Drosophila melanogaster) with a range of pathogen (Drosophila C Virus) doses and recording survival over time. The experiment was replicated for flies carrying the Wolbachia symbiont, which is known to reduce host susceptibility to viral infections. The entire dataset is fitted by a novel quantitative framework that significantly extends classical methods for microbial risk assessment and provides accurate distributions of symbiont-induced protection. More generally, our data-driven modeling procedure provides novel insights for study design and analyses to assess interventions.The biological effects of interventions to control infectious diseases typically depend on the intensity of pathogen challenge. As much as the levels of natural pathogen circulation vary over time and geographical location, the development of invariant efficacy measures is of major importance, even if only indirectly inferrable. Here a method is introduced to assess host susceptibility to pathogens, and applied to a detailed dataset generated by challenging groups of insect hosts ( Drosophila melanogaster challenged with different doses of Drosophila C virus. By replicating the procedure in groups of flies that carry the symbiont Wolbachia, we are able to estimate how the viral protection induced by this intracellular bacterium is distributed in the host population. Our results disentangle host infection status from observed mortality, accounting naturally for time since exposure. The multiple-dose design proposed challenges traditional study designs to assess interventions.While control options for plant, animal, and human pathogens are emerging rapidly, reliable assessment of the effect of interventions in biological systems presents many challenges. A major question is how to connect laboratory experiments and measurements with the relevant process in natural settings, where hosts are subject to pathogen exposures that vary in time and geographical location. With this aim, measures of protection that are invariant under varying exposure intensity need to be developed and integrated with mathematical models. In this article, we introduce a method to assess host susceptibility to pathogens, and apply it to survival of  Vibrio choleraeStreptococcus pneumoniaeHosts exposed to disease-causing agents respond in accordance to the challenge dose. Therefore dose-response curves contain information about disease processes that can be extracted by suitable analytic frameworks. Early examples concerning microbial risk assessment include counting lesions caused by tobacco mosaic virus on plant leaves Models that account for the sigmoidal shape in log-linear scale of the typical dose-response curve have been derived mechanistically, based on the assumption that each individual pathogen has a probability of infection independent of others, the so-called independent action hypothesis In the 1960s, Furumoto and Mickey Susceptibility distributions other than Beta have also been considered and are more commonly used in frailty models adopted in survival analysis The importance of accounting for time between challenge and observable toxicity responses to pathogens or other agents has been recognized. Recent models in ecotoxicology Wolbachia, found among many arthropod species including Drosophila melanogaster, is one such symbiont Wolbachia to D. melanogaster, we apply our inference framework simultaneously to two sets of time-dependent dose-response data: in one set the flies carry the symbiont bacterium Wolbachia (Wolb+); while in the other they do not (Wolb−). In this instance we extract the Beta distribution that best describes individual protection attributable to Wolbachia, as well as population statistics valid across entire dose ranges.Recent evidence for symbiotic interactions that reduce host susceptibility to pathogens has stimulated the development of quantitative frameworks to assess the levels of individual and population protection attributable to specific symbionts. The intracellular bacterium D. melanogaster lines with DrosDel w1118 background, with or without the endogenous Wolbachia strain wMelCS 2 and pricked flies with different doses of Drosophila C virus (DCV). We used tenfold serial dilutions – from 1010 TCID50/ml to 104 TCID50/ml – in Tris-HCl buffer, pH 7.5. Controls were pricked with buffer solution only. We used the pricking protocol described in We used virus free response, as a function of the concentration of infectious units given to hosts, i.e. the dose. We present a step-by-step derivation of descriptions that integrate dimensions that are usually treated separately as well as the motivations for doing so.Starting from established models, we refine the occurrence of mortality from infection, i.e. the p of causing an infection, while for d infectious units infection occurs with a probability described by d:Assuming independent action of infectious units, each unit has probability p fixed across individual hosts , the shape parameters for the Beta distribution that describes the susceptibility factor , and the probability of ineffective challenge , subject to two hazards: We first consider a survival model for a control group of flies pricked with buffer solution only  used for normalization purposes, the Beta distribution shape parameters to describe variation in susceptibility factor , the parameters that control death due to aging  and positive (Wolb+) D. melanogaster flies were challenged with a range of DCV doses and survival curves were traced as shown in Groups of + susceptibilities and the posterior samples for the Beta distribution shape parameters. For simplicity we have adopted the homogeneous model for Wolb− and focus on comparing susceptibility distributions of Wolb+ inferred at different days. Mean protection conferred by Wolbachia in this illustration is estimated as 79% and 56%, based on mortality measurements at day 30 and 50, respectively. Moreover, the distributions have fundamentally different shapes, with the appearance of a high susceptibility group as time progresses. This sensitivity to the day by which mortality data are collected is a concern that raises the need to disentangle infection status from the associated time-dependent mortality. In the following sections, infection and mortality are estimated explicitly using the integrated time-dependent model described in To emphasize the importance of day selection to infer distributions of susceptibility to infection by classic dose-response models − and Wolb+ flies pricked with buffer solution (no DCV) were compared with the Kaplan-Meier method using the log-rank test and no significant difference was found . By fitting the uninfected time-dependent model (4–6) to the control survival curves , the time-dependent dose-response model constructed in DCV challenge dose), fixing across doses the distribution of times to death from infection  that govern the dependence of response on challenge dose according to the adopted dose-response model. The estimated parameter values are listed in − group and the heterogeneous model for Wolb+ , we determine the mean protection conferred by the symbiont to its host as 85% (with a 95% HPD of 60–93%).The fitted dose-response curves that result from this analysis are shown in + in the case) we must have previously reduced the set of survival curves to a set of effectively infected proportions - one entry per challenge dose. To search for a range of days in which absolute mortality might provide an approximate indication of infection, we compare the estimated proportions effectively infected by each challenge dose with the mortality proportion measured at each day. Using a normalized Euclidean distance between these two measures, a day-selection score is provided by the red curve in To assess the best possible performance of classic methods Dose-response models have become standard quantitative frameworks in microbial risk assessment. Less recognized is their ability to estimate host trait distributions. Here we illustrate the concept by extracting host susceptibility distributions from mortality measured as a function of pathogen challenge dose, but similar procedures can be developed for measures of infection or infectiousness , and can be made a function of other environmental variables such as temperature or humidity (instead of dose). Understanding how to detach host trait distributions from environmental variables is crucial for the formulation of measures that can be transported between laboratory and natural conditions D. melanogaster) were experimentally challenged by viruses (DCV): one group consists of isogenic flies where no significant variability in susceptibility to infection is found; and another with the same genetic background but now carrying the symbiont bacterium Wolbachia known to reduce susceptibility to DCV We address this problem with an experimental design and inference framework that enables the estimation of distributions of host susceptibility to infection by analyzing simultaneously a collection of survival curves, each representing a different challenge dose . The proWolbachia confers on average 85% DCV protection to D. melanogaster under the specified laboratory conditions, and suggest significant variability in this effect. This variance in susceptibility is induced by the symbiont, since model selection criteria did not support heterogeneity in the susceptibility of flies not carrying Wolbachia. Since the Drosophila and Wolbachia populations used in this study are isogenic, the heterogeneity in susceptibility of Wolbachia-carrying flies uncovered here indicates variation in the host-microorganism interaction that lacks a genetic basis. A simple hypothesis is that variance in Wolbachia levels at the individual host level leads to variance in resistance to viruses. Although several lines of evidence support this hypothesis Wolbachia levels or, alternatively, a result of another environmental/physiological variance that is only expressed in the presence of Wolbachia.Our inferences indicate that Previous estimates of protection were based on survival analysis or viral titres in a dose-specific manner Wolbachia, other symbionts, or interventions that modify host susceptibility to infection, are introduced to induce desired transitions in populations. The introduction of Wolbachia into Aedes aegypti and other arthropod vectors is being considered as a promising strategy to control dengue and other infectious diseases of humans  has been proposed In epidemiological systems, the baseline transmission intensity is often not directly measurable but indirectly inferred in a model-based manner. Dose-response models, on the other hand, can account for experimentally controlled patterns of exposure Dataset S1− and Wolb+ groups of Survival data for WolbD. melanogaster challenged by various doses of DCV.(XLS)Click here for additional data file.Text S1Homogeneous vs heterogeneous model selection.(PDF)Click here for additional data file.


