There is a growing appreciation within the livestock industry and throughout society that animal stress is an important issue that must be addressed. With implications for animal health, well-being, and productivity, minimizing animal stress through improved animal management procedures and/or selective breeding is becoming a priority. Effective management of stress, however, depends on the ability to identify and quantify the effects of various stressors and determine if individual or combined stressors have distinct biological effects. Furthermore, it is critical to determine the duration of stress-induced biological effects if we are to understand how stress alters animal production and disease susceptibility. Common stress models used to evaluate both psychological and physical stressors in cattle are reviewed. We identify some of the major gaps in our knowledge regarding responses to specific stressors and propose more integrated methodologies and approaches to measuring these responses. These approaches are based on an increased knowledge of both the metabolic and immune effects of stress. Finally, we speculate on how these findings may impact animal agriculture, as well as the potential application of large animal models to understanding human stress. Early in the nineteenth century, Claude Bernard defined stress as a perturbation of the otherwise constant state of the “milieu interieur”. . Fifty yThese changing definitions reveal how difficult it is to precisely define stress responses, which now includes a much broader definition of the fight, flight or freeze response . ContribTwo different signaling axes have been identified by which mammals mount an integrated physiological response to perceived danger . The resGlucocorticoid receptors (GRs) are present in the cytoplasm of most cells and are low affinity receptors for cortisol. Following activation of the HPA axis, GRs are activated by the ensuing elevated concentration of cortisol . GlucocoGlucocorticoids and their receptors function as checkpoints for energy homeostasis and mediate many of the stress-related effects on metabolism. A recent study revealed that carbohydrate metabolism, including fructose and pentose phosphate pathways, was down-regulated in response to heat stress, resulting in decreased metabolism in bovine peripheral blood leukocytes . Mehla aet al., reported that individual stressors, such as transportation versus combined transportation and weaning, had significantly different effects when assaying lipopolysaccharide (LPS)-induced TLR4 signaling by bovine blood mononuclear cells [Glucocorticoids can influence a broad range of both innate and acquired immune responses. For example, it is well known that glucocorticoids have anti-inflammatory effects, but a recent study demonstrated that glucocorticoids can also induce pro-inflammatory responses . This isar cells . The incar cells . Thus, dar cells .in vitro and observations made following elevation of endogenous cortisol during a stress response suggest that the regulation of cell signaling may be much more complex in vivo. Unravelling this apparent contradiction may require new ways to analyze cell signaling responses initiated under the integrated influence of both the HPA and SAM axes.The expression of pro-inflammatory genes can be inhibited by glucocorticoids through an interaction of the GRs with transcription factors, such as NF-κB and AF-1 . A varieGlucocorticoids have also been reported to have multiple effects on acquired immune responses . These e2- adrenergic receptors [Norepinephrine and epinephrine are two catecholamine neurotransmitters released by the SAM axis that exert effects through both α- and β-adrenergic receptors, but mainly βeceptors . This ineceptors . At a ceeceptors .Epinephrine influences glucose metabolism through two mechanisms. Firstly, through protein kinase AMPK-mediated activation of the transcriptional factor, coactivator peroxisome proliferator-activated receptor-γ coactivator 1α, which stimulates mitochondrial biogenesis. This results in an increased capacity for glucose metabolism . The oth2 adrenergic receptor and induce activation of AKT signaling pathways. This signaling stabilizes the cytoskeleton, increases focal adhesion formation, and eventually inhibits karatinocyte migration. These epinephrine induced changes in epithelial cell function can result in impaired wound healing [Catecholamines have also been linked to alterations in a wide range of immune functions . Epineph healing .Research related to stress in all species has increased dramatically over the last 20 years with over 50,000 citations in 2013 A. In conThermal stressors, including both heat and cold stress, are commonly experienced by livestock living in a natural environment. Heat stress has been studied in dairy cows and a variety of outcomes have been documented, including negative consequences for both health and productivity . Heat stTransportation is one of the most common stressors experienced by cattle, especially with increased national and international movement of animals. Epidemiological studies have implicated transportation as a factor contributing to increased disease susceptibility and bovine respiratory disease for over 50 years ,42. A frFeed deprivation may occur at many levels, involving specific micronutrients or inadequate intake of protein or energy. Controlled protein-energy malnourishment over a 4 week period in newborn Holstein calves resulted in altered blood leukocyte counts, decreased lymphocyte function, and reduced antibody responses . HolsteiMaternal separation has been identified as a potent stressor in infants and young animals that may have long-term psychological and physical effects . Abrupt et al., used nose-paddles to differentiate between a calf’s dependence on maternal nutrition and the psychological bond with a mother. These studies demonstrated that dependence on maternal nutrition was an important factor contributing to the psychological response following maternal separation [The effect of calf age at the time of weaning on innate immune responses of beef calves has been studied. An endotoxin challenge study revealed that following weaning, 80-day old calves had lower TNF, IL-1, and IL-6 responses than 250-day old calves . In contparation . This moCattle are herd animals that establish social orders with dominant and submissive animals within each group. As such, social isolation or introduction to a new social group can be significant stressors for an individual animal  resultinversus low serum cortisol groups based on measurements during their first lactation. When cows from both groups were then socially isolated for 55 h, epithelial cell permeability was consistently greater in the high cortisol group. The authors concluded that social isolation altered epithelial tight junctions and mammary gland function. Collectively, these observations indicate that social isolation not only alters behavioral responses but may also have an impact on a wide variety of body systems.The effect of social isolation on physiological parameters has also been investigated using a respiratory disease model. Veal calves infected with bovine herpesvirus 1 (BHV-1) were randomly divided into two groups. One group was housed as individual animals while the other group was housed collectively. Socially isolated animals had lower basal cortisol concentrations and responded to ACTH injection with a decreased cortisol release relative to group-housed animals . These sCurrent husbandry practices frequently result in social mixing or regrouping of both young and old cattle. A number of studies have analyzed acute behavioral and biological responses following the introduction of a single animal to an established group. In dairy cows, the introduced animal displayed altered behavior, including reduced allogrooming and decreased feed access, and this was associated with a 24 h reduction in milk production . Similaret al., investigated the combined effect of restraint and social isolation on adrenocortical and nociceptive responses in dairy cows [et al., studied the effect of restraint stress in pregnant heifers [Restraint is a common procedure used when handling cattle and may have negative effects on productivity . Herskiniry cows . Plasma iry cows . Szenci  heifers . Plasma  heifers .et al., also investigated the effect of repeated restraint episodes in Brahman cattle. Entry order, serum cortisol concentrations, heart rate, and respiratory rate were recorded during the experiment [et al., [et al., also reported that the order in which calves were willing to enter the chute was consistent among experiments [Andrade periment . In addiperiment . These r[et al., , reflecteriments . Thus, teriments .et al., were able to demonstrate that weaning and transportation doubled mortality in 5 to 6-month old, Angus X Hereford calves. Increased mortality was associated with a significantly enhanced innate immune response to both the viral and bacterial infection. This enhanced innate immune responsiveness also resulted in a significantly decreased interval between infection and death, suggesting increased immunity resulted in immune pathology rather than immune protection [Epidemiological evidence has linked a wide variety of stressors, especially transportation, with respiratory disease which was referred to historically as “shipping fever” . Transpootection . This isotection ,19. A liotection , Johne’sotection , and salotection  in dairyQuantifying responses to experimental or natural stressors is an important first step in determining the potential effects of acute or chronic stressors. Developing tools that can measure complex physiological or psychological responses is also important if we are to begin unravelling the interactions among multiple stressors, which may include both psychological and physical stressors. We have observed that transportation alone has markedly different effects on innate immunity than a combination of weaning and transportation . The difet al., reported entry orders were consistent across replicate restraint experiments [Behavioural responses to stress have been well studied in mice but a limited number of stress-induced behavioural studies have been performed in cattle . Entry oeriments ,74. Theseriments . Anothereriments . Frequeneriments ,74,77. TActivation