow) pathways catalyzed by enzymes with a higher proteomic cost, lead to maximal growth rates under conditions of nutrient limitation  to utilize the amino acids being produced. Conversely, if there is not a large enough allocation of amino acid biosynthetic enzymes (or the enzymes feeding nutrient precursors to those biosynthetic enzymes), then the proteomic allocation to protein synthetic machinery would be excessive, and in a cellular environment where to total proteome is finite, the allocation would be suboptimal. The concentration of soluble protein in the cyanobacterial cytoplasm is estimated to be ~500 mg/mL  and accumulate potassium to maintain osmotic balance when challenged by high salt conditions. Immediately following an upshift in environmental salt concentrations, there is a decline in metabolic activities, such as in PSET, and there is a mobilization of many stress response genes. After this initial period, compatible osmolytes accumulate, damage is repaired and cyanobacterial cells regain nearly normal levels of metabolic activities and resume growth, albeit at a lower rate . Since most chlorophyll in cyanobacteria is associated with reaction centers, the prediction of higher PSET with increasing growth rates leads to the prediction that faster growing cyanobacteria should have higher chlorophyll contents on a per cell basis. Evidence for higher levels of chlorophyll per cell in cyanobacteria have indeed been observed for light-limited  for macromolecular synthesis. Natural selection has tuned the regulation to ensure optimal protein levels to achieve this balance. As noted, the ARM does not specify mechanisms, but it seems likely that metabolic intermediates that accumulate or are depleted under conditions of imbalance are good candidates to serve as allosteric modulators of gene expression. This type of regulation is observed, as one example, for control of the inorganic carbon uptake mechanism proteins (Nishimura et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fbioe.2015.00001/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


Salmonella disease in Africa is a major public health concern. With evidence of the transcontinental spread of the Salmonella Typhi H58 haplotype, improved estimates of the burden of infection and understanding of the complex interplay of factors affecting disease transmission are needed to assist with efforts aimed at disease control. In addition to Salmonella Typhi, invasive nontyphoidal Salmonella are increasingly recognized as an important cause of febrile illness and mortality in sub-Saharan Africa. Human experimental oral challenge studies with Salmonella can be used as a model to offer unique insights into host–pathogen interactions as well as a platform to efficiently test new diagnostic and vaccine candidates. In this article, we review the background and use of human challenge studies to date and discuss how findings from these studies may lead to progress in the control of invasive Salmonella disease in Africa.Invasive  Salmonella disease in Africa ) were diagnosed with enteric fever based upon clinical criteria alone (fever ≥38°C lasting ≥12 hours) without evidence of bacteremia. This finding highlights the relatively low sensitivity of automated blood culture even with repeated sampling in a high-income setting, supporting the view that reliance on blood culture alone may underestimate the true disease burden in African settings where multiple alternative causes for fever exist.Salmonella Typhi bacteremia without clinical signs of enteric fever. Two of these 4 participants had a significant increase in anti-lipopolysaccharide (LPS) antibody from a low baseline titer, suggesting that the humoral immune system was exposed to the bacteria at some point after challenge (unpublished observations). Subclinical or asymptomatic infection has long been suspected to occur following exposure to Salmonella Typhi and may play an important role in onward disease transmission. Human challenge studies permit the detailed assessment of this subset of people, who are difficult to evaluate in field settings, and these findings could in turn have important implications for transmission modeling.Conversely, 4 of 24 diagnosed participants (16.6%) in our model had detectable Salmonella Typhi in Southeast Asia [Knowledge of the pattern and extent of stool shedding, both during acute infection and chronic carriage, was found to be an important factor when modeling transmission dynamics for ast Asia . Prolongast Asia . CollectSalmonella Typhi in their stools for several weeks. The frequency of excretion peaked at 2 weeks after challenge in both those who did and those who did not develop typhoid disease, with stool cultures often remaining positive for 3 weeks, after which the frequency of positive stool cultures declined, such that all participants had negative stool cultures by 6 weeks after challenge.Data from the original Maryland challenge studies reveal a complex picture of stool shedding during the early phase of infection . A propoSalmonella Typhi shedding in any challenged participant.Whereas data on shedding during acute infection can be closely monitored, the Oxford typhoid challenge model offers limited scope to investigate shedding during chronic carriage as the latter is prevented by effective antibiotic therapy. Samples are collected a minimum of 2 weeks after completion of antibiotics to assess for chronic carriage and long-term shedding. In addition to this, participants with a history of gallbladder disease or ultrasonographic evidence of gallstones are excluded prior to study enrollment. To date, there has been no evidence of long-term Salmonella Typhi has resulted in the detection of asymptomatic primary Salmonella Typhi DNAemia from as little as 12 hours after challenge. Culture PCR was positive within 5 days of challenge in 10 of 40 participants, all of whom had negative routine blood culture during this period; 7 of 10 subsequently developed typhoid infection [Salmonella Typhi–specific anti-LPS salivary immunoglobulin A responses [Human challenge models provide a controlled environment to develop and test the sensitivity of new diagnostics assays. For example, use of a novel culture polymerase chain reaction (PCR) assay targeting the fliC-d gene of nfection , 28. Pubnfection  and Salmesponses , 31 haveSalmonella Typhi is associated with antibody responses to a broad range of antigens including outer membrane  or secreted proteins  [Natural infection with E, CdtB) , 33. HumE, CdtB) . Long-teSalmonella. Currently licensed typhoid vaccines, parenteral Vi polysaccharide and oral live attenuated Ty21a, have several limitations including moderate vaccine efficacy and contraindication for use in young children. Human challenge studies provide the opportunity to assess novel vaccine candidates in relation to vaccine efficacy and immunogenicity prior to assessment in costly field trials.Vaccines, together with other public health interventions, will play an important role in reducing the burden of disease caused by human-restricted typhoidal Salmonella Typhi [Proof-of-principle of this approach was provided by the Maryland studies where the attenuated strain Ty21a was shown to protect volunteers challenged with la Typhi . Our grola Typhi , and Ty2Salmonella exposure, dietary factors, intestinal microbiota, and the genetic profile of the host population, have on disease pathogenesis and host immune responses [Findings from challenge studies in a highly selected group of individuals in the United Kingdom may not directly correlate with those found in endemic regions. The response to enteric pathogens or oral vaccines, for example, may differ in an African setting due to preexisting natural immunity, genetic differences, or comorbid conditions , 36. Malesponses .Salmonella disease in Africa, in addition to the scientific and translational benefits provided by established human enteric fever challenge models, increasing attention has been given to the feasibility of developing an NTS human challenge model [Given the increasing role of NTS strains in causing invasive ge model . A contrge model , in addiSalmonella Typhi—a Vi-expressing strain isolated from a then-recently diagnosed chronic carrier (Mrs Quailes) [Salmonella Typhi, for example, would offer an opportunity to understand host–pathogen interactions using a strain of contemporary significance. Based upon current knowledge of NTS serovars and strains, Salmonella Typhimurium ST313 would be an appropriate choice to develop as a challenge agent, as it accounts for much of the iNTS disease seen in sub-Saharan Africa and, similar to Salmonella Typhi, appears to display