The efficiency of assisted reproductive technologies (ARTs) in nonhuman primates is low due to no screening criterions for selecting sperm, oocyte, and embryo as well as its surrogate mothers. Here we analyzed 15 pairs of pregnant and non-pregnant cynomolgus monkeys, each pair of which received embryos from one batch of fertilized oocytes, and found ratio of endometrial to myometrial thicknesses in abdominal ultrasonic transverse section of uterus is a reliable indicator for selection of recipients for embryo transfer. We performed 305 ovarian stimulations in 128 female cynomolgus monkeys and found that ovarian stimulation can be performed in a whole year and repeated up to six times in the same monkey without deteriorating fertilization potential of eggs until a poor response to stimulation happened. Fertilization can be efficiently achieved with both conventional and piezo-driven intracytoplasmic sperm injection procedures. In semen collection, semen quality is higher with the penile robe electrical stimulus method compared with the rectal probe method. Moreover, caesarean section is an effective strategy for increasing baby survival rates of multiple pregnancies. These findings provide a practical guidance for the efficient use of ARTs, facilitating their use in genetic engineering of macaque monkeys for basic and translational neuroscience research. A major milestone in biomedical research was the generation of the first gene knockout mouse through embryonic stem cell based gene targeting25789in vitro fertilized rhesus monkey babyAssisted reproductive technologies (ARTs) are essential for the production of genetically modified monkeys. Since the birth of the first Cynomolgus monkey is an attractive primate animal for genetic engineering of disease models due to its year-round reproductive capability, moderate size, complex behaviors and similarity to humans in physiology and pathology. Currently, there are few reproductive researches in cynomolgus monkeys, which result in a slow development of its ARTs. In this study, we systematically compared several key procedures in ARTs including stimulated ejaculation, seasonal variation of reproduction, superovulation, intracytoplasmic sperm injection (ICSI), embryo transfer and cesarean section. These results enhance the efficiency of ARTs and should increase the success of generating genetically modified monkeys, which helps for the creation of advanced animal models to make key breakthroughs on primate neuropathic diseases research.Eight adult male monkeys were used in this study. Four males were randomly selected to treat with REM and the rest four males were treated with PEM. Each animal received four stimulations at four day intervals. Then, the stimulus methods were exchanged between the two groups and each animal received another four stimulations with an interval of four days. Although REM took less time to achieve ejaculation, PEM was better in semen characteristics including seminal weight, sperm concentration , motilitCynomolgus monkeys can breed throughout the yearn = 30), after the first ovarian stimulation, were randomly divided into three groups for the second stimulation with the same regimen separated at intervals of two, three or four menstrual cycles, respectively  pregnancy compared with twin (25%) and single (25%) pregnancies in cynomolgus monkeys , althougIn the present study, we investigated some of the current problems of ARTs in cynomolgus monkeys and demonstrated successful solutions. Our key findings are: the thickness ratio of uterine endometrium to myometrium may be a reliable indicator for selecting embryo recipients; ovarian stimulation can be done year-round and repeated for six times in a monkey without dramatic decrease in oocyte number and quality, until a poor response happened; fertilization can be readily achieved with conventional as well as piezo-driven intracytoplasmic sperm injection procedures; in comparison of semen collection methods, seminal weight and sperm quality on the basis of motility, malformation and acrosomal integrity of sperm are higher in PEM than that in REM; moreover, cesarean section might be an effective strategy for increase baby survival rates of multiple pregnancy .Electro-stimulation ejaculation is commonly adopted to collect semen for reproductive research in nonhuman primates2526In rhesus monkeys, we and some researchers reported that age, body weight, season, the day of the menstrual cycle for starting ovarian stimulation, ovarian stimulation regimen, and repetition stimulation, all affected the outcome of ovarian stimulation122829303132in vivo compensatory mechanisms that optimize the developmental competence may be recruited, as in mice35It was suggested that consecutive ovarian stimulation could impair ovarian responsesBoth endometrium and myometrium undergo significant changes during reproductive cycle414245Gene editing with the recent developed ZFN, TALNE and CRISPR/Cas toolboxs have made the genetic manipulation of monkeys possible. As ARTs and gene editing technologies are indispensable compositions for generation of genetically modified monkeys. Less efficiency of ARTs of monkeys absolutely causes low efficiency of creation of genetically-modified monkeys. So ARTs are very important for generation of genetically-modified monkeys. Although, in this manuscript, we did not incorporate any gene editing data in monkeys, we did spend quite a lot of efforts in generating disease monkey model by the cutting edge TALEN and CRISPR/Cas toolsin vitro without difference in embryo development rate. We find that the ratio of endometrial to myometrial thicknesses in abdominal ultrasonic transverse section of uterus is the most reliable indicator for selection of recipients for embryo transfer. Furthermore, we find that caesarean section is an effective strategy for increasing baby survival rates for twin and multiple births resulted from embryo transfer. The findings provides extensive and detailed data for reproductive biology in nonhuman primates, which will be useful not only for enhancing ARTs efficiency in macaque monkeys, but also for facilitating their use in generation of genetically-modified monkeys for biomedical research.Taken together, here we show that compared to REM, PEM produces higher semen volume and better semen quality on the basis of motility, malformation and acrosomal integrity of sperm. Ovarian stimulation can be performed in all seasons, and recovery of oocytes and their fertilization ability remain fairly steady throughout the year. Moreover, cynomolgus monkeys can respond to repeated ovarian stimulation up six times separated at intervals of 2–4 menstrual cycles before the response to stimulation becomes poor. Both conventional and piezo-driven ICSI procedures can be used for fertilization All cynomolgus monkeys were provided and raised by Blooming-Spring Biotechnology Development Co., Ltd., of Guangdong (BBDC), which is an accredited member of the Association for Assessment and Accreditation of Laboratory Animal Care. Healthy, sexually-mature male monkeys  and female monkeys  were involved in the present study. All animal procedures such as semen collection, ovarian stimulation, oocyte recovery, abdominal ultrasonography, embryo transfer and cesarean section were approved by the Institutional Animal Care and Use Committee of BBDC and were carried out in accordance with the Guide for the Care and Use of Laboratory Animals. Unless stated otherwise, all chemicals were obtained from Sigma Chemical Co. .REM was conducted as previously describedPEM was conducted as previously describedEjaculates were collected directly from the tip of the penis after ejaculation into warmed tubes. Prior to measuring the characteristics of semen, ejaculates were kept in water bath at 37 °CpH, weight, and sperm density in semen were measured with reference to international standardsUrea in seminal plasma was measured with a biochemical analyzer  following the manufacturer’s instructions with some improvements. Accuracy testing was done four times with serum of male cynomolgus monkeys and recovery of urea added into the serum was more than 90% in actual concentrations of 8 mmol/L, 11 mmol/L, 14 mmol/L and 18 mmol/L . Urea coFemale cynomolgus monkeys were monitored twice a day (8:00 and 20:00) for vaginal bleeding to detect menstruation onset. Menstrual cycle period was defined as the duration from the first day of vaginal bleeding to the day before next vaginal bleeding.As shown in 182 in air under the mineral oil, 10–12 h later oocytes exhibiting two pronuclei were considered as fertilization. The fertilization rate meant the fertilized oocytes to all constituted oocytes in a trial.The conventional ICSI procedure was performed with commercially prepared injection and holding pipettes and an Eppendorf micromanipulation system attached to an Olympus IX 70 inverted microscope with Hoffman Contrast optics, as previously describedUterus and ovaries of recipients were examined with a real time B-mode ultrasound scanner  on the day of and just before embryo transfer, and 30 days after embryo transfer. Female monkeys were bound in a supine position under anesthesia on an operating table with the help of the manipulator’s finger inserted into the rectum to control the position of the uterus horizontally under the transducer. The transducer was placed on lower midline abdomen of the animal to locate the uterus in a transverse plane. The transducer was then moved slowly and laterally to obtain the transverse section of the uterine body and all images were recorded. According to the reflectivity of the uterus, the images were divided into five gradesMultiparous females with two consecutive normal menstrual cycles 26–32 days) and with normal ovaries containing dominant follicle or freshly ovulated follicle, and uterus with the normal echo in uterus cavity checked by ultrasound immediately just before embryo transfer were selected as embryo recipients. We drew blood of rhesus monkeys respectively and used rapid radioimmunoassays kit  to measure serum E2 and P4 levels. One-cell embryos were transferred into one oviduct of each recipient via laparoscopy with a fixed polythene catheter , threaded through a 25-gauge hypodermic needle days andApproaching parturition (140–160 days), pregnant monkeys showed either anxious, week of moving and loss of appetite, or appearance of blood and amniotic fluid from vagina, then they were anesthetized and cesarean section and postoperative care were carried out as per routine protocolHow to cite this article: Ma, Y. et al. Efficient production of cynomolgus monkeys with a toolbox of enhanced assisted reproductive technologies. Sci. Rep. 6, 25888; doi: 10.1038/srep25888 (2016).