of the HPA axis is frequently monitored through measurement of cortisol concentrations in either the plasma or serum , but theCortisol concentrations in cattle have also been measured in urine, saliva, hair, feces, and milk . CollectHPA and SAM axes activity has been measured by monitoring a diverse range of biological responses in cattle . For exaThe integrated study of the immune system and metabolism is an emerging research perspective. Historically, immunity and metabolism were treated as distinct biological processes. Immunity focuses on the recognition of and resistance to a pathogen or toxin and involves its own set of cells and tissues. Metabolism incorporates chemical processes that provide the energy to carry out the various functions of the organism. Energy-dependent functions include both innate and acquired immune responses. However, the overlap between immunity and metabolism has mostly been restricted to metabolism as a source of energy for the immune system. In view of the many effects of both the HPA and SAM axis on metabolism, it is then important to consider in greater detail the potential interaction between immunity and metabolism.The links between metabolism and immunity are extensive, and new interactions continue to be discovered. It is apparent that the responses of animals to different stress challenges are complex and multifaceted with unique phenotypic outcomes. To better understand these outcomes there is a need to better understand the molecular mechanisms that underlie stress responses. As already highlighted in this review, a number of investigative approaches, focused on different aspects and levels of molecular response have been employed to elucidate key events underscoring either metabolic or immune responses to stress. One emerging approach that is showing particular promise to provide a more comprehensive view of both metabolic and immune functions is the analysis of signal transduction at the level of kinase-mediated phosphorylation-dependent signal transduction. Across all eukaryotic species, protein phosphorylation is a central mechanism to regulate virtually every cellular process. These phosphorylation reactions are catalyzed by kinases and the collective activity of all the cellular kinases is referred to as the kinome.Protein phosphorylation mediated by kinases often represent the event initiating activation of a cellular process following the receptor–ligand interactions. This is of particular relevance when considering the release of mediators following activation of either the HPA or SAM axis. Understanding cellular responses at the level of the kinome offers an advantage over other omics approaches by providing insight at a biological level that immediately precedes phenotypic changes. As such, kinome analysis has the potential to offer a clear and unobstructed view of critical cellular responses while transcriptomics reflect cellular responses induced by this initial signaling. Kinome analysis is a relatively new technique but one of the first applications was to understand signaling responses induced in blood leukocytes of human volunteers immediately following an acute stress . From thOne challenge that often faces livestock researchers is the availability of new technologies that can be readily translated to the species of interest. A lack of cutting edge tools and reagents often limits the extent to which biological responses can be defined within food-producing animals. Fortunately, a variety of species-specific peptide arrays have been developed for kinome analysis. Arrays have been developed for cattle ,93,94, pStress has become a central issue in the growing debate on animal welfare and the psychological and physiological responses of animals to traditional husbandry procedures and new production technologies . This deThe management of pain and the stress associated with surgical manipulations, such as castration and dehorning, highlights the increasing concern regarding animal welfare . New tecThe co-evolution of domestic animals with humans has changed many of their behaviours and possibly their perception of danger or stress. If we can apply integrated tools, such as kinome analysis, to the measurement of physiological responses to stress it may be possible to begin the selection of animals with reduced stress responses. This integrated analysis of physiological responses will be important to not only understand the mechanisms mediating stress responses but to also understand the biological consequences of a reduced or altered stress response. Cannon defined stress as “the coordinated physiological processes which maintain a steady state in the organism” . TherefoAnxiety and stress-related disorders are debilitating psychiatric conditions that affect health, wellbeing and productivity of a large percentage of the population ,102. AnxAmong the many challenges to effectively diagnosing and treating stress is the wide spectrum of root causes and variation in individual’s responses to stress. The identification of molecular markers that effectively discriminate among various stresses, as well as adaptive and pathological responses to these states, would be of enormous benefit. For example, saliva cortisol levels are being examined as an indicator of various types of stress disorders as well as a read-out to monitor the efficacy of different treatment approaches . While aet al. demonstrated that the vast majority of research relating to immunological responses of mice did not have consistent results within human systems [Currently, as is the case for most animal research, rodents are the primary species for investigating stress responses and therapeutics. Mice offer considerable advantage in terms of cost, ease of handling, defined tools and tests for measuring cognitive responses, and availability of genetically manipulated mice to model human diseases. There are, however, growing concerns over the efficiency with which results obtained from mouse models can be translated to clinical application in humans. In particular, Seok  systems . This fiThe first advantage of a comparative approach to understanding stress responses across a number of species will be a greater evolutionary perspective on stress and the extent to which various responses are conserved across mammalian species. Understanding the conserved, core biology of stress responses, as well as unique species-specific differences, will help guide the selection of animal models based on conservation of molecular responses to similar stressors in humans. This will improve both the interpretation of results and their translation across species. The selection of animal models that best reflect or mimic human molecular responses would provide increased confidence in the selection or testing of therapeutics.Secondly, while research in mice enhances reproducibility by controlling genetic diversity, this does not accurately reflect the diversity of biological responses among humans to particular situations and/or treatments. A defining feature of many large animal experiments is the extent to which responses to a stimulus vary among individuals within an outbred population. While this complicates experiment design and interpretation, this provides a more accurate perspective on the challenges faced during the translation of results to clinical application. Furthermore, understanding the mechanisms mediating biological responses at the extremes of a specific phenotype is of central importance for defining biomarkers and treatments within human populations where these extremes are likely to represent priority patients. Finally, appropriately selected large animal models may be benefit in the identification of treatments that more effectively translate into human therapies. There are increasing examples of the use of large animal models, such as pigs, for the testing of therapeutics in the contexts of infectious disease , cancer The definition of stress has evolved to recognize that responses can vary widely among individual animals and that physiological responses to stress change rapidly over time. Furthermore, repeat exposure of an individual animal to a specific stress may result in adaptation to the stress or facilitation of an enhanced stress response. People involved in animal agriculture now face the challenge of accurately assessing individual animal responses to potentially stressful situations and determining whether homeostasis has been significantly perturbed. In the current review, we emphasized that perturbations of homeostasis have been measured in terms of both altered metabolism and immune function, which then impact animal health, well-being, and productivity. The emerging area of immunometabolism provides a framework for a much more integrated analysis of stress responses. Emerging methodologies, such as RNA-seq and kinome analysis, are species-specific tools that can be used to provide a much more comprehensive analysis of immunometaboism. These technologies provide not only a more comprehensive picture of physiological responses but their high throughput capacity makes it possible to profile dynamic changes that occur over time. The question remaining is how these technologies will be applied to ameliorate the potentially negative effects of animal stress. They will certainly be useful to better define the biological effects of individual or combined stressors and to define the duration of physiological perturbations following exposure to both acute and chronic stressors. A more controversial area may be the application of knowledge provided by these technologies to either the development of stress-inhibiting therapeutics or the genetic selection of animals. Will it be appropriate to ignore stressful animal production or handling systems if pharmacological agents can be used to inhibit physiological responses that negatively impact animal health or production? Alternatively, if selecting animals with a greater tolerance for stressful situations will it then be critical to determine if this selection compromises either animal health or production? The process of animal domestication has been dealing with this later question for thousands of years. We now have the tools, however, to ask more focused questions regarding the needs of both species involved in these mutually beneficial relationships.