features of human adaptation. In a similar fashion to the development of the Salmonella Paratyphi challenge strain (NVGH308), an isolate obtained from the blood culture of a clinical case of paratyphoid infection in Kathmandu, Nepal (NCT02100397), an ST313 isolate from the field could, in theory, also be isolated and manufactured to Good Manufacturing Practice standards for use in challenge studies. A series of quality control tests are performed before the strain can be used in human challenge studies including antimicrobial susceptibility and stability testing. In some settings, the substantial cost of strain manufacture may represent a major barrier to the development of an NTS challenge strain and, as such, significant financial investment and collaboration by funders and/or industrial partners would be required.Strain choice is integral to the design of a human challenge study. Ingestion of the challenge strain needs to result in the required disease endpoint, which is generally observable, reproducible clinical infection. The early Maryland studies, for example, failed to induce enteric fever after challenge with a variety of deposited Ty2 strains , but sucQuailes) , 43. ChaIn enteric fever challenge studies, fever and/or bacteremia have been used as objective measurable endpoints of infection. Defining an endpoint after NTS challenge that is both safe and clinically meaningful is particularly difficult. Fever or bacteremia would represent the most clinically relevant features of iNTS disease; however, it is uncertain if healthy, immunocompetent volunteers would develop inflammatory gastroenteritis or invasive disease after challenge. Even in the African setting, invasive disease is uncommon in healthy adults. Development of bacteremia may be associated with a high risk of complications such as endovascular infection, meningitis, or septic shock. Alternative endpoints could include diarrhea, isolation of challenge agent from stool, or a predetermined duration of stool shedding. Such endpoints have been used in other enteric challenge studies and would be associated with less risk to study participants. Although such a model would not address potentially important factors related to invasive disease, such as systemic host–pathogen interactions, it would assist with the study of mucosal immunity, stool shedding, and the effect NTS vaccine candidates have on preventing gastroenteritis and bacterial shedding, and as such, may have implications on preventing NTS transmission in the field.The overall mortality rate of iNTS disease is estimated at 20% in the African setting  and may Salmonella disease in Africa, many factors relating to disease transmission remain unknown. Human challenge studies can shed light on several of these unknown areas including strain infectivity, duration of stool shedding, and detection of early and late disease markers. Use of typhoid challenge models also provides a platform to evaluate novel vaccine candidates, which could play a key role in future disease control efforts given increasing rates of antimicrobial resistance among Salmonella Typhi isolates from Africa. We have shown that Salmonella Typhi human challenge models can be performed safely and offer a unique means of studying disease from the point of infection through to diagnosis, treatment, and convalescence. An NTS human challenge model could offer similar advantages of obtaining valuable human-specific data for a currently neglected and serious disease, but several knowledge gaps would have to be filled before such a model could be seriously contemplated. While a number of well-described risk factors for iNTS have been identified, invasive disease is a relatively infrequent occurrence in the African setting following exposure to NTS serovars in the gastrointestinal tract. Human challenge studies could allow further exploration of the host and pathogen factors associated with the iNTS phenotype; however, the unpredictable response to a controlled challenge in healthy volunteers is a potential hurdle. The low attack rate in the general population may preclude the successful development of a model, even at high challenge dose. On the other hand, there is a real potential for serious complications with iNTS. Careful consideration of ways to mitigate the risk to study participants may preclude development of a human challenge model in the short term, but it is hoped that insights from enteric fever challenge studies can benefit ongoing disease surveillance and contribute to the collaborative effort to control invasive Salmonella disease across Africa.Despite growing evidence demonstrating the significant morbidity and mortality caused by invasive


Our growing knowledge of viruses reveals how these pathogens manage to evade innate host defenses. A global scheme emerges in which many viruses usurp key cellular defense mechanisms and often inhibit the same components of antiviral signaling. To accurately describe these processes, we have generated a comprehensive dictionary for eukaryotic host-virus interactions. This controlled vocabulary has been detailed in 57 ViralZone resource web pages which contain a global description of all molecular processes. In order to annotate viral gene products with this vocabulary, an ontology has been built in a hierarchy of UniProt Knowledgebase (UniProtKB) keyword terms and corresponding Gene Ontology (GO) terms have been developed in parallel. The results are 65 UniProtKB keywords related to 57 GO terms, which have been used in 14,390 manual annotations; 908,723 automatic annotations and propagated to an estimation of 922,941 GO annotations. ViralZone pages, UniProtKB keywords and GO terms provide complementary tools to users, and the three resources have been linked to each other through host-virus vocabulary. Advances in high-throughput DNA sequencing have resulted in an explosion in the number of virus genome sequences deposited in public databases over the past decade Viruses are genetic entities that infect all kinds of organism. Their impact on living beings is huge, affecting human health, agricultural and other economic activity, global ecology, and even evolution. Exploration of the viral world is still very much in its infancy, with new species being discovered all the time - 138 new species have been officially recognized between 2011 and 2012, raising the total number of referenced viral species to 2618 (http://www.ebi.ac.uk/GOA/Keyword2GO).ViralZone is a database that links virus sequence data with knowledge of virus molecular biology curated from peer-reviewed literature using human-readable text and controlled vocabularies. This web resource was created in 2009 and has been continually developed since that time by the viral curation team of the SwissProt group. The core of ViralZone is the virus fact sheet that describes the virion, genome, replication cycle and host data for each of the known virus genera and families. Virus molecular biology pages describe viral processes such as viral entry by endocytosis and viral genome replication in detail, with graphical illustrations that provide a global view of each process and a listing of all known viruses which conform to the particular schema. ViralZone pages are linked to protein sequence records of the UniProt Knowledgebase (mostly reference proteomes). This provides facile access to expert-curated information on individual protein sequences and their functions in textual form and as controlled vocabularies and ontologies. Ontologies consist of hierarchized controlled vocabulary in computer-friendly format. They provide a frame for global annotation, and facilitate analysis of biological data. In the era of metagenomics and large-scale studies, ontologies are an extremely potent tool to link knowledge with gene products and help identifying common patterns. UniProtKB keywords constitute an ontology with a hierarchical structure designed to summarize the content of an entry and facilitate the search of proteins of interest. They are classified in 10 categories: Biological process, Cellular component, Coding sequence diversity, Developmental stage, Disease, Domain, Ligand, Molecular function, Post-translational modification and Technical term. A more complex and widely used vocabulary is that of the Gene Ontology (GO) in which relations between terms have a number of explicit meanings which can be used to make further inferences – such as eukaryotic transcription factors may be located in the nucleus. GO annotations are routinely used for the functional analysis  of many data types, such as differential expression data. GO provides almost 40,000 terms grouped in three categories: the molecular