The early-life environment, in particular maternal diet during pregnancy, influences a wide range of organs and systems in adult offspring. Mounting evidence suggests that developmental programming can also influence health and disease in grand-offspring. Transgenerational effects can be defined as those persisting into an F2 generation, where the F0 mother experiences suboptimal diet during her pregnancy. In this review, we critically examine evidence for transgenerational developmental programming effects in human populations, focusing on metabolic and reproductive outcomes. We discuss evidence from historical cohorts suggesting that grandchildren of women exposed to famine and other dietary alterations during pregnancy may experience increased rates of later health complications than their control counterparts. The methodological difficulties with transgenerational studies in human cohorts are explored. In particular, the problems with assessing reproductive outcomes in human populations are discussed. In light of the relative paucity of evidence available from human cohorts, we consider key insights from transgenerational experimental animal models of developmental programming by maternal diet; data are drawn from a range of rodent models, as well as the guinea-pig and the sheep. The evidence for different potential mechanisms of transgenerational inheritance or re-propagation of developmental programming effects is evaluated. Transgenerational effects could be transmitted through methylation of the gametes via the paternal and maternal lineage, as well as other possible mechanisms via the maternal lineage. Finally, future directions for exploring these underlying mechanisms further are proposed, including utilizing large, well-characterized, prospective pregnancy cohorts that include biobanks, which have been established in various populations during the last few decades. It is increasingly accepted that the environment during early development has a profound and lasting effect on many aspects of physiology and metabolism later in life. A large body of literature has gradually accumulated demonstrating such effects across generations in both human populations and animal models . It has been argued elsewhere that any developmental programming effect may be considered transgenerational by definition, as an intervention in the F0 generation  influences the outcome for the next generation (F1) , for which historical records are available that allow dietary intake data to be reconstructed over generations. Examination of the records shows that where the paternal grandmother (F0) experienced dramatic changes in food availability during her own growth, her female grandchildren (F2) were at higher risk of cardiovascular mortality  structural effects on tissues and organs, (ii) epigenetic programming of gene expression, (iii) glucocorticoid effects or (iv) accelerated cellular ageing. Evidence for maternal diet affecting the metabolic and reproductive outcomes of grand-offspring has been demonstrated via all three suggested mechanisms (Aiken et al. Maternal energy restriction in a rat model has been shown to lead to reduced pancreatic beta cell mass at birth in both the F1 and F2 generations (Blondeau et al. Transcriptional programming of gene expression, most often assayed by methylation status, is one of the most widely studied outcomes in developmental programming models. Methylation patterns as a means of transmission of developmental programming effects are often viewed as problematic, due to the concept that DNA methylation patterns are reset both in the primordial germ cell and the early embryo (Seisenberger et al. Although modification of DNA methylation patterns is the most widely studied, there are a number of other possible mechanisms of epigenetic modification that may contribute to transgenerational developmental programming. These include histone modification and differential expression of miRNAs. Debate is ongoing about the stability of histone modifications across generations in order to provide a mechanism for transgenerational inheritance of developmental programming effects, but there are some indications that histone marks may be stably inherited (Gaydos et al. Prenatal exposure to glucocorticoids, while not strictly a maternal dietary modulation, is nonetheless a commonly modified aspect of the early-life environment in both human pregnancy and animal models (Crudo et al. The final major mechanism that is often postulated for developmental programming effects is accelerated cellular ageing. Rapid cellular ageing and decreased longevity have been postulated as key elements of developmental programming phenotypes since the earliest landmark studies in animal models were published (Jennings et al. Although a decline in telomere length of second-generation offspring has not been directly observed in other metabolic and reproductive tract tissues, developmental programming phenotypes are characterized also by early accumulation of oxidative and nitrosative stress, and a significant upregulation of anti-oxidant defense mechanisms Ozanne . This isThere is a body of evidence from animal models that establishes the paradigm that metabolic and reproductive effects can be transmitted across generations in at least some maternal dietary models (Aiken et al. Determining whether such effects are truly present in human populations presents a variety of epidemiological challenges (Pembrey et al.


Speech research has recently seen a good deal of activity surrounding forward models  has been used to demonstrate the execution of prepared non-speech motor behaviors  with little or no interference from feedback regulation (Valls-Solé et al., Early analyses hypothesized that the rapid release of SAS-induced responses is the result of triggering subcortically stored information with faster neural transmissions (Carlsen et al., Following these studies of finger movement, Stevenson et al.  apply thThe observed StartReact effect in syllable production also suggests that SAS-induced motor tasks, including upper limb, and speech movements, may involve similar neural pathways. It is noteworthy that the StartReact pathways involve similar pathways for speech production. As summarized in Carlsen et al. , the StaSimilar to the calculation of the required time span for voluntary limbic movements, we can also conservatively calculate the time required for a speech response. First, Schroeder and Foxe  report aInsofar as programming is necessary for speech production, we believe that the SAS methodology provides a new perspective that can help us to uncover the kinematic and linguistic contents of forward speech plans. Neural correlates and pathways for SAS-induced responses also support the view that SAS-induced speech responses may contain unaltered details of speech plans, allowing researchers a window into forward speech planning that bypasses afferent feedback 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.


Acute pain triggers adaptive physiological responses that serve as protective mechanisms that prevent continuing damage to tissues and cause the individual to react to remove or escape the painful stimulus. However, an extension of the pain response beyond signaling tissue damage and healing, such as in chronic pain states, serves no particular biological function; it is maladaptive. The increasing number of chronic pain sufferers is concerning, and the associated disease burden is putting healthcare systems around the world under significant pressure. The incapacitating effects of long-lasting pain are not just psychological – reflexes driven by nociceptors during the establishment of chronic pain may cause serious physiological consequences on regulation of other body systems. The sympathetic nervous system is inherently involved in a host of physiological responses evoked by noxious stimulation. Experimental animal and human models demonstrate a diverse array of heterogeneous reactions to nociception. The purpose of this review is to understand how pain affects the sympathetic nervous system by investigating the reflex cardiovascular and neural responses to acute pain and the long-lasting physiological responses to prolonged (tonic) pain. By observing the sympathetic responses to long-lasting pain, we can begin to understand the physiological consequences of long-term pain on cardiovascular regulation. Of these “fight or flight” responses, only HR and pupil diameter may include changes in parasympathetic outflow; the remainder are subserved by changes in sympathetic outflow exclusively . While t…the reflex pattern in muscular sympathetic fibres is the same on the spinal and on the brain stem level” , now labeled complex regional pain syndromes (CRPS). Subjects with CRPS often complain of a burning spontaneous pain in the distal part of the affected extremity. Presumed sympathetically mediated effects may cause the affected limb to be warm or cold; there may also be trophic changes (the skin wet or dry) and motor changes . There iHowever, intraneural recordings of sympathetic nerve activity in human subjects have found no differences in sympathetic outflow to a painful limb compared to the contralateral non-painful limb, either in patients with CRPS suspected to be sympathetically maintained  or in heinfused the solution through an intramuscular cannula over a 45-min period, which resulted in pain lasting for ~60 min  this was shown not to hold, and there was no difference between any HRV parameters when comparing the increasing or decreasing group (Heart rate variability (HRV) is an often over-interpreted physiological marker purportedly reflecting the relative contributions of sympathetic and parasympathetic  outflow to cardiac control . Spectrareflexes . In our reflexes . Howeverng group .Given that the level of MSNA is consistent in a given individual on a day-to-day basis , we predpropensity of males or females to exhibit an increase or decrease in MSNA during tonic muscle pain  is sparse . It woulbefore the subjective onset of pain. This suggests that the neural response to a noxious stimulus is associated with a transient change in homeostasis – a transition from no pain to that of pain. Unlike animal studies, it remains unclear whether cutaneous vasoconstrictor neurones in humans are reflexly inhibited during noxious stimulation of the skin , suspected to be sympathetically maintained because of the marked cutaneous vasoconstriction, there was no difference in sympathetic outflow to the painful limb compared to the contralateral non-painful limb  than patients without pain (21%). Why this should be is not known.Unlike the short-lasting pain we had previously induced by bolus injections , 29, we ful limb . Perhapsful limb  showed tAB completed majority of the body of the review in all sections of manuscript. AF contributed to body of review particularly relating to all information regarding studies post 2009. Contributed all sections regarding sustained (infused) hypertonic saline pain models used in experiments. Intellectual contribution, clarification, and editing across entire manuscript. VM integrated contributions of AB and AF, and authored context for review and relevance to broader issues regarding pain. Major intellectual contribution and clarification of body of text, abstract, implications section, and general discussion section.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.