Circadian rhythms are oscillations in behavior, metabolism and physiology that have a period close to 24 h. These rhythms are controlled by an internal pacemaker that evolved under strong selective pressures imposed by environmental cyclical changes, mainly of light and temperature. The molecular nature of the circadian pacemaker was extensively studied in a number of organisms under controlled laboratory conditions. But although these studies were fundamental to our understanding of the circadian clock, most of the environmental conditions used resembled rather crudely the relatively constant situation at lower latitudes. At higher latitudes light-dark and temperature cycles vary considerably across different seasons, with summers having long and hot days and winters short and cold ones. Considering these differences and other external cues, such as moonlight, recent studies in more natural and semi-natural situations revealed unexpected features at both molecular and behavioral levels, highlighting the dramatic influence of multiple environmental variables in the molecular clockwork. This emphasizes the importance of studying the circadian clock in the wild, where seasonal environmental changes fine-tune the underlying circadian mechanism, affecting population dynamics and impacting the geographical variation in clock genes. Indeed, latitudinal clines in clock gene frequencies suggest that natural selection and demography shape the circadian clock over wide geographical ranges. In this review we will discuss the recent advances in understanding the molecular underpinnings of the circadian clock, how it resonates with the surrounding variables  and its impact on population dynamics and evolution. In addition, we will elaborate on how next-generation sequencing technologies will complement classical reductionist approaches by identifying causal variants in natural populations that will link genetic variation to circadian phenotypes, illuminating how the circadian clock functions in the real world. Among its many facets, the daily environmental cycles of light and temperature generated by Earth's rotation around its own axis are essential ecological factors shaping a variety of adaptations in living beings. In order to harmoniously follow these periodic changes, most organisms, ranging from bacteria to humans, have evolved a genetic multi-oscillatory timekeeping mechanism known as circadian clock  and temperature throughout the year Daan, . While nDrosophila melanogaster (see below), the lack of a pronounced seasonal response in this species prevented its use for photoperiodic clock studies  to harmonize the clock with the changing environment.To circumvent the high complexity of the natural environment, experiments in the late 1950's/early 60's moved toward the lab, where environmental variables could be strictly controlled and tested separately . However, more recent studies simulating conditions at higher latitudes revealed unappreciated clock plasticity at both molecular and cellular levels . The Trikinetics system has been widely used in D. melanogaster  and constituted the first behavioral gene identified using mutagenesis  and CYCLE  in cis-regulatory regions close to the promoter of genes whose expression they control   and Pdp1ε expression , this lag of abundance promotes Clk circadian expression  expression  and E75 , which not only autonomously co-ordinate gene expression, but are also able to respond to changes in environmental conditions to maintain cellular homeostasis .In Drosophila this is mainly achieved by the flavoprotein Cryptochrome (CRY); cryb mutant respond barely to light pulses and is still rhythmic under constant light, a condition that leads wild type flies to arrhythmicity  and the Ionotropic Receptor 25a (IR25a) genes are expressed in the ChO but exert different functions, the first being important to synchronization at lower temperatures and the other to perception of small temperature cycles -mediated degradation of TIM by the Small Optic Lobe (SOL) protease  and a period of less activity in the middle of day, a behavior that was termed the “siesta”  peak. Importantly, unlike “A” and “E” peaks that are clock-modulated, only the tim0 mutant showed temperature independence in morning onset activity, suggesting that the “M” component is driven by twilight-dependent temperature cycles and not the circadian clock. Finally, temperature was more important for entrainment than light/dark cycles since locomotor behavior from flies in Leicester (UK) and Treviso  were quite similar, despite a considerable difference of daytime length between these regions in summer , how can allopatric individuals conserve their taxonomic identity if they are governed by the commands of different environmental conditions?For many years, biologists have been trying to understand evolutionary forces influencing genetic variation within and among species  and circadian rhythmicity , with adaptive latitudinal clines in both of them  was not included in the polymorphism of D. littoralis clock genes  residues 17 alleles are found in Northern and Southern regions, respectively 20 variant shows a period that is not sensitive to temperature change and is on average slightly shorter than 24 h, appearing to be better buffered against temperature swings . Maybe one of the reasons for the less steep (Thr-Gly)20 cline in Australia was due to the fact the data were not analyzed for the same latitude range when comparing the Thr-Gly distributions in Europe and Australia. Figure 20 alleles are as steep in Australia as the frequencies are in Europe 17 were higher on the “tropical” slope . ls-tim probably arose in southern Italy < 10,000 years ago and causes attenuated photosensitivity of the circadian clock  in midsummer, a condition that leads them to arrhythmicity , but also on anticipating these changes. Thus, the proper resonation of the clock with the environment is of fundamental importance for an organism to adapt to its surroundings.Drosophila, such as the molecular basis of temperature compensation, could not be extrapolated for other insect populations, such as mosquitoes and sand flies. For example, the Thr-Gly polymorphism in the per gene is pivotal for natural temperature compensation and adaptation of European populations D. melanogaster . This suggests that other mechanisms for temperature compensation in these species might be encoded in other parts of genome and raises an important point that although conserved, the circadian mechanism has species-specific differences that demand dedicated unbiased approaches in order to reveal them.Clock genes in insects (and other multicelular eukaryotes) encode important factors that modulate hundreds of genes to drive cyclical changes in physiology and behavior. Correlations of circadian rhythms with the environment have been demonstrated in several species, providing mounting evidence that having a circadian system that matches the oscillating surrounds is adaptive  was established as a set of 204 fully sequenced inbred lines (Mackay et al., Drosophila to adapt and predict natural variable surroundings.In this regard, recessive alleles complicate genetic association studies and hence the identification of causal genes in natural insect populations. Alternatively, inbred lines can be used for genetic analysis, particularly in conjunction with genome-wide markers or full genome sequences, to enhance the power for genetic association analyses. For example, in the past, inbred populations have been successfully used in evolutionary genetics studies (Falconer and Mackay, Drosophila and other insect species. In addition to assaying different populations from the same species, multiple comparisons between evolutionary distant species in the same geographical locations might also reveal important phenotypic and genotypic aspects of adaptation related to convergent evolution, and thus provide a more generalist idea of adaptation of temporal niche in species through evolution.It is tempting to recommend that functional genomics should follow the behavioral assays in natural conditions in GBSR, LGSRB and ACAMF contributed to the conception, design, draft, and editing of this article.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.


An increasing number of RNAs have been recently shown to possess regulatory functions similar to those of proteins. In bacteria, these regulatory RNAs are usually noncoding and are short size (50–500 nts) transcripts that are often referred to as small RNAs (sRNAs) Salmonella Typhimurium with an enhanced expression when the bacterium was challenged with antibiotics such as tigecycline or tetracycline Salmonella Typhimurium viability when exposed to tigecycline. Therefore, SroA sRNA activation in a strain exposed to tigecycline may influence bacterial resistance to that antibiotic. In an additional study using the Staphylococcus aureus strain as a model, the expression of some sRNAs correlated with exposure to four antibiotics  In the last few years, several studies have shown a correlation between bacterial sRNA expression and antibiotic exposure. An initial study identified four sRNAs in Antibiotics can use miscellaneous mechanisms of action to target vital processes such as nucleic acid and protein synthesis, as well as envelope integrity. Strikingly, various bacterial sRNAs are involved in these functions  [3]. As Some antibiotics alter the transcriptional machinery, and sRNAs have been found to modulate this process. For instance, the 6S RNA functions through interaction with housekeeping forms of RNA polymerase holoenzyme in gram-negative and gram-positive bacteria. Even if 6S sRNA has not been shown to directly affect antibiotic resistance by regulating transcription, cells lacking 6S RNA are defective for persistence Protein synthesis involves translation of an mRNA. Many antibiotics interfere with protein synthesis, targeting various steps of the translation process Many antibiotics have to penetrate the bacterial cell envelope to be effective. For instance, the outer membrane of gram-negative bacteria functions as a selective barrier by combining a hydrophobic lipid bilayer with pore-forming proteins (porins). Therefore, any factor influencing bacterial membrane synthesis or permeability could lead to antibiotic resistance. There are two cell-penetrating pathways that can be used by antibiotics: the lipid-mediated pathway for macrolides  and other hydrophobic antibiotics, and the general diffusion porins for hydrophilic antibiotics  phoPQ mRNA in Escherichia coliLipopolysaccharides (LPS) are components of the outer membrane of gram-negative bacteria and provide a barrier against antibiotics using the lipid-mediated pathway Alteration of outer membrane composition, particularly outer membrane proteins (Omp), represents one of the major mechanisms for antibiotic resistance. Thus, sRNAs such as MicF or MicC, which target the major porins OmpF and OmpC respectively, are likely to mediate antibiotic resistance. Moreover, at least six other sRNAs  have already been described as regulator of Omps Neisseria gonorrhoea to resist various hydrophobic antimicrobials, including penicillin and erythromycin. Interestingly, NrrF sRNA reduces mtrF mRNA levels and therefore inhibits MtrF production The active pumping of antibiotics out of the cells by efflux pump systems also contributes to antibiotic resistance E. coliS (or RpoS), DsrA triggers the expression of genes encoding the MdtEF multidrug efflux pump and thus affects antibiotic susceptibility. Since other sRNAs  are involved in rpoS regulation One additional example is the overexpression of DsrA sRNA, which was found to induce multidrug resistance in cirA mRNA that encodes a receptor and translocator of the antibiotic colicin Ia cirA mRNA is not accessible for translation initiation. However, under iron starvation conditions, RyhB sRNA is expressed and pairs with cirA and that allows the accessibility to the SD sequence Similarly, the sRNA RyhB activates the translation of S. aureus, it was observed that a sRNA was down-regulated when cells were challenged with vancomycin and ceftobiprole, two antibiotics that target the cell wall mecA gene encoding the penicillin-binding protein 2a (PBP2a) which induces a higher level of resistance to β-lactams. This observation suggests a putative role of this sRNA in antibiotic resistance. Recently, another S. aureus sRNA, SprX , was shown to inhibit SpoVG protein synthesis, a protein encoded by the yabJ-spoVG operon involved in glycopeptide and oxacillin resistance Microbial cell wall is an essential structural component that is responsible for reproductive division and for maintaining cellular integrity. Furthermore, the cell wall limits entry of potentially harmful macromolecules into the cell. In a large scale analysis of RNAs represent an unexploited area of potential molecules for antibacterial design. The recent discovery of sRNAs as a general class of powerful regulators has revolutionized our understanding of gene regulation. sRNAs are involved in many cellular processes in response to stress or when cells are challenged with antibiotics. Unraveling the functions of sRNAs in virulence and host immunity will provide fundamental knowledge that can be used to develop next-generation antibiotics using sRNAs as original targets. However, many unanswered questions remain with respect to RNA biology, but we are convinced that sRNA-based therapeutic treatment of infectious diseases may become useful tools in the near future.