functions a gene product performs, the biological processes it is involved in and the cellular components it is located in This publication describes a project initiated by the SwissProt virus annotation team to study the complex interactions of viruses and their hosts and to encode this knowledge in a set of UniProt keywords, GO terms, and interlinked ViralZone pages. All organisms display an impressive battery of innate and acquired antiviral defenses. The viruses we observe today are ‘escape artists’ with a talent for evading these defenses, and whose ability to manipulate their host is essential for virus survival. Their genomes are generally extremely small, encoding few proteins, and these need to be very efficient – they often attack several key points of the host biology. For example, the Hepatitis B virus encodes only four proteins; the polymerase, capsid and surface proteins are essential for the basic life cycle of the virus while the HBX protein plays a major role in host immune defense evasion and cell transformation An extensive study of the recent literature was performed to identify essential and conserved host-virus interactions. The number of interactions to consider is significant: as an example, the 13 proteins of HIV-1 have been shown to interact with more than 2589 human proteins This work describes the creation of a common host-virus interaction vocabulary in ViralZone, UniProtKB and the Gene Ontology (GO), resulting in the creation of 14,390 manual and 908,723 automatic annotations (May 2014). At the inception of this project, only two UniProtKB keywords were applicable to protein sequences involved in interaction between virus and host. These were the keyword “Host-virus interaction” – used for any type of interaction between viral and host proteins – and “viral immunoevasion”, used to group viral proteins counteracting the host immune system. In this work we created 63 new keywords to capture all host pathways commonly usurped by viruses . To desiRepresentation of pathways remains a challenging task in the field of curation, and the relationship between individual proteins which are part of a larger process is often not easy to describe in text form. To facilitate understanding of how viruses impact their hosts we therefore developed 57 ViralZone pages corresponding to the majority of the newly created keywords . Each inThe GO editorial team at EMBL-EBI collaborated with the ViralZone team to update the Gene Ontology (GO) with terms corresponding to the new host-virus interaction ontology. This effort led to the development of 57 GO terms (most newly created) exactly matching new UniProtKB keywords . In someThe goal of this work was to curate viral reference proteomes in order to capture host-virus interactions. This curation has been performed in different ways . KeywordEach organism is the potential target of dozens of viruses Upon entering a host cell, viruses must deal with host innate immunity. Pathogen recognition receptors (PRRs) Upon virus pattern recognition, most vertebrate cells release interferon (IFN) alpha or beta. IFNs signal infection to neighboring cells Both PRR and interferon signaling trigger an antiviral state in which more than 300 interferon stimulated genes (ISGs) are up regulated The long history of viral and host coevolution has seen the development of a variety of evasive adaptations to circumvent or inactivate these antiviral effectors. One of the most early and effective means to do so is to prevent ISG transcription and the establishment of the antiviral state. This early attempt at evasion may fail though, and latent viruses will eventually have to face antiviral effectors. Therefore many viruses have evolved other ways to rescue their replication cycle by inactivating key effectors of cellular defense. For example the HIV-1 vif protein counteracts APOBEC3G activity In innate immunity, pathogen-specific receptors are encoded by the genome, whereas in adaptive immunity these receptors are “acquired” during the lifetime of the organism. This immunity is said to be “adaptive” because it can memorize the pathogen offence and prepare the body to fight future challenges.Adaptive immunity relies on the distinction between the bodys self-antigens and the foreign-antigens of unwanted invaders. Infected cells display viral antigens through major histocompatibility complexes (MHCs). These are recognized as non-self by T-lymphocytes, thereby triggering the adaptive response and inducing destruction of the infected cells and the synthesis of neutralizing antibodies. To escape this defense, many viruses inhibit MHC peptide presentation In response to viral infection, the NFkappaB transcription factor can be activated and induce the production of numerous cytokines and chemokines by different cell types including macrophages, dendritic cells or epithelial cells In higher plants and insects, post transcription gene silencing  operates as an adaptive antiviral defense mechanism After genome replication, and in the late phase of the infection, many viruses synthesize a large number of structural proteins and assemble a huge number of virions within the cell. The high demand for viral protein synthesis is often supported by shutoff of host gene expression, ensuring that all cellular resources are devoted to viral synthesis Inhibition of host transcription can be mediated by preventing host RNA polymerase II initiation. The TATA-binding protein (TBP) is targeted by the adenovirus E1A protein, which disrupts the interaction between the TBP and the TATA box Host messenger RNAs can be modulated at various stages including pre-RNA processing, nuclear export or modification of RNA decay. Whatever the means, viral messenger RNAs rely on alternative ways to be expressed. For example, viruses shutting off host nuclear gene transcription or mRNA export are often transcribed in the cytoplasm.The main mechanism by which viruses inhibit host gene expression is by specifically targeting the translation of host mRNAs – which requires proteins not used for translation of viral proteins. Viruses inhibiting cap dependent translation use an internal ribosome entry site (IRES) or similar structures for translation initiation of their proteins Viral replication is limited by the ability of the host metabolic machinery to produce the resources – nucleic acids and proteins – necessary for the assembly of viral progeny Several pathogens interfere with or exploit the host autophagic pathway for their life-cycle or in order to evade or immune responses In order to prevent viruses from spreading within the infected host organism, cells can commit apoptosis, a genetically controlled program of cell death. Many viruses have evolved strategies to inhibit apoptosis by blocking both intrinsic and extrinsic host-initiated cell death pathways. Several adenoviruses, herpesviruses and poxviruses encode proteins that are homologous to the cellular anti-apoptotic Bcl-2 protein. These viral proteins sequester pro-apoptotic Bcl-2 family members including Bak and Bax, thereby inhibiting apoptosis. Viral FLIPs (vFLIPs) block the interaction of the death receptor-adapter complex with the cellular effector FLICE (caspase-8) to prevent the initiation of the downstream caspase cascade.The host-virus vocabulary presented here consists of 57 ViralZone terms, 65 UniProtKB keywords and 57 corresponding GO terms, describing most of known interactions between viruses and their hosts. The terms provide comprehensive coverage of the mechanism used by the virus families known to infect eukaryotic hosts. While most of current knowledge on host-virus interactions is covered by these terms, our systematic approach will allow expanding and updating the system. Indeed this area of knowledge has grown much in the past ten years, and will presumably continue to develop in the future. Our efforts to create eukaryotic host-virus interaction ontology have led to three levels of implementation: global knowledge and facts in ViralZone pages; viral protein annotation in UniProtKB through keywords; and viral gene and protein annotation through GO terms. At the time of writing the keywords provide a total of 923,113 annotations in UniProtKB while the GO terms provide 922,941 annotations. Together these three implementations provide a global view of viral biology, and a means to annotate knowledge, for a wide user community. Several research institutes and public databases have initiated projects involving the annotation of viral genomes, and we hope that the terms and ontologies presented in this article, which are available from the ViralZone, UniProtKB and GO websites, will help them in these efforts.


Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction, difficulties with language, and repetitive/restricted behaviors. The etiology of ASD is still largely unclear, but immune dysfunction and abnormalities in synaptogenesis have repeatedly been implicated as contributing to the disease phenotype. However, an understanding of how and if these two processes are related has not firmly been established. As non-inflammatory roles of microglia become increasingly recognized as critical to normal neurodevelopment, it is important to consider how dysfunction in these processes might explain the seemingly disparate findings of immune dysfunction and aberrant synaptogenesis seen in ASD. In this review, we highlight research demonstrating the importance of microglia to the development of normal neural networks, review recent studies demonstrating abnormal microglia in autism, and discuss how the relationship between these processes may contribute to the development of autism and other neurodevelopmental disorders at the cellular level. Despiteng brain –6. Furthng brain –10. Therin vivo imaging techniques have repeatedly demonstrated that microglia are highly active during normal brain development and that they interact directly with neurons during critical period of neurogenesis  of postmortem brain tissue from children with ASD as compared to controls (DAP12 lead to Nasu–Hakola disease (OMIM# 221770), a rare autosomal recessive disorder characterized by bone abnormalities and adult-onset neuropsychiatric features, such as social disinhibition, distractibility, and lack of appropriate emotionality (In addition to providing trophic support to developing neurons, it has also been shown that microglial cells play a central role in the normal postnatal apoptosis and phagocytosis of neurons and their connections, which are naturally over-produced and then “pruned” away based on experience-dependent usage . Specificontrols . Additioionality .CX3CR1 knockout mice had delayed synaptic pruning, resulting in excessive and electrophysiologically immature synapses. Moreover, related studies have even suggested that the behavioral phenotype of these mice is altered. For instance, it was demonstrated that mice lacking CX3CR1 had decreased functional brain connectivity, deficits in synaptic pruning, and behavioral changes associated with the autism phenotype, such as deficits in social interaction and increased repetitive behaviors (Apart from directly impacting the number of neurons in the developing brain, microglial cells have been shown to be involved in more nuanced aspects of synaptogenesis, such as in controlling the number of synapses, and in regulating synapse maturation and function –28. Synaehaviors .Although microglia’s role in normal neurodevelopment is beginning to be well-recognized, parallel research into the role of microglia in autism and other neurodevelopmental disorders apart from their classic inflammatory function is only in its infancy. However, there is a large body of literature assessing aberrant immune function in peripheral tissue of children with ASD. For instance, numerous studies have suggested the innate immune response is globally abnormal in autistic patients –34. SpecIn 2005, Vargas and colleagues demonstrated that postmortem brain tissue from patients with autism exhibits an increased microglial density in gray matter and an activated microglial morphology; additionally, altered cytokine profiles were found in both ASD postmortem brain tissue and cerebrospinal fluid . Since tIn addition to cell-level studies of microglia in autistic postmortem brain tissue, a number of whole-genome expression studies have investigated RNA expression patterns in ASD postmortem brain tissue as compared to neurotypical controls –42. A reMeCP2 null mice, a model of Rett syndrome, produced a conditioned media that damaged synaptic connectivity via a glutamate-excitotoxicity mechanism (MeCP2 gene, suggesting microglia play a key role in the pathogenesis of this disease. Moreover, it was recently demonstrated that the Rett phenotype may be partly related to MeCP2 direct modulation of microglial inflammatory gene transcription (While postmortem brain studies have begun identifying abnormal microglia in ASD, by their nature, they are inherently limited in attempting to ascertain if abnormal microglia are a reaction to, or the cause of, abnormal neurodevelopment. However, recent studies in rodent models of autism have begun to explore this question in more mechanistic detail. For instance, it was found that microglia from echanism . Importacription . Similarcription –50. Thiscription .Much work remains to definitively link abnormal microglia with the broad autistic phenotype; however, the evidence presented provides strong support to the notion that microglial cells may be able to reconcile the two most consistent gene expression and cellular findings in autism – changes in synaptogenesis and immune pathways. This hypothesis is particularly exciting as inherent “hypomorphic” abnormalities in microglia at the genetic or epigenetic level and their resultant impaired functioning in neurodevelopment may help to explain the substantial contribution to autism risk that is thought to arise from interactions among intrinsic and environmental factors .In summary, mounting evidence suggests that normal neurodevelopment represents a complex interplay between microglial cells and synaptic wiring. Furthermore, non-inflammatory roles for microglia in normal neurodevelopment are becoming increasingly identified. In parallel, abnormal immune signaling and microglial function are consistently demonstrated in postmortem tissue from autistic individuals as well as in mouse models of ASD. Previous research into altered immune function in autism has focused on the notion that microglia may cause destruction of already developed neural networks, perhaps in response to external insults, through their traditional inflammatory role. However, as their critical non-inflammatory role in normal neurodevelopmental becomes increasingly recognized, it is equally likely that this is what may be perturbed in ASD, yet was traditionally considered to be “immune dysregulation.” Future research should attempt to integrate the genetic and epigenetic etiology, neuronal synaptic disconnectivity, and abnormal microglia/immune findings in autism in an attempt to determine the precise role non-immune functions of microglia may play in the pathogenesis of ASD and other neurodevelopmental disorders.CAE and MNZ contributed equally to the conception, writing, and editing of the manuscript. OMR was involved in the conception and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vascularization remains one of the most important challenges that must be overcome for tissue engineering to be consistently implemented for reconstruction of large volume bone defects. An extensive vascular network is needed for transport of nutrients, waste and progenitor cells required for remodelling and repair. A variety of tissue engineering strategies have been investigated in an attempt to vascularize tissues, including those applying cells, soluble factor delivery strategies, novel design and optimization