Genetically controlled resistance provides plant breeders with an efficient means of controlling plant disease, but this approach has been constrained by practical difficulties associated with combining many resistance genes together and strong evolutionary responses from pathogen populations leading to subsequent resistance breakdown. However, continuing advances in molecular marker technologies are revolutionizing the ability to rapidly and reliably manipulate resistances of all types – major gene, adult plant and quantitative resistance loci singly or multiply into individual host lines. Here, we argue that these advances provide major opportunities to deliberately design deployment strategies in cereals that can take advantage of the evolutionary pressures faced by target pathogens. Different combinations of genes deployed either within single host individuals or between different individuals within or among crops, can be used to reduce the size of pathogen populations and generate patterns of disruptive selection. This will simultaneously limit immediate epidemic development and reduce the probability of subsequent evolutionary change in the pathogen for broader infectivity or increased aggressiveness. The same general principles are relevant to the control of noncereal diseases, but the most efficacious controls will vary reflecting the range of genetic options available and their fit with specific ecology and life-history combinations. Puccinia striiformis in wheat  and that which is nonspecific being expressed against all races of a pathogen. Race-specific, major gene resistance generally complies with the gene-for-gene paradigm Flor  which isPathogen infectivity and aggressiveness profiles are essentially the result of co-evolutionary interactions between pathogens and their host plants. Infectivity determines whether or not a pathogen will infect a given host and generate a fully compatible (susceptible) reaction. Pathogen isolates that generate a full susceptible reaction are considered infective while those that do not, (either failing to generate macroscopic symptoms or only limited infection and sporulation) are classed as noninfective. Host plants in these reactions are classed as susceptible, (fully) resistant or partially resistant respectively.The genetic basis behind these phenotypic patterns is variable. Interactions that involve pathogen race-specific interactions with host lines typically involve gene-for-gene interactions with resistance control by single (major R) genes. Partial resistance on the other hand may be generated by any of (i) incompletely expressed R genes, (ii) adult plant resistance (APR) genes, or (iii) minor resistance genes. In the case of incompletely expressed major R genes, the interaction between host and pathogen is still race-specific; whereas for APR genes and resistance genes it is race nonspecific with the resistance being expressed against all pathogen isolates.On the pathogen side, once infection occurs, aggressiveness is a quantitative component of measure of pathogen development on the host. While interactions may be race nonspecific, differences between pathogen isolates in a range of quantitative traits affecting epidemiologically relevant components of the pathogen's life-cycle  may, when integrated over time, generate different levels of disease.Partial resistance imposes different selective pressures on the pathogen than does R gene resistance. Furthermore, because it does not display the spectacular failures seen with R gene deployment, partial resistance is often regarded as ‘durable’ and therefore preferable. However, studies of such interactions have demonstrated than partial resistance does not prevent pathogen evolution rather, there is growing evidence for increased aggressiveness and adaptation by pathogen isolates to partially resistance host lines. The extent to which this occurs in important crop – pathogen interactions  – is unclear as is the fully integrated consequences of differences in different traits (e.g. latent period versus spore production per pustule) on epidemic development.Major gene (R) resistance has largely been manipulated in breeding programmes through tracking of its phenotypic expression when challenged by individual pathogen races, bulk race inocula or exposure to natural field infection. However, the generally dominant nature of R gene expression imposes a major limitation on this approach as the phenotypic resistance expression of one gene may mask the resistance expression of another. This makes phenotypic tracking of two or more R genes only possible through the use of pathogen isolates capable of discriminating between the individual infection types of each R gene  highly effective marker tagging systems for R genes and (ii) increasingly efficient means of manipulating multiple minor genes. As a consequence, in principle, few restraints are placed on the combinations of R genes, APR genes and minor gene resistances that are possible. However, what is lacking are careful assessments , using ecological and evolutionary principles, of the most effective disease resistance deployment strategies  that will maximize both the short-term epidemiological and the longer-term evolutionary benefits of different combination strategies. McDonald and Linde ,b2002b aThe current review particularly focuses on the use and deployment of different resistance mechanisms to control fungal pathogens of cereals. However, genetically based resistance mechanisms are widely used to counter the effects of a broad range of plant pathogens in agricultural, horticultural and forestry settings including field  and vegetable crops , pasture and amenity species , perennial horticultural fruit crops , and a range of trees grown for timber or pulp . As a consequence, while this review focuses on the control of cereal diseases, the application of an evolutionary approach to long-term disease control is equally applicable to most, if not all, agricultural disease control. Despite this, the nature of the specific controls invoked is likely to vary depending on the ecology and life history of both host and pathogen. For example, the extent of auto- versus allo-infection will alter between different hosts depending on their structural architecture. In turn, this will affect the balance of selection for infectivity versus aggressiveness. Furthermore, APR genes are an important part of the defence armoury of wheat and barley but have not been identified outside the Poaceae.Glycine species challenged by Phakopsora pachyrhizi; Burdon and Speer Disease symptoms, manifest as differences in lesion size and type, are the phenotypic expression of genetic interactions between host resistance and pathogen infectivity and aggressiveness modified by environmental conditions, including the host germplasm (‘genetic background’) in which resistance genes are expressed. Major R genes, mostly characterized by nucleotide-binding site leucine-rich repeat motifs , although others are effective against a single pathogen only . It should be noted that this resistance is very different to the partial resistance generated by incompletely expressed R genes; see Box Puccinia coronata in Lolium perenne explain between 2.6% and 24.9% of the phenotypic variation  will favour different pathogen traits depending on the degree to which pathogen sporulation is restricted. In situations where sporulation is almost completely restricted [e.g. pathogen isolate–host combinations resulting in low ITs (0–2)], there may be strong selection for novel infectivity rather than increased aggressiveness. In contrast, partial resistance that permits higher levels of sporulation (e.g. ITs = 2+ to 3) is epidemiologically more similar to minor gene-controlled resistance and hence is more likely to promote selection for increased pathogen aggressiveness. Complexities of this type are often encountered in host–pathogen interactions occurring in natural ecosystems , flowering time requirements, increasing water-use efficiency, and pests and diseases are traits important in enhancing yield that may be positively Burrows  or negatper se may also be associated with physiological costs that negatively affect agronomic performance. The extent of these ranges widely with a striking contrast between high costs of resistance often found in model systems and low costs in most crop systems  to 9% (Sr26) were detected. Both of these genes had been derived from wild crosses involving less or more distant relatives (Thinopyrum ponticum and Agropyron elongatum respectively). A similar association of reduced yield has been detected with resistance genes for eyespot and powdery mildew derived from Aegilops speltoides and A. ventricosa , realized dispersal patterns are highly environment dependent. For example, wind speed and gustiness will interact with features such as crop phenology and architecture (e.g. leaf area and degree of tillering) and the spatial position of the pustule in the crop to affect the frequency of allo- versus auto-infection Burdon ; Fig. 2.Melampsora lini occurring in a native association with its host Linum marginale  was used in conjunction with major R genes. This is important when considering the durability of APR genes, because the R genes acted to prevent reproduction of all uninfective pathogen isolates  been deployed alone . For example, the wheat variety ‘Hope’, the first commercial variety released carrying the APR gene es Knott  thereby This is not to say that there is no potential for APR genes to provide long-term durability. Both major gene and APR are controlled by the action of single genes, but their molecular nature and role in the biochemical functioning of the plant are very different. Such functional differences may have important consequences for pathogen evolutionary potential Box . AnswersDeploying host resistance mechanisms that are functionally difficult to overcome promises one potential avenue towards durable resistance [see Boyd et al.  and Dangper se, and in the absence of genetic constraints or trade-offs, their durability is likely still dependent on the evolutionary potential of the pathogen in question  confers previously susceptible plants resistance to a wide range of bacterial pathogens. This gene recognizes the bacterial translation elongation factor EF-Tu, an essential factor for protein synthesis that cannot be simply deleted or modified. However, whether such engineering efforts will confer durable resistance remains to be seen and will likely depend on the complexity of the pathways required to circumvent the PRR, and the ability of pathogens to acquire new infectivity factors via horizontal gene transfer.One approach to raise the level of functional constraint facing the pathogen is to mine or engineer resistance genes that recognize molecules essential to the pathogen that cannot be deleted or modified without major consequence. For example, all pathogens emit a conserved set of signals that play an essential housekeeping role in pathogen growth and survival [known as pathogen-associated molecular patterns . Furthermore, APR resistance is partial and race nonspecific (often across multiple pathogen species). In the case of Lr34, resistance is conferred via three amino acid mutations at the ABC transporter gene  does the pathogen directly ‘highjack’ the cell's sugar transporting mechanism for its own purposes?Partial resistance genes found in cereals may prove to be a source of resistance that pathogens find functionally difficult to overcome. For example, in wheat, both major (R) gene and adult plant resistance (APR) are controlled by the action of single genes, but their molecular nature and role in the biochemical functioning of the plant are very different. Unlike R genes that are an intimate part of an effector-receptor interaction process, APR seemingly genes mediate resistance through modifying sugar regulation or signalling (e.g. Bipolaris sorokiniana (spot blotch) than do lines having drooping and semi-drooping leaves, possibly because erect leaves retain less free water, thus making germination of pathogen spores less likely , and or alternative or alternate hosts, the long-term protection provided will be determined by the product of the effective population size on susceptible varieties, the mutation rate at relevant pathogen infectivity loci and the probability that a novel pathogen mutant survives and increases in the population. The survival of a novel mutant will be determined by competitive fitness interactions and the real possibility of rapid extinction through genetic drift . However, in reality, such multigene combinations often arose through sequential insertion of additional R genes into advanced germplasm that, as a consequence of on-going pathogen evolution, was effectively unprotected (only ineffective R genes present) . The R gene is still subject to challenge by the pathogen, but the presence of the APR gene prevents a full loss of resistance when the R gene is overcome. There is thus a shift in the type of selection pressure being experienced by the pathogen as the R gene is overcome  wheat cultivars demonstrated that partial resistance selected for more aggressive isolates leading to greater leaf areas infected than did susceptible host lines  on the three host multilines.Although the major focus of studies of pathogen–host interactions in mixtures has been on disease development, detailed empirical studies of the genetic structure of pathogen populations have detected changes in both infectivity and aggressiveness within crop mixtures compared to that observed in relevant pure stands. Thus, pioneering work on barley mildew populations demonstrated that under the selective regime imposed by a three-component mixed host population, the range of infectivity possessed by pathogen genotypes depended on the relative fitness of each pathotype across all host lines , while others will be dependent on the population context (e.g. the fitness of competing pathogen races) or be isolate specific (e.g. the extent of fitness costs associated with specific infectivity factors). Mundt  succinctThe evolutionary response of pathogens to selection by mixtures and multilines involving limited host resistance has been extensively modelled for situations involving R genes. Depending on the level of ecological realism involved , such models generally predict pathogen populations in mixtures to be smaller with a pathogenically more diverse (more races) but simpler (lower infectivity pattern) population structure than those occurring in relevant pure stands Barrett , while lThese and earlier global dispersal models demonstrated that the way in which R genes were deployed in mixtures  could affect the evolution of complex races  and have demonstrated reductions in epidemic development , of crop–climate models and of planting and harvesting technologies raise the possibility of future farming approaches that are far more amenable to dealing with close intercropping or even mixtures (separation technologies) than is currently possible. In summary, we argue that development of a robust predictive framework for evaluating the epidemiological and evolutionary consequences of different genetic and spatio-temporal deployment options in the context of what is feasible from a farming perspective represents a major research goal with real application potential for managing disease.