Small populations are predicted to perform poorly relative to large populations when experiencing environmental change. To explore this prediction in nature, data from reciprocal transplant, common garden, and translocation studies were compared meta-analytically. We contrasted changes in performance resulting from transplantation to new environments among individuals originating from different sized source populations from plants and salmonids. We then evaluated the effect of source population size on performance in natural common garden environments and the relationship between population size and habitat quality. In ‘home-away’ contrasts, large populations exhibited reduced performance in new environments. In common gardens, the effect of source population size on performance was inconsistent across life-history stages (LHS) and environments. When transplanted to the same set of new environments, small populations either performed equally well or better than large populations, depending on life stage. Conversely, large populations outperformed small populations within native environments, but only at later life stages. Population size was not associated with habitat quality. Several factors might explain the negative association between source population size and performance in new environments: (i) stronger local adaptation in large populations and antagonistic pleiotropy, (ii) the maintenance of genetic variation in small populations, and (iii) potential environmental differences between large and small populations. The management of small populations remains a major focus of conservation biology. Habitat fragmentation due to ongoing anthropogenic activities has resulted in the depletion of many species, such that many now exist only as small, isolated populations. Population size is thought to be associated with risk factors that impact the capacity of populations to persist in a changing environment  outperformed large populations . By doing so, this analysis controlled for any potential confounding relationships between population size and the strength of local adaptation or habitat quality on performance not accounted for in the ‘home-away’ contrast above.Finally, the third ‘habitat quality’ analysis used data exclusively from reciprocal transplants to determine whether large populations tended to inhabit better-quality environments. By comparing the survival of individuals from the same set of populations within the same set of environments, this analysis could assess whether survival across these environments was associated with the size of the populations naturally inhabiting them, while controlling for the effect of local adaptation and source population size on survival.We conducted keyword searches on the academic search engine ISI Web of Science™. A complete keyword search of ‘local* adaptation*’ + ‘reciprocal* Transplant*’ was performed, as well as for the phrases ‘phenotyp*’ + ‘plastic*’ + ‘Transplant*’. References within studies were then used to obtain studies missed by keyword searches, with emphasis on other reciprocal transplants and meta-analyses.Survival was chosen as a relative fitness component for our three analyses due to its relatively unambiguous relationship with fitness and ease of standardization across studies. Only populations for which survival data and measurements of adult census population size could be found were included in the analysis. While suitable transplant experiments were quite common in plants, few of these experiments have been conducted on vertebrates outside of salmonid fishes; all suitable vertebrate studies found were conducted on salmonids.Many transplant studies reported survival in both native and novel (‘away’) environments, but lacked data on source population size, whereas others reported population size but lacked survival data. For many studies, source population size data were found using other resources , particularly for well-studied salmonids. If relevant fitness or population size data were unobtainable in the original paper or through secondary sources, primary and secondary authors were directly contacted to obtain the information. When survival and/or population size information was contained in figures, the program ImageJ  the transplanted population's taxa  and (ii) the life-history stage of the transplanted organism , as this can affect subsequent performance in plants and salmonids  model with ESSpecies, population, and transplant site were included as random effects in all models to control for issues of nonindependence (pseudoreplication) arising from multiple comparisons. Many species and populations included in our study were examined at multiple LHS, so random effects were conditioned on life-history stage. Although study is typically included in meta-analysis as a random effect, it was omitted here because of its almost complete correlation with species (few studies examined the same species) and because most studies examining the same species were conducted by the same researchers.V = 1, ν = 0.002) did not significantly affect model conclusions.To assess the effect of source population size on performance in novel environments, a formal meta-analysis was conducted using the MCMCglmm package Hadfield  in R 3.0LOR) is solely used, it is possible that one population might exhibit greater performance in all environments relative to another transplanted population but exhibit a reduced effect size . That is, comparing a population's performance in transplant environments relative to its performance in its home environment does not control for a population's overall performance relative to others. We therefore also collated and analyzed the survival of individuals from multiple source populations of known size that were transplanted to novel common garden natural environments, including reciprocal transplants.If the previous statistic   Akaike , correctTo assess the potential relationship between habitat quality and population size that may have confounded previous estimates of population size and fitness Oakley , a thirdlme4 , of which 102 populations were from plants and 9 from salmonids . Evidence was also found that the effect of source population size depended upon the life-history stage of the transplants .The best fit model describing overall performance in natural common garden environments included all random effects, fixed effects, and two-way interactions . When transplanted to their native habitat at early LHS, all populations, regardless of source size, performed equally well . When organisms were transplanted at later LHS to their native environments, source population size had a positive effect on performance that was significantly different from zero . Although a trend was observed that small populations exhibited maladaptation and large populations exhibited local adaptation at later LHS, neither large nor small populations exhibited significantly different overall performance in native relative to novel environments. In novel environments, the effect of source population size on performance at later LHS was small and not statistically different from zero .The effect of source population size differed for organisms transplanted at later LHS relative to those transplanted at earlier stages , but this was dependent upon the life-history stage of the transplant . The effect of source population size also depended upon the life-history stage of the transplants  and whether they were transplanted to a novel environment or their native environment .When only reciprocal transplants were examined, the relationships between performance and source population size, life-history stage, and local adaptation remained consistent with the previous analysis or increased in strength. Populations exhibited local adaptation . The effect of source population size on transplanted organisms differed between native and novel environments , with source population size having no effect on performance at early LHS within native environments . Source population size had a positive effect on performance in native environments at later LHS in reciprocal transplants . However, organisms transplanted at later LHS exhibited no effect of population size on performance in novel environments . No evidence was also found that the performance of organisms transplanted at later LHS differed between native and novel environments.In reciprocal transplant experiments, only early life-history stage transplants exhibited local adaptation. Similar to the previous analysis, this was a due to a negative effect of source population size on performance in novel environments at early LHS , not adult census population size, is the most appropriate measurement to use , below which populations should experience significantly low levels of genetic variation and detrimental levels of inbreeding . Furthermore, the large number of populations sampled relative to the number of species may help control for variation in the response to novel environments.Why did we find evidence that small populations exhibited similar or slightly better performance relative to large populations when transplanted to novel natural environments, when previous analyses based on observational studies or artificial common gardens have found significant positive relationships between source population size and fitness  during later LHS, we did find a statistically significant association between source population size and performance in later LHS that was exhibited within native environments. This finding is consistent with previous results from observational studies that found positive associations between population size, fitness, and local adaptation in wild populations in their native habitats Reed  and coulSmall populations exhibited similar or better performance relative to large populations in novel common garden environments, providing no evidence of genetic Allee effects resulting from reduced genetic diversity, increased inbreeding, and increased genetic load (Willi et al. If large and small populations inhabit environments that vary systemically, previous observational studies examining the relationship between population size and fitness may potentially be confounded. While we did not find any association between habitat quality and population size, habitat may vary systematically between large and small populations in other ways. Habitats inhabited by small populations may tend to be more variable, for example (Wood et al. Our meta-analysis raises important questions about the nature of commonly observed fitness trade-offs in local adaptation studies Hereford  and how We found some evidence that source population size was associated with decreased performance in novel environments during life-history stages at which local adaptation is strong. However, because of limited data in the literature, we cannot presently conclude whether the performance of large populations in their native environments was compensated by increased local adaptation, although we postulate that it is likely based on related findings in previous studies (e.g., Reed We also found no evidence for potential genetic Allee effects associated with small population size in novel environments. Under some novel selection regimes, small populations appear to cope with short-term environmental change as well as – or better than – large populations. Whether this also translates into enhanced long-term persistence is unknown: the potential for increased genetic diversity in larger populations may allow them to better adapt to novel change over subsequent generations than small populations, despite an initially larger demographic impact. Many organisms can respond to environmental change through rapid adaptation, in which case large population size may play a significant and important role (i.e., Samani and Bell Furthermore, the widespread distribution and/or generalist nature of most of the species in our study could affect the influence of population size on performance in new environments. Generalist species that are capable of tolerating a wide range of environments may be buffered against environmental change through phenotypic plasticity and/or could be capable of persisting at small population sizes due to nonevolutionary responses. Conversely, specialist species that occupy narrow niches and limited geographic ranges are already vulnerable to environmental disturbance and prone to extinction (Kotiaho et al. Future research into the effect of source population size on the strength of local adaptation and performance in novel, natural environments should endeavor to focus on the magnitude of trade-offs associated with local adaptation at multiple LHS. Additional research into the performance of subsequent generations in transplant environments could assess the long-term adaptive consequences of source population size and its effect on genetic variation, an issue of particular relevance for both the conservation of threatened species and invasive species biology (Theoharides and Dukes Reciprocal transplants represent the best research designs available to control for potential confounding effects that could influence estimates of the effect of source population size and may also allow researchers to disentangle the magnitude of trade-offs associated with local adaptation. We would encourage future reciprocal transplant experiments to include, when possible, population size estimates.