of bio-active materials, vascular assembly pre-implantation and surgical techniques. However, many of these strategies face substantial barriers that must be overcome prior to their ultimate translation into clinical application. In this review recent progress in engineering vascularized bone will be presented with an emphasis on clinical feasibility. Vascularization and bone formationCellular crosstalkDesign criteria for clinical successIntroductionCells and scaffoldsCells and soluble factorsBioreactorsCell based strategiesGrowth factor deliveryScaffold designCell-free strategiesSurgical approachesConclusionLarge volume bony defects resulting from trauma, congenital defects or cancer remain a significant challenge for reconstructive surgeons. Autologous tissue transfer is the standard treatment for such defects, but is hindered by donor site morbidity, risk of infection, poor cosmetic and functional outcome, and reduced graft integrity. Allografts are an insufficient solution due to immune response and a lack of sufficient donor tissues. Synthetic materials suffer from erosion, infection and poor outcome. The ability to engineer vascularized bone graft implants with patient-specific geometries has the potential to be an alternative source for tissues used in reconstruction.Vascular networks are vital to the development, healing and function of bone. The vasculature supplies oxygen and nutrients to the tissue and is a source of osteoprogenitor cells necessary for healing or regeneration in response to local injury. Long bones have a complex hierarchal vascular structure consisting of diaphyseal, metaphyseal, epiphyseal and periosteal arteries. Due to their high metabolic need, osteocytes are typically within 100 μm of a blood vessel Vascularization and bone formation are highly linked. It is widely agreed that vascularization occurs prior to osteogenesis during both embryonic development and healing of adult bone. During foetal development, mesenchymal precursors in the embryonic limb bud differentiate into chondrocytes, assemble into an avascular cartilaginous bone template and then secrete extracellular matrix and the angiogenic protein VEGF in vitroin vivoThe vasculature regulates bone formation in a variety of ways. It serves as a source of oxygen, nutrients and progenitor cells but also regulates bone behaviour through direct interactions between endothelial cells (ECs) and bone cells. Endothelial cells induce differentiation of osteoprogenitor cells A complex network of communication occurs between ECs and osteoblasts, osteoclasts and osteoprogenitor cells. One of the primary mechanisms by which this communication occurs is through the secretion of soluble factors. Mesenchymal stem cells secrete a number of soluble factors that can influence nearby ECs, including VEGF, angiopoietin-1 (Ang-1), basic fibroblast growth factor, platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) via Cx43 is essential for the maintenance and function of bone tissue.Gap junctions allow for direct cytoplasmic connections between two cells and are essential for cellular communication within bone. Connexin43 (Cx43) is the most abundant gap junction protein in bone tissue and plays a critical role in its development and maintenance. Deficiency in Cx43 results in general osteoblast dysfunction and delayed ossification Bone regeneration continues to be one of the most active areas of tissue engineering research. It is well-established that vascularization is critical to the field and there are a number of excellent reviews that discuss strategies for engineering vascularized bone in situ. The support structure would then be removed once the implanted bone developed sufficient strength.In most cases, one primary role of the skeletal system is mechanical support. The strength of any bone tissue implant is fundamental to maintaining appropriate function. The mechanical properties vary significantly within a given bone Engineering bone of sufficient volume to treat large defects commonly encountered in the clinic is one of the most significant barriers to application. Critical-sized defects in simple fractures in humans often result in a 2–3 cm gap in vitro and in vivoThe large majority of tissue engineering strategies apply cells to scaffolds to enhance tissue formation. For these techniques to be realized clinically, cell sources would need to be readily available. When evaluating vascularization of tissues, many studies use ‘model’ cell types, such as HUVECs which may form extensive vascular networks etc.) must be willing to adopt a new procedure. This may require that the treatment have significantly improved outcome in comparison to current standards of care. The decision is also influenced by trends, personal preferences, patient opinion and cost. Cost-effectiveness is an increasingly important consideration in any clinical treatment. Strategies that require extended in vitro culture, complex scaffold materials or preparation, or expensive proteins may place a significant financial burden on patients and/or the healthcare system. With continuously evolving healthcare systems, tissue engineers must proceed with careful consideration of approaches that may ultimately be cost-prohibitive.Surgical practicality and cost-effectiveness are necessary for a technique to become standard of care. Strategies involving multiple surgeries or implant locations increase the risk of complications and associated costs. In addition, the medical community  and a bone source, often osteoblasts or stem cells . The scaffolds may be supplemented with soluble factors or matrix proteins in an attempt to further enhance tissue formation.in vivoSeveral groups have investigated the use of polymer scaffolds combined with a single cell source to engineer vascularized bone. There have been numerous studies in which MSCs (bone marrow or adipose derived) have been seeded on a scaffold, resulting in increased osteogenesis in model systems in vivovia the release of angiogenic factors. Interestingly, combining these ECs with ASC-derived OBs in the same model resulted in less bone volume and microvessel density than ASC-derived ECs alone In these approaches, vascularization may also result from direct assembly of MSCs into vascular structures in vitro after 7 days and exhibited earlier osteogenic differentiation than in monoculture in vitroA more common strategy is to seed cells from both osteogenic and endothelial lineages into a scaffold to coordinate new bone formation and vessel assembly. Demineralized bone matrix scaffolds seeded with both bone marrow-derived EPCs and MSCs resulted in a significantly higher blood supply, biomechanical strength and bone mineral density than scaffolds without EPCs when implanted in a segmental defect model Cell spheroids have been investigated in a variety of tissue engineering applications due to increased cell-cell interactions in vitro prior to implantation. The goal of this approach is to achieve rapid and enhanced perfusion of the networks following implantation in vivoin vitro results in increased bone volume and vascular structures in vivoin vivo in under 2 weeks in vitroin vitro culture resulted in enhanced vessel formation and higher