Freshwater ecosystems are important for global biodiversity and provide essential ecosystem services. There is consensus in the scientific literature that freshwater ecosystems are vulnerable to the impacts of environmental change, which may trigger irreversible regime shifts upon which biodiversity and ecosystem services may be lost. There are profound uncertainties regarding the management and assessment of the vulnerability of freshwater ecosystems to environmental change. Quantitative approaches are needed to reduce this uncertainty. We describe available statistical and modeling approaches along with case studies that demonstrate how resilience theory can be applied to aid decision-making in natural resources management. We highlight especially how long-term monitoring efforts combined with ecological theory can provide a novel nexus between ecological impact assessment and management, and the quantification of systemic vulnerability and thus the resilience of ecosystems to environmental change. This is a subtle but important departure from the idea that ecosystem processes are mostly reliant on structural community attributes, like species richness  were present in all analyzed lakes. While the scale-specific distribution of individual feeding groups varied between subarctic and hemiboreal lakes, they shared overall similar functional attributes . The functional redundancy within and among the observed temporal scales was similar across lakes, highlighting the similarity in resilience characteristics across both subarctic and hemiboreal lakes. Another important finding from this case study was that cold-stenothermic species have been lost and replaced with warm-tolerant species in the subarctic lakes. However, this did not yield any observable loss in the resilience of subarctic lakes. Thus, the functional compensation of feeding group attributes over time, despite structural community change, currently seems to maintain the functional underpinnings of ecosystem processes, conferring robustness to subarctic lakes.The subarctic lakes study aimed at identifying resilience characteristics between lake types that have potentially different vulnerabilities to environmental change . However, many cases exist where humans have already had a negative effect on ecosystems. Such a case is anthropogenic acidification, leading to biodiversity loss in many lakes that were sensitive to acidification due to their low acid buffering capacity. There is evidence that acidification caused a regime shift in many Scandinavian lakes. Despite the implementation of international policy to mitigate the impact of acidification, many lakes have shown weak chemical and biological recovery and thus resisted returning to previous conditions  compared littoral invertebrate communities to changing abiotic conditions in acidified (degraded state) and circumneutral  Swedish lakes with long-term data 1988–2012), using the time series modeling approach described above. They again identified dominant and distinct temporal frequencies in the data, which in the time series models are associated with different canonical (RDA) axes  and esthetic services. Some of these services might be at stake if acidified lakes are more vulnerable to further regime shifts with environmental change.Both case studies make clear how long-term monitoring efforts, combined with an ecological complexity approach that is often neglected in assessing environmental change problems, can facilitate an evaluation of systemic vulnerability. However, long-term monitoring is lacking for most ecosystems. The next case study shows an alternative approach to quantify resilience with limited data.Using the vertebrate fauna of the Everglades wetland complex of south Florida (USA), Forys and Allen  quantifiBy combining the case studies with our understanding of resilience theory, we present a conceptual model to empirically assess systemic vulnerability of freshwater ecosystems to environmental change Fig. . Our modOur conceptual model is novel in that it emphasizes the need to identify the scale-inherent structures in data sets for assessing systemic vulnerabilities Fig. . It espeResources for managing ecosystems are always limited, requiring the identification of trade-offs and priorities. Freshwater ecologists and managers are challenged to identify and protect ecosystems that provide rare and vital services but are vulnerable to regime shifts. Our conceptual model provides a starting point for reducing uncertainty by identifying systems that are vulnerable to environmental change, and allowing for standardized comparative analyses of systemic vulnerabilities within and across ecosystems. We believe this approach will facilitate the efficient and effective identification of ecosystems requiring management priority. By quantifying and comparing scaling patterns and the distribution of functional traits within and across scales, inference about the relative resilience of freshwater ecosystems can be made.We illustrate this with the following hypothetical scenarios Fig. . In thesIt is not our aim to provide an exhaustive list of how our model could inform management; environmental change can have context-dependent effects and will therefore require site-specific approaches. However, the following considerations can provide guidance for tailoring structured and site-specific management plans within the context of a growing understanding of general ecosystem response to various aspects of environmental change.It is increasingly clear that environmental change has scale-specific impacts  data with good spatial resolution. Management must continue to emphasize long-term monitoring efforts (Maberly and Elliott Additionally, some level of monitoring must occur in concert with the application of our conceptual model to create an iterative approach capable of capturing ecological complexity and variability over time. Fortunately, existing data do allow us to empirically study vulnerability patterns in ecosystems. These, in combination with specifically designed experiments (Ledger et al. This paper demonstrates how long-term monitoring, combined with other approaches, can be used to create a nexus with ecological theory to refine ecological impact assessment and improve environmental management. A better mechanistic understanding of the ecological complexity that is inherent to ecosystems is needed to improve our knowledge of ecosystem responses to environmental change. This work shows how this complexity can be quantified through the use of monitoring data.


Cancer & Metabolism, Andrzejewski et al. present evidence that metformin acts directly on mitochondria to inhibit complex I and limits the ability of cancer cells to cope with energetic stress. Here, we discuss evidence that supports the role of metformin as a cancer therapeutic.Metformin has been a first-line treatment for type II diabetes mellitus for decades and is the most widely prescribed antidiabetic drug. Retrospective studies have found that metformin treatment is associated with both reduced cancer diagnoses and cancer-related deaths. Despite the prevalence of metformin use in the clinic, its molecular mechanism of action remains controversial. In a recent issue of http://www.cancerandmetabolism.com/content/2/1/12.See research article:  The biguanide metformin is an antihyperglycemic agent used to treat type II diabetes. Metformin decreases blood glucose levels by suppressing liver gluconeogenesis and stimulating glucose uptake in skeletal muscle and adipose tissues. Metformin is prescribed to over 120 million people, providing a wealth of epidemiological data. Retrospective studies have found that metformin treatment is associated with diminished tumorigenesis, with a recent meta-analysis of these studies reporting a 31% reduction in cancer incidence and a 34% reduction in cancer-specific mortality across many tumor types [Although the molecular underpinnings of metformin action remain an area of active investigation, the best described mechanism is inhibition of complex I, the first component of the mitochondrial electron transport chain Figure . ComplexThough AMPK was once considered the primary executor of metformin antidiabetic action, genetic loss-of-function studies in mice have indicated that hepatic expression of AMPK and its upstream activating kinase LKB1 may not be absolutely required for suppression of gluconeogenesis by metformin . An AMPKIn addition to targeting complex I, metformin has recently been shown to inhibit mitochondrial glycerol-phosphate dehydrogenase . Increased mTOR activation has been implicated in a wide variety of different cancers, and AMPK-mediated attenuation of anabolic metabolism and/or mTOR signaling by metformin may contribute to the antitumor effects of metformin in some situations. However, LKB1-deficient tumors, which are compromised in their ability to activate AMPK, are particularly sensitive to biguanides . This suet al. [in vivo have suggested that some tumor cells also oxidize glucose in the mitochondria [The findings of Andrzejewski et al.  support chondria . Since min vivo. Stabilization of hypoxia-inducible factors (HIFs) is a critical part of the metabolic adaptation to low-oxygen and activation of HIFs is reported in many cancers [Decreased vascularization of tumors affects the availability of both glucose and oxygen and may influence how glucose is used by cancer cells  cancers . Metform cancers , suggestin vitro to elicit a biological effect in cancer cells. How the concentrations used in vitro translate to the effects on tumor tissues remains controversial, but some studies have shown that metformin induces cancer autonomous metabolic changes in vivo. Metformin has been shown to activate AMPK in intestinal tumors, even in the absence of changes in organismal blood glucose or insulin levels [in vivo [Despite increasing evidence that metformin can have profound effects on cancer cells, it is not well understood if the drug accumulates to sufficient levels to cause these direct effects in patient tumors. The liver is a clear site of drug action, as it is exposed to high levels of oral metformin and expresses the metformin transporter OCT1, facilitating uptake and allowing for drug accumulation in this tissue. At metformin doses prescribed to diabetic patients, the circulating levels in plasma and peripheral tissues are orders of magnitude below the concentrations used n levels . In anot[in vivo . These d