Vulnerability assessments have often invoked sustainable livelihoods theory to support the quantification of adaptive capacity based on the availability of capital—social, human, physical, natural, and financial. However, the assumption that increased availability of these capitals confers greater adaptive capacity remains largely untested. We quantified the relationship between commonly used capital indicators and an empirical index of adaptive capacity (ACI) in the context of vulnerability of Australian wheat production to climate variability and change. We calculated ACI by comparing actual yields from farm survey data to climate-driven expected yields estimated by a crop model for 12 regions in Australia’s wheat-sheep zone from 1991–2010. We then compiled data for 24 typical indicators used in vulnerability analyses, spanning the five capitals. We analyzed the ACI and used regression techniques to identify related capital indicators. Between regions, mean ACI was not significantly different but variance over time was. ACI was higher in dry years and lower in wet years suggesting that farm adaptive strategies are geared towards mitigating losses rather than capitalizing on opportunity. Only six of the 24 capital indicators were significantly related to adaptive capacity in a way predicted by theory. Another four indicators were significantly related to adaptive capacity but of the opposite sign, countering our theory-driven expectation. We conclude that the deductive, theory-based use of capitals to define adaptive capacity and vulnerability should be more circumspect. Assessments need to be more evidence-based, first testing the relevance and influence of capital metrics on adaptive capacity for the specific system of interest. This will more effectively direct policy and targeting of investment to mitigate agro-climatic vulnerability. Crop yields are vulnerable to climatic variability and change –6. WhileMost previous relevant work on the adaptation of crop production to climatic variability has been framed in terms of crop-drought vulnerability. A plurality of concepts, methods, data, and indicators have been used in this context, reflecting the broader vulnerability field in general . TypicalVulnerability and adaptive capacity assessments have often been grounded in sustainable livelihoods theory (SLT) ,22–25. Ttheory-driven, studies assume that selected capital indicators influence adaptive capacity and vulnerability based on the tenets of SLT  an an13] anThere are several limitations to the data used in our study and these will have influenced the results. While our empirically-based vulnerability indicator combined two of the best and most widely used agricultural information sources in Australia—they both have limitations. The ABARE AgSurf farm survey data used to quantify actual wheat yields include low sample sizes relative to the large spatial extent and diverse farming systems of each region, resulting in substantial uncertainty . The APSOur results lead us to draw somewhat different conclusions about regional vulnerability and adaptive capacity in Australian farming than previous deductive studies that built indices of adaptive capacity based on the general logic of SLT but did not test whether capital measures influenced observed vulnerability. For example, the index of vulnerability of rural communities in Australia constructed on SLT logic by Nelson et al.  designatThis finding suggests that caution and further empirical effort should be exercised in applying the SLT in assessing adaptive capacity and vulnerability. The approach is growing in popularity and we concur that it is an effective way to organize thinking about how endowments of multiple forms of capital may influence vulnerability and adaptive capacity of households, farms, and regions to disturbances such as climatic variability and change . HoweverCommon practice in vulnerability assessments involves constructing indices of social, human, physical, natural, and financial capital measures assumed to affect adaptive capacity based on the deductive logic of sustainable livelihoods theory (SLT), and then using the indices to identify vulnerable regions. To date, however, there has been little testing of the assumption that capitals influence adaptive capacity in the assessment of agro-climatic vulnerability using the SLT. We developed an empirical measure of vulnerability and adaptive capacity based on indices of actual and expected wheat yield for 12 regions in the wheat-sheep zone of Australia from 1991–2010. We then assembled data for 24 capital indicators and statistically tested their effectiveness in explaining adaptive capacity. Only six of 24 indicators were significantly related to adaptive capacity in ways predicted by theory, four were significantly related but with the opposite sign to what would be predicted by theory, and the other 14 were unrelated. We conclude that the use of SLT should be more circumspect and that further empirical work is needed on the selection of capital indicators which have an empirically-established relationship with adaptive capacity. Failure to do this may result in misguided policy and targeting of investment for increasing adaptive capacity and reducing vulnerability to climate variability. While we concur that SLT is a useful construct, we believe that there is still significant work required to make it operationally useful. In agro-climatic vulnerability analyses, more work to understand farm scale diversity and other drivers of the differential between actual and expected yield would also be useful.S1 Dataset(XLSX)Click here for additional data file.S1 Supporting InformationTransformations with the lowest chi square value are best.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Our experience, memories, and knowledge have modulatory influence on how we perceive the world. Top-down expectancies are supposed to be implemented as templates in our minds. Mental templates are compared against the current sensory input, which can match or mismatch with the aim of minimizing prediction error Friston, .But what are the underlying neuronal mechanisms leading to the activation of mental templates and their comparison with sensory input, i.e., predictive coding? Biasing sensory processing by expectancies has been strongly associated with prefrontal brain activity influencing responses in visual cortex  is an indicator for locally highly synchronized neuronal activity. If two distant brain areas are functionally coupled, it is assumed that a higher level of coherent, synchronous neuronal activity can be found between these distant areas than one would expect from chance. Comprehensive work by von Stein and co-workers suggests that long-range interaction between prefrontal and posterior cortices necessary in top-down control of cognitive processes is reflected by neural activity resonating in large-scale networks and therefore oscillating at rather slow frequencies: so called theta and alpha oscillations , alongside more distributed cortical activity, and high frequency oscillatory brain activity occurring particularly in visual areas. Evoked gamma activity as a signature of predictive coding is very compelling in visual perception of semantically congruent information .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.


Development of an effective HIV/AIDS vaccine remains a big challenge, largely due to the enormous HIV diversity which propels immune escape. Thus novel vaccine strategies are targeting multiple variants of conserved antibody and T cell epitopic regions which would incur a huge fitness cost to the virus in the event of mutational escape. Besides immunogen design, the delivery modality is critical for vaccine potency and efficacy, and should be carefully selected in order to not only maximize transgene expression, but to also enhance the immuno-stimulatory potential to activate innate and adaptive immune systems. To date, five HIV vaccine candidates have been evaluated for efficacy and protection from acquisition was only achieved in a small proportion of vaccinees in the RV144 study which used a canarypox vector for delivery. Conversely, in the STEP study (HVTN 502) where human adenovirus serotype 5 (Ad5) was used, strong immune responses were induced but vaccination was more associated with increased risk of HIV acquisition than protection in vaccinees with pre-existing Ad5 immunity. The possibility that pre-existing immunity to a highly promising delivery vector may alter the natural course of HIV to increase acquisition risk is quite worrisome and a huge setback for HIV vaccine development. Thus, HIV vaccine development efforts are now geared toward delivery platforms which attain superior immunogenicity while concurrently limiting potential catastrophic effects likely to arise from pre-existing immunity or vector-related immuno-modulation. However, it still remains unclear whether it is poor immunogenicity of HIV antigens or substandard immunological potency of the safer delivery vectors that has limited the success of HIV vaccines. This article discusses some of the promising delivery vectors to be harnessed for improved HIV vaccine efficacy. Thirty years after the discovery of HIV/AIDS, the search for a safe and effective vaccine has intensified, as a number of promising candidate vaccines progressing to phase IIb/III clinical trials have failed to show efficacy. One of the greatest barriers to HIV vaccine development is the enormous virion diversity (depicted by the existence of numerous clades and subtypes in distinct geographic demarcations) and the continuous evolution which generates numerous quasi-species within an infected individual  employing a broad range of vaccine delivery vectors have been tested and some have progressed to evaluation of potential efficacy in phase IIb/III trials. Of significant relevance as far as safety is the STEP trial that used human adenovirus serotype 5 (Ad5) to deliver a well-designed HIV immunogen expressing Gag/Pol/Nef, which was associated with increased risk of HIV acquisition in uncircumcised male vaccinees with pre-existing immunity to Ad5  and lysosome associated membrane protein (LAMP1), all of which significantly enhance transgene expression and trafficking, thus leading to increased vaccine immunogenicity , a protein found in blood and known to bind strongly to DNA  in the macaque model have reported protective immune responses which controlled SIV/SHIV replication or protected from infection  did not differ significantly from those in a simplified ChAdV63-prime and MVA-boost (CM) regimen. The superior immunogenicity of this regimen is not unique to HIV immunogens, as it has also been demonstrated in preclinical and clinical studies of experimental malaria vaccines  cellular and antibody responses to malaria antigens, either on its own or when used to boost responses primed by vectors such as DNA, fowlpox or AdHu5  is their ability to induce mucosal immune responses when administered via mucosal routes  showed no efficacy in earlier trials (VAX003 and VAX004), the success of RV144 points to the delivery vector (ALVAC) and possibly the benefits of a combined viral vector and protein immunization