vessel density following implantation in vivo, with the MSCs exhibiting a pericyte-like stabilizing role These studies show that the combination of ECs and bone cells can increase both bone formation and vascularization. A more complex approach involves assembly of ECs into a vascular network in a scaffold The periosteum is a rich source of vasculature and osteoprogenitor cells that is known to enhance bone formation and healing. A tissue engineered periosteum is an emerging strategy for enhancing the formation of vascularized bone. HA/poly(ester urethane) scaffolds surrounded by a platelet-rich plasma (PRP) gel and seeded with MSCs have been investigated as an engineered periosteal substitute e.g. HUVECs) that are not a realistic cell source for clinical treatment of patients. In addition, the cells that are actually available in the potential patient population have substantial variability in osteogenic or angiogenic potential that could hinder translation into clinical application in vitro culture expansion could decrease the proliferation, differentiation potential and bone formation potential of MSCs Despite the success of cell-based bone tissue engineering strategies in research settings, many of the techniques used model cell types  scaffold assembled into vascular-like structures within bone tissue in a rotating wall vessel bioreactor Bioreactor strategies have seen some success in clinical studies, but these efforts have not yet transitioned to clinical use for bone tissue engineering. A primary concern is cost. The cost of a clinical bioreactor bone graft has been estimated to be $10,000–$15,000, taking into account the cost of cells, labour, testing, miscellaneous expenses and a portion of the initial cost to set up a bioreactor system  Not all tissue engineering strategies focus on the application of isolated cells. Cell-free strategies avoid issues of cell sourcing by focusing on the ability to induce surrounding cells to invade and generate bone of sufficient volume in time frames appropriate for clinical success. Typically, this is done through the delivery of growth factors or the application of specially designed bio-active scaffolds.in vitroVEGF is one of the most widely investigated growth factors for controlled stimulation of angiogenesis. An injectable alginate hydrogel releasing VEGF was shown to enhance angiogenesis in a rodent cranial defect model and concomitantly enhanced bone regeneration in the absence of additional cells via gelatin microparticles within a porous poly(propylene fumarate) scaffold resulted in increased bone and blood vessel volume in a rat cranial defect model  composition demonstrated evidence of remodelling by osteoclasts as well as supported EC tube formation in vitrovia cobalt ions 2+ ions to replace parts of Ca2+ ions, then seeded with bone marrow stromal cells and cultured for 7 days in vitroin vivoBioactive glass in vitroin vivo, scaffolds seeded with ASC aggregates exhibited increased cellularity and vascular density, particularly within the centre of the scaffold in vitro prior to implantation Scaffolds have been designed with physical and chemical features designed to enhance bone formation. The degradation rate of biomaterials can influence tissue development. Hyaluronic acid hydrogels with controlled degradation properties have been investigated to encourage oriented bone growth when combined with soluble factors Using bioactive materials to induce bone formation may be an effective treatment method that avoids the cost and risks associated with cells and soluble factors. However, many of these strategies may need to be supplemented with cells or soluble factors to optimize bone volume. Designing scaffolds to best mimic the structure of bone and its vasculature shows potential to speed bone formation by increasing the availability of nutrients and progenitor cells. The recent widespread popularity of 3D printing may make these techniques more accessible and clinically feasible, and could lead to further improvements in the design of patient-specific scaffolds.Tissue engineering has often progressed with the goal of engineering ready-to-implant, fully functional tissues. These strategies sometimes neglect the inevitable remodelling process that occurs following implantation in vivo bioreactor was created in rabbits by implanting a tissue engineering strategy around the saphenous vessel bundle and wrapping it with the muscularis membrane in vitro and allowed to adhere on scaffolds overnight prior to implantation in critical-sized segmental femoral defects in rabbits A vascular bundle inserted within a scaffold can help prefabricate the construct to improve vascularization and bone formation. An A surgically induced periosteal membrane takes advantage of the body's natural healing processes and has been characterized in humans Existing periosteum can be exploited as a source of osteoprogenitor cells and vasculature to enhance ectopic bone formation. Following ectopic growth and development of a tissue engineering chamber implanted against the periosteum, the new tissue can then be transferred to the defect site as a vascularized tissue. Chambers containing autologous morselized bone graft (MBG) implanted against rib periosteum in sheep generated significant calcified tissue within the chambers, with maximal after 8 weeks of implantation One approach that has been studied extensively for other engineered tissues is the arteriovenous-loop model Space maintainers can be used in conjunction with these strategies to preserve and enhance a defect site while the bone graft is grown at an ectopic location in vitro culture. Two-step surgical procedures may be the required strategies for further translational clinical applications, however, these strategies introduce additional risks and hospitalization costs.Surgical solutions for vascularized bone tissue engineering may help to enable the translation of basic research strategies into the generation of large bone volumes. However, these techniques have not been exploited significantly in the field of tissue engineering. In some cases, these techniques utilize autologous cells and proteins without extensive Many recent advances have been made towards engineering vascularized bone. The widespread utilization of autologous, clinically available cells is encouraging for the clinical translation of these methods. Additional insight into the complex interactions between osteogenic and ECs may lead to future success in cell-based strategies. Another major hurdle in developing tissue engineered vascularized bone grafts is scaling up to the appropriate volume. Many of the strategies discussed here result in small volumes of tissue on the order of millimetres, whereas defects are often on the centimetre scale. Surgical strategies are able to create larger volumes, but often require multiple surgeries or surgical sites, increasing risk of infection and other complications. Bioreactor-based approaches are designed to result in larger volumes, but high costs and long culture times could hinder their clinical implementation. The ultimate clinical success of tissue engineered vascularized bone requires novel strategies to overcome these challenges.