The physiological relationship between abiotic stress in plants and polyamines was reported more than 40 years ago. Ever since there has been a debate as to whether increased polyamines protect plants against abiotic stress  or cause damage to them (perhaps due to hydrogen peroxide produced by their catabolism). The observation that cellular polyamines are typically elevated in plants under both short-term as well as long-term abiotic stress conditions is consistent with the possibility of their dual effects, i.e., being protectors from as well as perpetrators of stress damage to the cells. The observed increase in tolerance of plants to abiotic stress when their cellular contents are elevated by either exogenous treatment with polyamines or through genetic engineering with genes encoding polyamine biosynthetic enzymes is indicative of a protective role for them. However, through their catabolic production of hydrogen peroxide and acrolein, both strong oxidizers, they can potentially be the cause of cellular harm during stress. In fact, somewhat enigmatic but strong positive relationship between abiotic stress and foliar polyamines has been proposed as a potential biochemical marker of persistent environmental stress in forest trees in which phenotypic symptoms of stress are not yet visible. Such markers may help forewarn forest managers to undertake amelioration strategies before the appearance of visual symptoms of stress and damage at which stage it is often too late for implementing strategies for stress remediation and reversal of damage. This review provides a comprehensive and critical evaluation of the published literature on interactions between abiotic stress and polyamines in plants, and examines the experimental strategies used to understand the functional significance of this relationship with the aim of improving plant productivity, especially under conditions of abiotic stress. Polyamines (PAs) are small, positively charged, organic molecules that are ubiquitous in all living organisms. The three common PAs in plants are putrescine (Put), spermidine (Spd) and Spm, with some plants also having thermospermine (tSpm) in place of or in addition to Spm. The simplicity of their structure, their universal distribution in all cellular compartments, and presumed involvement in physiological activities ranging from structural stabilization of key macromolecules to cellular membranes make them an attractive group of metabolites to assign a multitude of biological functions. Their accumulation in large amounts in the cell could presumably sequester extra nitrogen (N) thus reducing ammonia toxicity and also balance the total N distribution into multiple pathways. It is not surprising that fluctuations in their cellular contents are often related to varied responses of plants to different forms of stress and to different phases of growth activity. As much as their cellular functions are diverse, and sometimes contradictory, so are their roles in plant stress. They have been deemed important in preparing the plant for stress tolerance and to directly aid in ameliorating the causes of stress, and at the same time, their own catabolic products are responsible for causing stress damage. Several aspects of the relationship between PAs and abiotic stress in plants and their seemingly contradictory roles in the process have been reviewed over the years , and with every cell division, cell enlargement and differentiation activity within the organism. Over its lifetime, there are significant changes in the growth environment; some caused by weather events (like rain or drought), and others part of seasonal changes in temperature and day length. For perennials, there still are the longer-term climatic changes that are relevant to their life. Despite difficulties of precisely defining stress, thousands of experimental studies have involved a variety of stress treatments  and analysis of the physiological, biochemical and molecular responses of plants to such treatments when they were growing under otherwise “normal” conditions—thus in most cases significant deviation from status quo may be considered stressful.Before delving into the specific roles of PAs in plant stress responses, a few details are important to consider regarding the phenomenon of “abiotic stress.” The first and the foremost is the lack of a precise definition of this term. Each plant constantly faces a changing microenvironment from the moment it starts its growth, be it from a seed or a vegetative cutting. On a daily basis, these changes occur from sunrise to sunset  may involve avoidance of the imposed stress or short-term adaptation to it with the ability to revert back to the original growth and metabolic state. This is in contrast to the evolutionary adaptation  and the long-term physiological adaptations, e.g., those in shade loving plants vs. those that grow better in full sun, and plants requiring large quantities of fertilizer vs. those that can thrive on marginal lands. In most cases the genetics and physiology of a plant allow it to live in a wide range of environmental conditions (as defined by the climate) while in others the range of acceptable environments may be rather narrow. The developmental stage of the plant also plays a significant role in its response to changing environment.Abiotic stress exposure in plants can be divided into three arbitrary stages: stress perception, stress response and stress outcome Figure . DependiThe transmission of the stress signal also involves a multitude of mechanisms; some of which are common for different types of stress. For example, drought, flooding, salt, heavy metals, ozone, and sometimes heat or cold all show a common set of physiological responses, which involve regulatory metabolites like abscisic acid (ABA), salicylic acid and jasmonate or methyl jasmonate (MeJa). Frequently, these modulators of stress may affect metabolites that are common for tolerance and/or amelioration of a variety of stresses  or they may be specialized . Polyamines, in combination with Pro and GABA belong to the former group with almost universal involvement in a variety of stress responses.2O2; (v) regulators of several ion channels; and, finally (vi) participation in programmed cell death. To this list can be added their role in metabolic regulation of ammonia toxicity, nitric oxide (NO) production, and balancing organic N metabolism in the cell  could cause major homeostatic shifts in cellular metabolism. Therefore, under conditions of stress, PAs could perform these functions better when changes in their metabolism are transient and within narrower limits, thus avoiding catastrophic perturbations in the overall cellular homeostasis of C and N  or its quality .Measurements of stress response have included visual symptoms of water loss or wilting, changes in fresh weight, dry weight, ion release, gene expression, and analysis of enzyme activities and cellular metabolite, etc.ADC or ODC in plants have resulted in a significant increase in Put content  with relatively smaller (<2-fold) changes in Spd and Spm. Transgenic manipulation of SPDS, SPMS, and SAMDC causes smaller (compared to Put) increase of Spd and Spm contents (~2–3 fold).Transgenic manipulations of The fold increase in Put content often varies with the plant species, homologous or heterologous gene sources, nature of the promoter, developmental stage of the plant, and the tissues analyzed.In reviewing the literature on the improvement of stress tolerance in transgenic plants over-expressing a homologous or a heterologous gene encoding a PA biosynthetic enzyme, a few conclusions stand out  positively affected defense-related (biotic/abiotic stresses) genes, signaling pathway genes , and genes associated with ABA-, JA-, and SA- biosynthesis related enzymes. The commonalities of up-regulated stress-related gene expression in Put and Spm-overproducers  suggest overlapping functions of Put and Spm (by interacting with ABA or modulating Ca++ homeostasis), which are common to tolerance against drought, salt or low temperature.The detailed functional enrichment analyses have been reported for differential gene expression in high Put-producing transgenic Oryza sativa L. ssp. indica and japonica) kept under 18 days of drought stress, Do et al. , using a heterologous oat (Avena sativa) ADC gene under the control of a drought/ABA inducible promoter RD29A  the expression of NCED as compared to the wild type plants, suggesting the possibility of transcriptional regulation of ABA synthesis by Put.Recently the role of endogenously produced Put affecting the expression of drought responsive gene 9-cis-epoxycarotenoid dioxygenase (NCED) - can enzyme that controls ABA biosynthesis under stress, was studied in lotus  compromised for tSpm and Spm biosynthesis showed hypersensitivity to NaCl and KCl but not to MgCl2  or by increasing endogenous Spm by constitutive over-expression of a homologous SPMS  with significantly reduced Put content showed increased sensitivity to low temperature . Complementation and reciprocal complementation with ABA and Put, respectively improved low temperature tolerance of the mutants.An Arabidopsis single mutant of A potential role of ABA  at the time of anthesis and prior to application of drought stress, significantly increased photosynthetic attributes, increased contents of Pro, total amino acids and soluble sugars, improved water status, reduced membrane damage, and significantly increased total grain yield as compared to the control plants .➢ Using an s et al.  found thT. aestivum cv. Giza 168) plants. At 5 days after spray, the plants had higher amounts of endogenous Put, Spd and total amino acids, and lower amounts of ammonium and ethylene. Total yield at 155 days in the Put-treated plants was higher vs. the controls  in 30–35 day old wheat (Oryza sativa L. ssp. indica) prior to treatment with NaCl (that continued till maturity) significantly increased grain yield, Ca++ content in the grains, and a higher K+/Na+ ratio as compared to the non-treated control plants  at early booting stage of rice  plants were seen by Tanou et al.  