regimen as opposed to homologous boosts. This might suggest that combined live vector-priming and protein-boost immunization modalities could be further refined to achieve greater potential for increased efficacy. Alternatively, protection by the combined vaccines could be attributed to T cell help for the antibody responses. It must however be noted that unlike the RV144 study, VAX003, and VAX004 were conducted in high-risk populations, which might be a strong confounding factor, although this might as well be reflective of the very limited efficacy of stand-alone protein subunit vaccines for HIV. Despite the modest efficacy of RV144, the immune responses waned within a short time indicating that ALVAC may not be a particularly suitable vector to induce long-lived anti-HIV immunity, unless it is combined with other powerful vectors. In direct comparison of immunogenicity, ALVAC was found to be less immunogenic than MVA, possibly due to MVA's enhanced antigen expression within dendritic cells . As such, BCG provides a platform to co-deliver HIV immunogens in neonates to potentially protect against mother-to-child transmission of HIV-1. The potential use of BCG as an HIV vaccine vector was explored in preclinical studies of adult and new-born BALB/c mice using the HIV-1 clade A Gag immunogen (HIVA)  was shown to persist in vaccinated rhesus macaques and conferred durable protection from disease progression owing to induction of high magnitude effector memory CD8+ T cells, despite pre-existing CMV immunity  administered intranasally as a boost following intramuscular priming with an envelope-independent DNA vaccine (CMV-SHIVdEN) demonstrated very strong suppression of intravenous SIVmac239 challenge in rhesus macaques, which was extended over a 3-year period  and achieved significant protection against intra-rectal challenge of rhesus macaques with SIVsmE660 that was a mismatch of the vaccine strain  would have been different if other delivery vectors  had been used to deliver the same immunogens in these trials is key for further progression in the field. An Alternative way to look at this is to ask whether the results of RV144 trial would have been worse if Ad5 was used instead of ALVAC, assuming that the prevalence of Ad5 neutralizing antibodies in the RV144 population does not differ significantly from the STEP and Phambili study populations.The finding that the vaccine was not at all efficacious amongst men who were circumcised or in uncircumcised men who did not have pre-existing Ad5 immunity raises doubts as to whether efficacy was genuinely hindered by Ad5 serostatus. This is further supported by the results of HVTN505 study which tested only circumcised individuals without Ad5 antibodies, yet no protection was observed. Moreover, the absence of Ad5 antibodies in the HVTN505 study participants  was not associated with any significant enhancement of the magnitude and quality of immune responses over those seen in the STEP and Phambili studies. Therefore, Ad5 serostatus can be safely removed from the equation, leaving the only plausible explanation for vaccine failure to be the quality and quantity of immune responses. If this can be fully documented beyond doubt then it implies that either the Ad5 delivery vector or the HIV-1 antigens used were not immunogenic enough to afford protection from infection or post-infection virus control. However, considering that Ad5 is one of the most immunogenic vectors currently available, (and that the immunogen used in these studies was comprehensive and well-designed), this would have serious implications for vaccine design, as it sets the bar really high for new candidate vaccines which would be expected to stimulate responses of extremely higher magnitudes and superior qualitative properties in order to achieve even the minimal efficacy. On a brighter side, this would perhaps instigate intense scrutiny of the current methods used for assessing vaccine immunogenicity in order to standardize and synchronize with those for efficacy measurements.One other interesting question is whether (and how) Ad5 sero-positivity is intrinsically associated with HIV acquisition. Although studies of uncircumcised men document increased risk of natural HIV acquisition due to a high frequency of CD4+CCR5+ target cells in the foreskin  had the vaccines been administered mucosally. This question might have two sides to it, in the sense that mucosal delivery would probably have generated higher frequencies of activated HIV targets at the genital mucosae, hence increasing the potential of fuelling infection. On the other hand, induction of robust and polyfunctional effector immune responses at mucosal portals of HIV entry would probably have cleared the incoming HIV before infection became established. Although these questions have no clear cut answers and cannot be addressed retrospectively in the context of the clinical trials they relate to, they however highlight the extreme challenges in HIV vaccine delivery, and new studies designed to directly tackle these issues will be quite informative for future vaccine development research. Studies looking at whether the most promising delivery vectors (and the respective immunogens) can concurrently induce activated HIV-1 target cells that preferentially home to and persist in the genito-rectal and GALT mucosae, and whether or not such vaccine-induced cells become highly permissive to HIV infection will be of particular interest in efforts aimed at limiting the risk of vaccine-induced HIV-1 acquisition and accelerated disease progression.Ideally, vectors for HIV-1 vaccines should directly target antigen presenting cells (APCs) or other immune cells to induce long-lived, strong antibody and cellular responses that can broadly disseminate to systemic and mucosal compartments. The vaccine-specific T cells in particular should be broad and contain activated effector, effector memory and central memory phenotypes in various proportions in order to achieve a proper balance between immediate virus clearance and sustained immune-surveillance for long-term protection, as demonstrated by the RhCMV-SIV vaccine which controlled and cleared pathogenic SIV infection  vectors or vectors with slow immunogen release features would induce durable immunity. Nonetheless, replicating vectors with lower sero-prevalence and minimal pathogenicity  lengths of time. Of utmost importance, however is generation of vaccine-specific immune responses in the genito-rectal mucosae, the major portals of HIV entry. Emerging evidence strongly suggests that non-pathogenic, low-level replicating viral vectors which can mimic live attenuated vaccines, but with low sero-prevalence might be the best way to achieve HIV vaccine efficacy. As these vectors persist long after immunization, they are capable of inducing and maintaining effector/memory CTLs for continued immune surveillance that is necessary to protect from infection, disease progression and to clear or prevent establishment of latent reservoirs. Thus, to achieve protective efficacy HIV vaccine development will need ingenious state of the art technologies to create the very best of T cell and antibody immunogens, delivered by the most potent but safe vectors possessing remarkably high capacity to induce both systemic and mucosal immunity, but without significant immune activation likely to fuel HIV acquisition. Recent significant advances in vaccine delivery technologies and HIV immunogen design provide hope that this is not far from reality.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.


In recent decades, new research into the developmental defects and pathophysiological basis of congenital diaphragmatic hernia (CDH) has revealed opportunities for the development of innovative therapies. Importantly, the use of animal models to represent this anomaly in the laboratory has resulted in the discovery of many important genetic, epigenetic, and other molecular contributors to this condition. In this review, the most commonly used and newly devised animal models of CDH are presented to familiarize the reader with the latest innovations in the basic sciences. Pediatric surgeons continue to face the challenge of treating a variety of congenital anomalies and neonatal disorders for which the etiology and pathophysiology are poorly understood and under-studied. In addition to the fact that many of these defects are rare in overall incidence and lack a known genetic cause, there has been a general dearth of biological or animal models to specifically study the conditions that result from developmental failures. However, for congenital diaphragmatic hernia (CDH), there has been much research effort and productivity. Over the past four decades, an increasing number of animal models – small and large animal, teratogenic, and experimental, have been employed to give scientists and clinicians the basis toward a better scientific understanding of this condition, we treat surgically. In this paper, I will provide a brief and updated summary of the currently available animal models for CDH, the history of their use, discoveries made using these models and highlight new insights for future studies.Congenital diaphragmatic hernia is a rare birth defect affecting 1 in 2500 live births , 4 compaTo perform this research, scientists have utilized rodent models (teratogenic and genetic) and the experimental large animal  model. These two models have been functionally although not exclusively complementary in their research applications. The teratogenic rodent model has a long history and is widely used to determine the fundamental developmental and physiological defects of CDH, specifically in dissecting the molecular and cellular features of CDH in order to understand their effects on diaphragm, lung, and vascular development . In paraThe most commonly used teratogenic model for CDH is the nitrofen model. Nitrofen , is 2,4-dichloro-1-(4-nitrophenoxy)benzene, a Protox-inhibiting herbicide of the diphenyl ether class. Its use has been banned in Europe, Canada, and the United States since the mid-1990s for its documented carcinogenic effects on rodents (liver and pancreatic adenocarcinomas) and is considered a Group 2B class carcinogen (possibly carcinogenic to humans) , 15. It −/−) or the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) gene (see the section below) showed similar changes in PPF morphology, there appears to be good evidence that similar changes underlie the human defect.Initial studies using the nitrofen model were particularly fruitful in debunking embryological myths surrounding CDH pathogenesis, with the most detailed studies coming from John Greer’s group. While it had been speculated that CDH development resulted from the failure of the pleuroperitoneal canal to close in mid-gestation thereby causing the diaphragmatic sequelae, the nitrofen model revealed that CDH occurs early  from devWith access to the earliest stages of CDH pathogenesis in this teratogenic model, multiple developmental pathways have been implicated as the mechanism(s) driving nitrofen-induced CDH. Initial observations suggested that nitrofen was thyromimetic and exerted its teratogenic effects by disrupting the maternal–fetal