In addition to the obvious differences in body shape, there are substantial differences in lipid metabolism between men and women. These differences include how dietary fatty acids are handled, the secretion and clearance of very low-density lipoprotein-triglycerides, the release rates of free fatty acids (FFA) from adipose tissue relative to energy needs, and the removal of FFA from the circulation, including the storage of FFA into adipose tissue via the direct uptake process. We will review what is known about these processes and how they may contribute to the sexual dimorphism of body fat distribution. Where we store fat in our bodies is a significant predictor of metabolic disease risk. Metabolic diseases predominantly affect people with an upper body/android fat distribution compared to those with a lower body/gynoid distribution . Jean Va2 often have 25–35% body fat, whereas men of the same BMI have 10–20% body fat; highly athletic women and men can have less body fat  fatty acids (FA) is stored via lipoprotein lipase (LPL) into adipose tissue. In this pathway, endothelial LPL hydrolyzes FA from the glycerol backbone of chylomicron-TAG, allowing them to enter the adjacent adipose tissue. Some of the FAs from chylomicron-TAG are not taken up locally, but instead “spillover” into systemic circulation. A less appreciated pathway of FA storage occurs via the direct re-uptake of free fatty acids (FFA) from the systemic circulation independent of the LPL mechanism. Although we typically envision that the FFA released as a result of adipose tissue lipolysis are destined to supply fuel to lean tissues for fat oxidation, a portion is taken up and stored in distant adipocytes. We have referred to this process as direct FFA storage.In addition to LPL, there are a number of other proteins and enzymes that are involved in adipose tissue FA metabolism. When it comes to FA entry into cells, FA can be taken up by passive (flip–flop) diffusion or via membrane protein facilitated diffusion . One of The relative and absolute size of the different adipose tissue depots is ultimately determined by the balance between lipolysis on one hand and storage of FA from chylomicrons, very low-density lipoprotein (VLDL), and direct FFA uptake on the other. Although other factors, such as the gain or loss of lean tissue and changes in endocrine signaling, impact body composition, this review will focus on studies that examine the contributions of FA metabolism in determining regional body composition. Several studies have been conducted to expand our understanding of how the variation in body fat distribution in men and women relate to systemic and regional fat metabolism.Figure de facto have greater energy expenditure per kilogram FFM than those with greater amounts of FFM.In the postprandial and postabsorptive state, FA oxidation does not appear to be different between men and women whereas during exercise fat oxidation is higher in women. This is because there are several factors other than sex that are potential independent contributors toward the variations in fat oxidation between individuals including energy balance, age, heredity, and body composition . BecauseWhen the results of tracer studies were integrated, it was suggested that over a day that includes 2 h of exercise, 14 h in the postabsorptive state, and 8 h in the postprandial state, fat oxidation was similar in men and women . This coIn the fasting state, most circulating plasma TAG are in VLDL, which are synthesized and secreted by the liver . SimilarPatterns of meal FA storage only somewhat mirror the patterns of regional fat distribution. We initially found no differences in patterns of meal FA storage in men and women; more meal FAs were stored (per gram lipid) in upper body SAT than in leg SAT in both groups –32. HoweEven more so than meal FA storage, patterns of circulating FFA storage  correspond to the sexual dimorphism in fat distribution. When the direct FFA storage pathway was first reported, FFA storage into SAT was consistent with body shape in men and women. More specifically, direct FFA storage in SAT was less in men than women, and men stored more FFA in abdominal vs. femoral SAT depots whereas women did not . Follow-If rates of lipolysis were a major contributor toward sex differences in AT deposition, we would expect sex differences in the relative rates of total and regional lipolysis in men compared to women. For example, since women have greater lower body fat mass, lower rates of leg adipose tissue lipolysis would be observed in women compared with men. However, we found that the opposite is true. We found that leg adipose tissue lipolysis rates were 44 μmol/min in lean and 107 μmol/min in obese men, and 61 μmol/min in lean and 107 μmol/min in obese women. Thus, lipolysis in leg SAT of men and women was approximately equal, and even a little higher in lean women vs. men . MoreoveThe sexual dimorphism in adiposity along with differences in fat metabolism found in men and women allude to a compelling role of sex steroids in determining regional fat distribution. Several studies have examined the effects of sex steroid suppression and/or supplementation on fat metabolism. Table Periodic 24 h measurements over a 4-year period showed that fat oxidation did not change in premenopausal women, whereas in postmenopasual women, fat oxidation decreased 32% . HoweverWe believe the balance of data suggests that testosterone increases fat oxidation in men. One study showed that acute hypogonadism did not change fat oxidation over a 3-week period compared to men replete with estrogen and/or testosterone . In contStudies examining the effects of sex hormones on VLDL-TAG kinetics in men and women do not appear to clarify the lower VLDL-TAG concentrations in women. VLDL-TAG secretion in premenopausal women was found to be twice that of postmenopausal women despite higher VLDL-TAG concentrations in postmenopausal women . In anotFew studies have been conducted to examine the effects of estrogen on meal FA storage. We found that postmenopausal had greater meal FA storage than premenopausal women . We alsoThere have been a limited number of studies on the effects of acute or chronic testosterone hormonal manipulation on meal FA metabolism. We found that chronically hypogonadal men stored more meal FA in the femoral region than eugonadal men . WhereasWe matched pre- and postmenopausal women for age and body composition and found that direct FFA storage was greater in postmenopausal than premenopausal women . HoweverAlthough regional differences in the relative rates of lipolysis are unlikely to be central in determining regional fat differences in men and women, there are major differences in rates of lipolysis relative to energy expenditure , 39. TheTestosterone treatment has been shown to decrease total and abdominal fat mass and thus, would be expected to increase lipolysis. Testosterone treatment has been shown to stimulate lipolysis in the abdominal region  and in VThe objective of this review was to describe the differences in lipid metabolism between males and females and to link, when possible, these differences to how they may cause or create the differences in body shape. Subsequently, we also sought to explore how sex steroids regulate this relationship. Fat oxidation, lipolysis, VLDL-TAG, meal FA metabolism, and direct FFA storage may collectively contribute toward total and regional adiposity. Several studies have been conducted to examine these aspects of fat distribution in the context of differences in sex and sex steroids. Regional meal FA and direct FFA storage appear to contribute toward the sexual dimorphism in fat distribution. While the extent to which fat oxidation and VLDL-TAG kinetics contribute to body composition are unclear, it is evident that lipolysis has little effect in contributing toward differences in body shape between men and women.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.