Shi et al.  and antioxidants is perhaps among the most complex and apparently contradictory physiological and biochemical interactions in plants. A functional association between ROS and abiotic stress has been known from the time of their discovery since ROS are capable of causing widespread damage to a variety of cellular metabolites as well as macromolecules  are also up, hence their roles in preventing damage from stress are beneficial as well as deleterious. The role of PAs in augmenting antioxidant based defense systems to impart tolerance against heavy metals, UV and other stresses that are potent inducers of superoxide molecules causing oxidative damage to the living cells have been reported in several studies  is needed for lignin production in the apoplast adjacent to the plasma membrane  in the tonoplast membrane apparently helps the vacuole to contain Na+ within it, thus changing the effective K+/Na+ ratio in the cytoplasm under conditions of stress. Other interactions of PAs with the ion channels have been discussed in several recent reviews  can effectively block cation channels  or from Orn by Orn aminotransferase (OAT). A third metabolite whose cellular content seems to follow the same pattern as Put and Pro is GABA, which, like Pro, is also synthesized by two alternate pathways—from Glu by Glu decarboxylase (GAD) and from Put by diamine oxidase (DAO). An additional product of the Glu/Pro/Arg/Put/GABA pathway is nitric oxide (NO), whose role in various developmental and physiological processes in plants has recently drawn serious attention  showed up to 9-fold increase in foliar Orn, which was accompanied by a small but significant increase (10–29%) in citrulline and a decrease (~20%) in Arg levels. Transgenic lines showed increased germination % and higher root tolerance index when grown in media containing 250 mM NaCl; there also was greater tolerance to salt or drought in mature plants as indicated by their bigger leaf size, and higher growth and chlorophyll contents under stress situations. Additionally, the transgenic plants showed better recovery after stress withdrawal.Ornithine is a non-protein amino acid that is synthesized from Glu (major metabolic entry point of inorganic N in plants) via several steps Slocum, . It is a2O2 vs. the control cells. Similar stress ameliorating properties of Orn were observed in tobacco cells exposed to high osmoticum (polyethylene glycol); interestingly though, a differential role of D-Orn and L-Orn was observed where the former was more effective under salinity and the latter under drought conditions , a perennial weed, a significant increase in free amino acids and soluble protein were observed as an overwintering strategy  plants, exogenous Orn (but not Glu) along with salt stress showed a 2–3-fold increase in Pro contents, suggesting Orn as an effective precursor for Pro accumulation  has received only limited attention. This application is quite relevant to monitoring the health status of perennials in commercial plantations as well as in managed and unmanaged natural forests. Several studies on analysis of foliar metabolites and soluble inorganic ions in mature forest trees have shown a strong positive correlation between PAs (particularly Put) and chronic effects of environmental stress from acid precipitation or excessive N fertilization of soils. The results suggest a potential for developing guidelines to include such biochemical analysis in forest management practices for stress amelioration.a.k.a. “acid rain”). These acids solubilize Ca++ from its bound form in the soil, enabling the plant to absorb it easily, thus initially the increase in mobile Ca++ may help trees to grow better. However, the acidity also mobilizes aluminum (Al), which does two things: (i) it blocks the uptake of Ca++ by roots and (ii) it binds very tightly to soil particles, thereby displacing Ca+ from the soil, which eventually leaches from the watershed to surface water bodies (e. g. lakes). The net result is a serious accumulation of soluble N in the inland water bodies , thus causing algal blooms and harm to other biota. Consequently, some forest soils have become depleted of Ca++ to the point where select tree species have developed Ca++ deficiency; this has happened in the US  and N, emitted into the environment from industrial processes  react with water to form strong inorganic acids, which make up the major component of acidic deposition  in plants whose concentrations change in response to a single or multiple stressor(s) in a predictable (and stable) manner can be useful in monitoring the status of stress response and recovery due to stress ameliorating treatments. Only a handful of metabolic markers linked to specific functions have been identified in plants growing under chronic stress conditions. Phytochelatins are one example of such markers for heavy metal exposure. To be of practical value in assessing community/forest health, the biochemical markers should have the following characteristics: (a) the cost to develop and test them should be reasonable, (b) they should be relevant to ecosystem function that is under study, (c) they should be sensitive and dose responsive so that change in their cellular content should be higher in magnitude relative to “normal” background fluctuations, (d) they should maintain a longer-term new homeostatic level under persistent stress conditions such as those found in forests, (e) their concentration should revert to normal range when the stress inducer is removed from the environment, and (f) they should have broad applicability over temporal and spatial ranges  within US, we have found PAs , along with some of the related metabolites  and inorganic ions to be reliable markers of plant health under conditions of environmental stress or soil nutrient deficiency in forest trees. Their contents in plant tissues like foliage in forest trees show strong correlations with a variety of abiotic stress conditions before the damage due to these conditions is visible.The signs of environmental stress in trees often develop slowly, i.e., in comparison with those from insect or disease damage. For example, visual symptoms of drought stress or nutrient deficiencies may take several years to appear. Unfortunately, once the symptoms of stress and damage become apparent it is often too late to stop or reverse the decline in forest productivity. As mentioned above, it is often difficult to diagnose the source of stress on trees because multiple factors work together  to cause the decline in forest productivity over a period of many years. Moreover, different tree species have different tolerance limits for each type of stress. Thus, a comprehensive analysis of the growing environment in an ecosystem  by multidisciplinary teams that include physiologists, ecologists, pathologists, microbiologists, biogeochemists, and hydrologists must be carried out to assess the complete situation. The joint data collected concurrently would help us to develop links between tree function and environmental disturbances, and risk assessment and stress remediation strategies for forest trees prior to the onset of obvious decline. In this regard, based on studies spanning over multiple years in several ecosystems, including some National Science Foundation (NSF) funded Long-Term Ecological Research (LTER) Sites (4NO3) to simulate the effects of chronic N addition to the soil  stands in three NE US states, which had suffered long-term exposure to acidic deposition from industrial sources, and where the soil solution pH in the organic soil horizon (Oa) was <4.0. The presence of a large number of dead and dying trees at some of the sites indicated that they were apparently under some form of environmental stress. Analysis of soil chemistry and PAs in needles from apparently healthy trees over a 2-year period revealed: (i) a strong positive correlation between Ca++ and Mg++ in the needles and in the Oa horizon of the soil; and (ii) that needles from trees growing on relatively Ca++-poor soils with a high exchangeable Al concentration in the soil solution had significantly higher concentrations of Put than those growing on Ca++-rich soils with a low exchangeable Al concentration  and soil solution Al: Ca++ ratios  of the Oa soil horizon. The study revealed that foliar Put concentration could be used as a reliable biochemical marker for early detection of stress due to soil Ca++-deficiency in natural forests before the appearance of any visual symptoms of stress damage.In our attempts to test the utility of these metabolites as biochemical markers for abiotic stress, our studies have involved multiple sites in the Northeastern US that were either chronically impacted by environmental acid precipitation due to NOx and SOx or experimentally fertilized with N (NHAcer saccharum) increased with the elevation which was accompanied by rise in ambient level of N deposition. Accompanying changes in soil chemistry included decrease in soil Ca++ with increasing N  at Hubbard Brook Experimental Forest NH, USA. In this case, amelioration of soil Ca++ improved the overall health of sugar maple trees and increased seedling regeneration, which was accompanied by a concomitant decrease in foliar Put content. The data further revealed that changes in Put were species-specific in that increased Put indicated stress effect only in species that showed Ca++ deficiency in regard to the ambient levels of Ca++ in the soil. For example, sugar maple, which is known to be more sensitive to Ca++ deficiency showed bigger changes in Put relative to yellow birch  that was less sensitive to existing soil Ca++ levels , foliar Put concentration in sugar maple 2SO4] at Harvard Forest MA, USA (HF) and Bear Brook Watershed ME, USA (BBWM), where N fertilization began in 1989, have shown that N-amended pine (Pinus strobus) and hardwood (red maple - Acer rubrum and red oak - Quercus rubra) stands at HF and mixed-wood  stand at BBWM contained higher concentrations of PAs and amino acids in their foliage  studies on chronic N fertilization [NHVaccinium myrtilloides, Ledum groenlandicum, and Chamaedaphne calyculata) at Ottawa, Canada  can tolerate short term exposure to a multitude of stress factors, only a handful of studies on the survival and yield (fresh or dry biomass of usable product) in these plants under prolonged stress conditions or repeated exposure to the same stress, have been reported. Most importantly, no viable plant variety has yet been created or selected based upon genetic modification of PAs either via breeding or via transgene expression, which could be evaluated in comparison with other varieties showing similar characteristics. It must also be pointed out that a similar situation exists with respect to a plethora of other genetic manipulation approaches that have been shown to be effective in imparting short-term stress tolerance in various plant species. It is expected that the advanced high through-put techniques of genomics, transcriptomics and proteomics, coupled with better techniques of monitoring the live plants under stress and their metabolic status (the metabolome), would provide a better holistic picture of the consequences of up-regulation or down-regulation of genes likely to be involved in stress tolerance in relation to metabolites like PAs. The ability to identify unique or common regulatory nodes of metabolic pathways, and the cross-talk among the different pathways that are affected by genetic manipulation of PA metabolism, will provide us effective targets to genetically engineer plants that are tolerant to different abiotic stresses. The best-case scenario for such genetic manipulation will be that PA metabolism can be controlled in a transient and cell/tissue/organ specific manner in response to the earliest perception of stress exposure before the stress reaches its peak to cause damage. This would generate plants, which will produce additional PAs to protect themselves from stress only when needed without significant alterations in PA and amino acids metabolism under normal growth conditions.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.