thyroid hormone pathway through suppression of maternal TSH levels . However2 receptor antagonist originally developed to inhibit platelet aggregation , is a spermatogenesis inhibitor whose teratogenic effects in addition to CDH include cardiovascular anomalies , a metabolite of AH23848, a thromboxane-Aregation . Bisdiamnomalies . SB-2106nomalies . Importanomalies , leadingWhether by intention or serendipity, genetic models of CDH became available as molecular techniques for gene silencing or over-expression grew into wider application. While the majority of human CDH cases are not associated with known genetic defects [I will refer the reader to reference  for an eGenes encoding hormone receptors overlapping with the retinoid pathway have also yielded the CDH phenotype when their expression has been disrupted. The COUP-TFII gene, also known as NR2F2, is a transcriptional protein belonging to the steroid/thyroid hormone receptor superfamily whose expression is regulated by retinoids and, in turn, include among its functions the regulation of gene transcription by modulation of retinoic acid receptor heterodimerization , 47. Of WT1, in two cases of human CDH-associated with Denys–Drash syndrome have been reported (WT1 in mice was found to be associated with fetal CDH development (slit3 gene develop a central CDH similar to Morgagne hernias rather than Bochdalek hernias (fog2 mutation were found to have diaphragmatic defects in the posterior and peripheral aspects (gata4 developed heart, lung, and diaphragm defects (The role of genes less obviously associated with human CDH has also been revealed through genetic models. Mutations of the Wilm’s Tumor 1 gene, reported . Abrogatelopment  with itselopment . In some hernias . Friend  hernias  that pri hernias . FOG2–GA aspects  while mi defects . Interes defects .While the CDH rodent models are scaled and optimized to uncover early developmental and genetic derangements contributing to CDH, large animal models for CDH are scaled for life-sized interventions of the affected lung or diaphragm for direct translation to the clinical domain. As large animal models of CDH require diaphragmatic defects to be created during early stages of fetal development, this model cannot be used to study the earliest origins of the CDH diaphragmatic defect that is better served by the rodent models. The fetal lamb is the most commonly used experimental model but some researchers have also used rabbits for similar studies .One of the pivotal reasons for choosing the fetal lamb model for CDH was that there was already a large body of literature on fetal lamb pulmonary physiology prior to its adoption as a CDH model. With this background, the fetal lamb model for CDH has aided the study of ventilator-related lung damage observed in CDH and the development of fetal interventions aimed at arresting or reversing CDH-associated pulmonary hypoplasia. As the fetal lamb model was more expensive and time-consuming for CDH research compared to rodent models, most investigations focused exclusively on defining the pulmonary effects of CDH or fetal ventilation strategies –61. To cTwenty-five years after he first started his laboratory studies in the fetal lamb model for CDH, the first randomized controlled trial of fetal tracheal occlusion for the treatment of high-risk CDH was reported by Harrison et al. in 2003 . While tAnimal models for CDH have generated vast troves of data in diverse areas of science and medicine. Rodent and fetal lamb models for CDH have encouraged crosstalk between geneticists, developmental biologists, and clinicians to discover new knowledge and create new treatment targets for fetal interventions, particularly in arresting or reversing the devastating pulmonary sequelae of CDH. The field of fetal surgery owes a debt of thanks to CDH disease and the research teams who first imagined fetal surgery as a treatment option. The past use of these animal models has yielded exciting results, spawning clinical trials and novel therapies for CDH. There is hope and excitement for other congenital anomalies that similar use of animal models will also bring such fruitful results for our patients.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.


There has been increasing interest in the possibility that behavioral experience—in particular, exposure to stress—can be passed on to subsequent generations through heritable epigenetic modifications. The possibility remains highly controversial, however, reflecting the lack of standardized definitions of epigenetics and the limited empirical support for potential mechanisms of transgenerational epigenetic inheritance. Nonetheless, growing evidence supports a role for epigenetic regulation as a key mechanism underlying lifelong regulation of gene expression that mediates stress vulnerability. This Perspective provides an overview of the multiple meanings of the term epigenetic, discusses the challenges of studying epigenetic contributions to stress susceptibility—and the experimental evidence for and against the existence of such mechanisms—and outlines steps required for future investigations. Can behavioral experience—and, in particular, exposure to stress—be passed on to subsequent generations via heritable epigenetic modifications? This Perspective assesses the evidence for and against. The term epigenetics is used in at least three different ways, each referring to a fundamentally different mode of biological regulation, which has contributed to considerable confusion in the field. Epigenetics, in its broadest meaning, is used by some to denote stable changes in gene expression that are mediated via mechanisms that do not involve modification of DNA sequence. In the field of stress responses, such epigenetically induced stable changes in gene expression likely result from any number of environmental stresses that occur throughout a lifetime. For example, chronic stress may induce epigenetic mechanisms that alter gene expression in the adult brain . LikewisThe term epigenetic is also used to describe two additional phenomena for which the evidence base is less solid. One concerns stochastic changes during development, whereby random epigenetic modifications in the developing brain generate variations in an individual’s traits, including differences in stress vulnerability, without changes in either genomic sequence or environmental exposures. The other relates to epigenetic inheritance across multiple generations, whereby epigenetic modifications induced in an individual’s germ cells in response to stress or other environmental exposures are transmitted to offspring to control their stress vulnerability. The degree to which these two forms of epigenetic regulation contribute to the lasting consequences of stress, including stress-related syndromes such as depression, remains more controversial.DNA is wrapped around histone octomers to form nucleosomes—the unit of chromatin . A gene’Post-translational modifications of histones remain the best studied form of epigenetic regulation. Each of the four canonical histone subunits in mammals is modified at specific amino acid residues in diverse ways, including acetylation, methylation, polyADP-ribosylation, and ubiquitination, among many others. DNA is also modified, without affecting nucleotide sequence, most prominently through its methylation, hydroxymethylation, or related changes . MeanwhiEnvironmental stimuli, such as stress exposures over a lifetime, regulate these various epigenetic processes in two main ways . First, As the human brain develops, generating ~100 billion neurons and ~100 trillion synapses, enumerable stochastic events occur that generate diversity even though genetics and environment remain constant ,19. The An alternative approach would be to compare stress-induced changes in epigenetic state in a given brain region or cell type of a susceptible versus resilient adult mouse after social defeat stress to the epigenetic states seen in the same brain region or cell type of mice with or without exposure to stress or other challenges early in life. In fact, preliminary evidence suggests that a form of early life stress, which renders a mouse more susceptible to social defeat stress later in life, induces patterns of gene regulation in several brain regions—in the absence of adult stress—that resemble patterns induced by adult stress in susceptible mice . HoweverIn any event, stochastic, epigenetic differences in brain function could be one reason why it has been difficult to identify genes that confer risk for depression and other stress-related disorders. This use of the term epigenetics comes closest to Waddington’s original definition in 1942, which posited—before we knew that DNA carried genetic information—that the effect of genetics in determining phenotype is subject to random variations during development, akin to several marbles taking distinct paths rolling down a hill .The term epigenetics is also used to refer to the transgenerational transmission of traits without a change in DNA sequence ,24. It iHowever, the mechanisms underlying this clear transgenerational transmission of stress vulnerability remain controversial. Stress produces DNA methylation changes at particular genes in the sperm of stressed mice , yet it Such skepticism for the transgenerational epigenetic inheritance of stress susceptibility is based on current schemes whereby virtually all epigenetic modifications that occur throughout life are “erased” during meiosis ,29. EvenAdditionally, increasing attention has been given to miRNAs as possible vehicles of transgeneration epigenetic inheritance. In worms, for example, phenotypes as varied as viral immunity, nutritional status, and aging can be influenced across several generations via induction of specific miRNAs in parents –32. GrowThe failure of in vitro fertilization to demonstrate robust epigenetic inheritance of social defeat-induced susceptibility (stated above)  may not The final consideration in studying transgenerational epigenetic inheritance is the fact that most groups have examined paternal transmission. This is due to two practical considerations. First, studying paternal transmission is one generation easier, since the eggs of the F1 generation are present in mothers exposed to stress or any other stimulus, and the eggs of the F2 generation are present in those F1 pups. As a result, the first generation that could show true transgenerational inheritance is F3. Second, it is well established that different amounts or types of maternal care can have a profound, lasting effect on stress-related behavior in offspring animals ,36, whicFurther work is needed to understand whether and to what extent true epigenetic inheritance of stress vulnerability adds to the well-established and powerful influence of genetics and environmental exposures in determining an individual’s susceptibility versus resilience to stress throughout life. Despite the two extremes of disbelief versus wild speculation, there is growing evidence for at least some contribution of epigenetic regulation—perhaps achieved by miRNAs—in mediating part of the ability of parental behavioral experience to influence stress vulnerability in their offspring.