Biomonitoring using birds of prey as sentinel species has been mooted as a way to evaluate the success of European Union directives that are designed to protect people and the environment across Europe from industrial contaminants and pesticides. No such pan-European evaluation currently exists. Coordination of such large scale monitoring would require harmonisation across multiple countries of the types of samples collected and analysed-matrices vary in the ease with which they can be collected and the information they provide. We report the first ever pan-European assessment of which raptor samples are collected across Europe and review their suitability for biomonitoring. Currently, some 182 monitoring programmes across 33 European countries collect a variety of raptor samples, and we discuss the relative merits of each for monitoring current priority and emerging compounds. Of the matrices collected, blood and liver are used most extensively for quantifying trends in recent and longer-term contaminant exposure, respectively. These matrices are potentially the most effective for pan-European biomonitoring but are not so widely and frequently collected as others. We found that failed eggs and feathers are the most widely collected samples. Because of this ubiquity, they may provide the best opportunities for widescale biomonitoring, although neither is suitable for all compounds. We advocate piloting pan-European monitoring of selected priority compounds using these matrices and developing read-across approaches to accommodate any effects that trophic pathway and species differences in accumulation may have on our ability to track environmental trends in contaminants.The online version of this article (doi:10.1007/s10646-016-1636-8) contains supplementary material, which is available to authorized users. Falco peregrinus), bald eagle , white-tailed sea eagle  and sparrowhawk (Accipiter nisus) populations have been widely documented in Europe and North America in the 20th Century  were among the first wildlife species known to be affected by anthropogenic pollutants. Pesticide and/or contaminant related declines in peregrine falcon , regulation of industrial chemicals, plant protection products, pharmaceuticals, and biocides is governed by directives such as Regulation (EC) No 1907/2006 and ammendements , Regulation (EC) No 1107/2009 concerning the authorisation of plant protection products, Regulation (EC) No 726/2004 concerning the authorisation of human and verterinary pharmaceuticals, and The Biocidal Product Regulation  528/2012). These directives apply across all EU Member States but lack any monitoring of their effectiveness in protecting against environmental pollution. This may be partly because such monitoring is challenging given the large spatial scales involved. However, a recent survey  non-destructive sampling. This includes blood and biopsies taken from live birds, plucked feathers and preen oil, and samples taken without contact with a living bird such as moulted feathers, addled or deserted eggs, regurgitated pellets, excrement, and tissues from carcasses that have been found and collected. Collection of samples can be demanding logistically since nests are often difficult to access and collection usually requires skilled and trained personnel, specialised equipment and legal permits. Therefore, if monitoring is to be conducted across continental and other large spatial scales, it is only likely to be feasible if it utilises samples that are already being collected as part of existing monitoring programmes or after careful prior selection of the suitable sample material and species. Two recent inventories collected by questionnaires  or continuously replaced feathers reduces the issues both of temporal displacement and, to a large extent, external contamination  and, depending on the timing of sampling relative to exposure, pollutant concentrations are generally lower and more variable than in body tissues. For lipid soluble contaminants such as POPs, low concentrations in blood are primarily related to relatively low lipid content compared to other commonly sampled tissues such as eggs and tissue  are to be measured. Advantages and disadvantages on using each compartment are summarised in Table Dried blood spots (DBSs) have been proposed as an alternative to fresh blood for monitoring chemical residues in raptors  of 0.94 (whole blood) and 0.98 (plasma) have been suggested, based on relative density  suggest that the highest concentrations of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), two PFASs widely used in industry  pellets were estimated to contain 25–29 % of ingested dose  were correlated with concentrations in internal tissues and the association was stronger than that between feathers and soft tissues. This was believed to be because talons, unlike feathers, grow continuously, and talon Hg concentrations are more likely to be representative than feathers of the current Hg body pool. Hopkins et al.   has been found to be exposed to and apparently killed by the NSAID flunixin  and carbamate (CB) compounds are used as antiparasitics in livestock and secondary exposure can result in raptor mortalities  exposed to imidacloprid suffered sublethal effects including altered biochemical parameters in plasma and oxidative stress in red blood cells  of interest, but we have attempted to show the relative merits of each matrix for major groups of contaminants. Of all the matrices, blood and liver are probably used most extensively for determining recent and longer-term exposure, respectively. However, blood samples are less widely and frequently collected across Europe than several other matrices Table  and voluAlthough a wide range of samples are collected across Europe, (failed) eggs and feathers are the two matrices for which collection effort appears to be greatest Table . These mSupplementary material 1 (DOCX 1062 kb)Below is the link to the electronic supplementary material.


Cystoid macular edema is a condition that involves the macula, caused by an accumulation of extracellular fluid in the macular region with secondary formation of multiple cystic spaces. This condition is provoked by a variety of pathological conditions such as intraocular inflammation, central or branch retinal vein occlusion, diabetic retinopathy and most commonly following cataract extraction, hereditary retinal dystrophies, and topical or systemic assumption of drugs. Niacin is a vitamin preparation usually used for the treatment of lipid disorders. The treatment with niacin, alone or in combination with other lipid-lowering agents, significantly reduces total mortality and coronary events and slows down the progression of and induces the regression of coronary atherosclerosis. Several cases of niacin-induced cystoid macular edema have been reported with different dosages. Cystoid macular edema (CME) refers to swelling of the central part of the retina. The macula is responsible for detailed central vision. When the macula experiences swelling (edema), central vision is reduced. CME represents a common pathologic sequel of the retina and occurs in a variety of pathological conditions such as intraocular inflammation, central or branch retinal vein occlusion, diabetic retinopathy and most commonly following cataract extraction, hereditary retinal dystrophies, and topical or systemic assumption of drugs –2. Several cases of niacin induced CME have been reported –6. The aCME is a disorder that involves the central retina, caused by an accumulation of extracellular fluid in the macular region with secondary formation of multiple cystic spaces . HistoloThe exact pathogenesis of CME remains uncertain. CME following disruption of the blood–retinal barrier (BRB) . When thCME is the consequence of diabetic retinopathy . The edeNiacin (nicotinic acid) is a vitamin preparation usually used for the treatment of lipid disorders . Niacin Niacin is available in a variety of formulations: immediate-release (IR), slow-release (SR), extended-release (ER) . StudiesNiacin improves endothelial function –54 and rThe problem with niacin is the high incidence of flushing (57). The flushing is cutaneous and usually restricted to the face and chest and is also associated with a burning sensation and often generalized pruritus. It lasts 20–30 min and habituates with exposure declining in both severity and frequency with time –59. Niacin-induced flushing appears to be due to the subcutaneous release of PGD2, which is mediated by niacin’s action as a pharmacologic ligand for the adipocyte and macrophage G protein–coupled nicotinic acid receptor GPR109A and appears to involve the formation of vasodilatory prostanoids –61. EpidThis effect is often temporary  but in 5Many authors reported ocular side effect after therapy with niacin. Fraunfelder et al.  reportedThe second and the most reliable hypothesis supports that the niacin has a direct toxic effect on Müller cells, without disruption of the blood-retinal-barrier. Alterations in cellular metabolism cause intracellular fluid increase and swelling of these cells, with secondary formation of cysts between the glial spaces. After the discontinuation of niacin therapy, there is a complete regeneration of the Müller cells and their normal function –76. RepoGass described reversible bilateral CME with no angiographic fluorescein leakage in 3 patients taking doses of niacin greater than 1.5 g daily to treat hyperlipidemia . AlthougIt was shown that the incidence of niacin maculopathy has been estimated to occur in 0.67% of patients being treated for hyperlipidemia. Studies have shown that even small doses of niacin cause CME. Patients with ocular symptoms such as blurred vision, decrease in visual acuity and metamorphopsia, should be immediately go to an ophthalmologist and stop the assumption of nicotinic acid before the onset of maculopathy. Niacin should be used with caution and under medical monitoring or periodic examination. Further studies would be desirable to investigate the safety dose, pathophysiologic mechanisms or predisposing risk factors and the possible interaction with others agents that may cause niacin maculopathy.The authors report no conflicts of interest in this work.