Over 12 million people in the United States are surviving after being diagnosed with cancer; this number is expected to rise as baby boomers age and emerging targeted therapies create less toxicity . Yet in The cancer survivor’s transition from being cared for by an oncologist to a PCP was formally assessed by the Institute of Medicine (IOM) in its 2005 report "From Cancer Patient to Cancer Survivor: Lost in Transition" . The repSome of the recommendations for the medical community included (1) implementing a comprehensive survivorship care plan summarizing cancer treatments and details for follow-up, (2) creating and disseminating evidence-based guidelines for screening and managing late complications, (3) seeking reimbursement from third-party payers for survivorship services, and (4) cooperating with both public and private agencies in the research and awareness of survivorship programs . Since tMost oncologists agree (at least in theory) that creating a care plan is important for meeting the IOM recommendations, but they often struggle to choose a concise, workable format and to find the time, personnel, and resources to complete an individual plan for each patient . ComprehThe term "cancer survivorship" can encompass many different phases of treatment, starting from the time of diagnosis through hospice care. An understanding of the patient population being served is fundamental when discussing the initiation of survivorship care. Resources for understanding general guidelines and different types of survivorship programs can be found in the Patients receiving treatment for cancer represent less than 1% of the commercially insured population but account for approximately 10% to 12% of health care expenditures . The extThe use of technology is one way of working toward making the delivery of care more cost-effective. Although more complex systems have incorporated either electronic medical records (EMRs) or personalized care plans to facilitate care for cancer survivors, educating survivors through Web-based tools is becoming standard. But how can emerging and established technologies be used in survivorship programs to incorporate efficiency with patient satisfaction yet remain economically feasible?To better understand how to enhance and improve survivorship programs through technology, a review of the literature was done. This process attempted to illuminate (1) how patients and clinicians currently use the Internet, (2) how specific software is used to improve oncology care, and (3) what strategies survivorship programs have used to incorporate the Internet to improve care. This understanding could lay the foundation for future research and implementation of newer models for survivorship programs.During the literature review, the analysis encompassed a narrower definition of patients: those actively being treated for cancer, including those being transitioned back to the referring physician. This population required facilitation of care among the oncologist, the referring physician, and the patient, where the Internet or other forms of technology had shown an impact in patient outcomes. The focus of this literature review did not consider facilitating treatment while the patient was in hospice or receiving palliative care.Internet Use by Cancer Survivors and CliniciansInternet use among patients with cancer has increased during the past decade. Dissatisfaction with information and communication during clinical encounters with health-care professionals, among other factors, has propelled cancer survivors to search for information on the Internet. This dissatisfaction may have resulted from a lack of face-to-face time with their providers or the fact that the information provided by their clinicians was outdated . In one Several oncology practices have implemented technology into their outreach programs, with varying degrees of success. Educational materials can be found on clinic websites, including transcripts of oncology seminars, YouTube interviews, podcasts, and links to accredited oncology resources . PatientOther clinics have used social media to facilitate conversations between patients. The Mayo Clinic, for example, created a Facebook page to assist patients newly diagnosed with Barrett’s high-grade dysplasia and esophageal cancer as they anticipated surgical and postoperative challenges. Initially, 65 patients were enrolled; the page provided mutual support for managing treatment effects or nutritional challenges after surgery. The physicians did not give medical advice through the website but were able to update patients with newer technologies and newsletters while using information from patient posts to facilitate treatment strategies during clinic visits. After the initial success of this support group, the oncology staff enrolled more patients, who participated in sharing best practices and insights about coping with treatments and outcomes .Oncology Practices Using Software to Improve CareOne of the more comprehensive implementations of technology discussed in recent literature involved a group of physicians who integrated an oncology-based software system within their private practice . This niThe implementation of this software allowed a more personalized approach, and clinicians were able to identify and treat patients at higher risk for complications. A nurse phone triage system, for example, was created to identify, track, and manage symptoms such as dehydration, diarrhea, and insomnia. Identifying such symptoms allowed nurses and patient navigators to facilitate referrals with community services more proactively, thus avoiding patient morbidity and clinic resource utilization. Another essential feature this software integration was physician documentation. Consulting physicians had portal access to facilitate prompt communication, with up-to-date information about treatments and other comorbid issues .After the practice used this software for 5 years, it had lowered patient emergency room (ER) visits by an estimated 68%, reduced hospital admissions treated with chemotherapy by 51%, and reduced length of stay for admitted patients by 21%. The practice also had seen a 22% reduction in outpatient visits/year in the hematology/oncology population and a 12% reduction in outpatient visits/year in the chemotherapy subpopulation .One component critical to this model, as noted by the author, was adequate third-party reimbursement for what was considered more efficient care. In the present fee-for-service reimbursement system, fewer ER visits or less chemotherapy administered means less revenue for the hospital and oncology staff. Without a change in the reimbursement structure, this type of innovative care delivery system would not be financially viable. The author mentioned ongoing discussions with insurance companies regarding hybrid concepts that would incorporate bundle reimbursement plans to make this type of care financially feasible .However, other experiences of incorporating newer technologies within oncology private practices have not been as successful . For insInternet Use With Survivorship ProgramsCancer survivorship programs have incorporated treatment models using Internet- or software-based technology to facilitate a better quality of life for patients. After the IOM recommended individualized care plans for cancer survivors, the University of Pennsylvania launched a Web-based tool using survivorship care plans to provide comprehensive individualized plans for those with breast cancer . During Similar to other studies, this trial showed a higher trend in users being Caucasian, younger, and more educated than other cohorts of breast cancer patients. Only 12% of these women had previously received survivorship information, and 17% of this same cohort could identify an established survivorship program . There wOne academic center assessed patients’ ability to enroll in and use an Internet educational program after receiving a bone marrow transplant BMT; . CurrentThis academic center formed a cross-functional team, which took 2 years to create and program an Internet site, including online registration, consent, assessment, and study implementation . In thisThe results focused on the technical aspects of enrolling and maintaining an interactive website for those on study. Almost 60% of the participants required one phone conversation for technical questions about the online process, whereas almost 30% required multiple phone conversations. Once patients had mastered the technical aspects of the site, there was improved compliance in answering the questions and using available resources. The authors expect to broaden this interventional tool for patients from different cancer centers who need ongoing assessment after BMT, with further tailoring to include video and social-networking features .Survivorship programs have a unique opportunity to incorporate the Internet and other software technologies, as this new area of managing cancer patients is in its infancy, with no concrete "brick-and-mortar" policies in place. Practitioners can learn from common themes, as results from these initial programs have been published .Because clinical trials using Internet-based intervention are relatively new, internal review boards must establish standard practices proactively to ensure efficient implementation of future similar clinical trials . This shBuilding Internet-based programs (compared with institution-based ones) using evidence-based medicine will be critical as we move forward in pioneering new ways of monitoring, educating, and intervening on the behalf of patients . As mentTransitioning Back to Primary CareThe model of using technology to transition patients from their oncologist back to their referring physician could be a prototype for managing other comorbidities such as cardiac disease or diabetes . ImplemeSprandio’s pilot study using oncology-specific software has allowed his clinic to improve its efficiency in caring for its patient population, as evidenced by decreased ER visits and shortened inpatient stays . By usinSprandio’s aforementioned dialog with insurance companies toward replacing fee-for-service reimbursement with a form of bundled payments is a conversation being repeated in multiple settings, as our reimbursement system is undergoing changes under the Affordable Care Act . Once thTechnology Comfort LevelsHelping patients use Internet-based programs will continue to be a challenge, as there has been a wide range of patient participation (12%–54%) in Web-based care systems . AlthougClinicians should also be mindful of some patients’ limited capabilities in using technology or the Internet. More efficient care with effective interventions may, in part, depend on features of the Internet site or software programs. It may be necessary to provide technology support for patients who are not "technologically savvy" to aid in navigating any Internet-based program that requires participation, including accessing password-protected systems . Varied Another disparity observed among cancer survivors using the Internet is that non-Caucasian ethnic groups may be less likely to use or have access to the Internet compared with other groups, perhaps as a result of a lower level of education, living in a rural area, or having poorer physical health . EffortsThe Impending Shortage of OncologistsThe use of technology in caring for oncology patients may be hastened by the impending shortage of oncologists. Studies have shown that even 3 years after a diagnosis of cancer, 50% of patients are still being followed by their oncologist, 10% by their primary care physician, and approximately 40% by both . By 2020Given the impending shortage of oncologists, an option for efficient delivery of care in transitioning the patient back to the primary care physician is to maximize the educational level of the clinical staff when assigning routine tasks . The useFor example, one survey of nurses reported that 16% provided employment assistance or legal issues while setting aside other nursing responsibilities , including nurse practitioners and physician assistants. As survivorship programs are developed, more centers appear to incorporate APs in the patient care role, as physicians continue to see patients in the active treatment setting while remaining available for consultation .The American Society of Clinical Oncology (ASCO) commissioned a study showing the emerging importance of incorporating APs in the oncology setting . ProductStaff Training and SupportBecause survivorship programs require a team approach, adequate education of both treatment-based complications and the proper use of any Internet site or software-based programs is needed. In a study from the Oncology Nursing Society, only 27% of 399 nurses surveyed worked in settings with a formal survivorship program . The greOne recommendation to provide educational programs for nurses new to adult survivorship programs is to learn from established pediatric programs, where there is a larger experience base in monitoring long-term complications. Only 33% of nurses working exclusively in adult settings said that patients were provided a written summary and follow-up care plan, vs. 70% of those in pediatric settings . These pThe creation of survivorship clinics could not come at a better time, as the overall incidence of cancer is estimated to increase by 40% among women and 55% among men by 2020 . As acadCreating Internet-based survivorship programs to facilitate the transition of care back to referring physicians is one facet of care that is still in its infancy. Until data elements conform to common standards, are communicated easily from oncologists to primary care providers, and are placed in other providers’ EMRs, the goal of fully integrating technology and care of cancer patients will not be achieved. Clinical trials currently under way may reach a broader group of patients, as more of them become comfortable using technology to complement their present care during this transition period. Moving away from institution-based technology systems toward Internet-based systems will also allow more patients to participate, thus increasing data banks where stored information is retrieved; emerging standards of care can be implemented with more efficient clinical pathways for cancer survivors transitioning back to their referring physicians.