Zizeeria maha, which coexists with humans. The butterfly larval ingestion of contaminated leaves found in areas of human habitation, even at low doses, resulted in morphological abnormalities and death for some individuals, whereas other individuals were not affected, at least morphologically. This variable sensitivity serves as a basis for the adaptive evolution of radiation resistance. The distribution of abnormality and mortality rates from low to high doses fits well with a Weibull function model or a power function model. The offspring generated by morphologically normal individuals that consumed contaminated leaves exhibited high mortality rates when fed contaminated leaves; importantly, low mortality rates were restored when they were fed non-contaminated leaves. Our field monitoring over 3 years (2011–2013) indicated that abnormality and mortality rates peaked primarily in the fall of 2011 and decreased afterwards to normal levels. These findings indicate high impacts of early exposure and transgenerationally accumulated radiation effects over a specific period; however, the population regained normality relatively quickly after ∼15 generations within 3 years.One important public concern in Japan is the potential health effects on animals and humans that live in the Tohoku-Kanto districts associated with the ingestion of foods contaminated with artificial radionuclides from the collapsed Fukushima Dai-ichi Nuclear Power Plant. Additionally, transgenerational or heritable effects of radiation exposure are also important public concerns because these effects could cause long-term changes in animal and human populations. Here, we concisely review our findings and implications related to the ingestional and transgenerational effects of radiation exposure on the pale grass blue butterfly,  The Fukushima Nuclear Power Plant (NPP) accident is the second largest nuclear accident next to the Chernobyl accident in the history of mankind. Epidemiological and physiological studies on humans following the Chernobyl accident that assessed infant leukemia –6, infanThe biological impacts of the Fukushima accident have been demonstrated in several studies. Census studies indicate that the number of birds and insects, especially butterflies, has decreased in contaminated areas –24. For Oxalis corniculata, which is distributed worldwide and is difficult to eradicate from farmland. Thus, the pale grass blue butterfly is a beneficial insect in agriculture. It is the most prosperous butterfly in Japan, both in rural and urban areas, including Tokyo .


Pharmacogenomics aims to shed light on the role of genes and genomic variants in clinical treatment response. Although, several drug–gene relationships are characterized to date, many challenges still remain toward the application of pharmacogenomics in the clinic; clinical guidelines for pharmacogenomic testing are still in their infancy, whereas the emerging high throughput genotyping technologies produce a tsunami of new findings. Herein, the potential of whole genome sequencing on pharmacogenomics research and clinical application are highlighted. Pharmacogenomics (PGx) aims to develop strategies for individualizing therapy to optimize drug efficacy and minimize toxicity on the basis of our improved understanding of how genomic variants influence drug response. PGx research focuses on both pharmacodynamic and pharmacokinetic effects . PharmacPharmacogenomics research has exhibited a profound acceleration during the past decades and the discovery and investigation of gene variants have followed several paths . This waObtaining a full picture of a person’s variome is necessary, particularly in the case of novel unique variants that may either render a drug metabolizing enzyme and/or transporter inactive, or disable its expression and, hence, the carrier of the variant intermediate or even poor metabolizer. NGS approaches are gradually being adopted for PGx, involving either whole exome  or wholeThere are very few whole genome sequencing studies in PGx, despite the fact that genetic disorders have already begun to greatly benefit from whole genome sequencing applications. Recent evidence, although limited at the present time, supports the idea that whole genome sequencing is capable of revealing unique (or rare) PGx markers that would otherwise go undetected, if conventional genetic screening methods were employed.de novo genomic variants needed to manage all current and future treatment modalities lies in the range of US$3000 and is declining. Hence, it is anticipated that soon its cost effectiveness will be appreciated, when compared to the costs of testing for a single or several markers in few pharmacogenes  the costs of sequencing technologies, (ii) the regulation scheme regarding the use of (next-generation) sequencers as medical devices, (iii) setting up a  whole genome sequencing facility, and (iv) training clinicians to interpret PGx data . The curwww.genomicmedicinealliance.org). The latter would only be possible through a whole genome sequencing approach.In light of the above, design and implementation of advanced informatics solutions that ease to fill-in the gap between PGx research findings and clinical practice emerges as a major need. In 2014, major – although, limited – milestones have been set. eMERGE-PGx aims to (i) deploy PGRNseq, assessing sequence variation in 84 pharmacogenes in 9,000 patients and several clinical sites, (ii) integrate clinically validated PGx genotypes into electronic health records (EHRs) with associated clinical decision support, and (iii) develop a PGx variants’ repository of unknown significance linked to a repository of EHRs-based clinical phenotype data for on-going PGx discovery . SimilarAs whole genome and/or whole exome sequencing approaches begin to take hold in clinical care, not only how sequencing technologies evolve, but also how they get integrated into a clinical setting are of utmost importance regarding the development of PGx clinical tests and the understanding of the genetic effects on a prescription and treatment level that need to become more readily available to the clinician. So far, the translation of PGx research findings into clinical practice has been slow. The advent of whole genome sequencing technology will offer an outstanding potential toward the clinical application of PGx. Some key components regarding successful clinical implementation have been already addressed and actions have been already taken, whereas others are to be met.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.


Preservation of health from infectious diseases depends upon both mucosal and systemic immunity via the collaborative effort of innate and adaptive immune responses. The proficiency of host immunity stems from robust defense mechanisms—physical barriers and specialized immune cells—and a failure of these mechanisms leads to pathology. Intriguingly, immunocompetence to pathogens can be shaped by the gut microbiome as recent publications highlight a dynamic interplay between the gut microbiome and host susceptibility to infection. Modulation of host immunity to enteric pathogens has long been studied where gut bacteria shape multiple facts of both innate and adaptive immunity. Conversely, the impact of gut commensals on host immunity to extra-gastrointestinal (GI) tract infections has only recently been recognized. In this context, the gut microbiome can augment host immunity to extra-GI tract bacterial, viral, and parasitic pathogens. This review explores the research that affords insight into the role of the gut microbiome in various infectious diseases, with a particular emphasis on extra-GI tract infections. A better understanding of the link between the gut microbiome and infectious disease will be critical for improving global health in the years ahead. Conversely, certain metabolites confer resistance to CDI. Bacteria such as Clostridium scindens that encode the 7α-hydroxysteroid dehydrogenase enzyme, which is needed for secondary bile acid biosynthesis  inhibit the growth of C. difficile  to treat recurrent CDI has shown great promise in the clinic. For example, FMT has been shown to be well tolerated with an overall clinical cure rate of 89% , which promote Th17 immune responses in the intestine  is produced by T helper cells and leads directly to expression of antimicrobial peptides by Paneth cells that help control C. rodentium; these peptides include RegIIIβ and RegIIIγ  such as LPS and muramyl dipeptide and inducing inflammatory conditions in the gut  fluid  and Enterobacteriaceae . Populations that are significantly changed as a result of an influenza infection included a decrease in . PopulatLymphocytic choriomeningitis virus (LCMV) is an Arenavirus that is a naturally occurring rodent pathogen and is often used to study host-pathogen interactions. There are several strains of LCMV that differ in their ability to cause acute vs. chronic infections. Using a strain of LCMV that takes about 1-month to clear post-infection Abt et al. determined how the gut microbiome impacted the host immune response following systemic infection , which is caused by a retrovirus that invades and infects immune cells of its host, specifically helper T cells, macrophages, and dendritic cells . Overall, significant changes in the resident microbiome occurred between infection and death; in particular, there was a significant loss in gut bacteria diversity initially that was followed by a recovery in diversity  is an infectious disease caused by rculosis . OverallPlasmodium species. In 2013, there were at least 198 million infections and 584,000 deaths attributed to Plasmodium infections, with the majority occurring in sub-Saharan Africa   such as propionate can circulate to the bone marrow and induce generation of antigen-presenting cells (APCs) that induce diminished Th2 response and thus ablates allergic airway disease (Trompette et al., Plasmodium, that have evaded the development of effective vaccines. Therefore, it is essential we gain a greater understanding of how the gut microbiota communicates with the immune system to affect the outcome of systemic infections.Although, decades of research have linked the gut flora with susceptibility to enteric pathogens, research in recent years has now demonstrated that the gut microbiota also impact the host immune response to extra-GI tract infections. This effect may not be universal, yet we should not be surprised to learn of other systemic infections that are modulated by gut commensals. It is also becoming increasingly clear that systemic infections can also alter the composition of the gut microbiota. Collectively, these observations point toward novel therapeutic interventions for pathogens, such as All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.