E. coli [NTSEC] and Mannheimia haemolytica), between 2 strategies for sampling feces , and between 2 strategies for determining which cattle to sample (cattle that were culture-positive for Mannheimia haemolytica and those that were culture-negative).The purpose of this study was to objectively compare methodological approaches that might be utilized in designing an antimicrobial resistance (AMR) surveillance program in beef feedlot cattle. Specifically, four separate comparisons were made to investigate their potential impact on estimates for prevalence of AMR. These included investigating potential differences between 2 different susceptibility testing methods (broth microdilution and disc diffusion), between 2 different target bacteria  and broth microdilution (Sensititre®) for both M. haemolytica recovered by nasopharyngeal swab is recommended if monitoring an animal health pathogen is an objective of the surveillance program as results of surveillance using fecal E. coli cannot be extrapolated to this important respiratory pathogen. If surveillance of E. coli was pursued in the same population, study populations could target animals that were culture-positive for M. haemolytica without biasing estimates for AMR in E. coli. Composite pen-floor sampling or sampling of individuals per-rectum could possibly be used interchangeably for monitoring resistance in E. coli.We found that for general purposes, the susceptibility test selected for AMR surveillance must be carefully chosen considering the purpose of the surveillance since the ability to detect resistance appears to vary between these tests depending upon the population where they are applied. Continued surveillance of AMR in  While several authors have suggested that there are questions that need to be addressed in order to optimize the structure of an antimicrobial resistance (AMR) surveillance program, many approaches have become accepted standard practices without investigating whether they are associated with differences in the ability to detect resistant isolates -3. ScienDecisions regarding antimicrobial susceptibility testing methodology and sampling approaches are crucial to obtaining relevant estimates of AMR in any population . Disk diEscherichia coli has often been assumed to be a reasonable surrogate for pathogens impacting cattle and people. However, feedlot cattle are not treated with antimicrobial drugs (AMDs) for this typically innocuous enteric organism. Rather, AMDs are most often used in feedlots to treat or prevent respiratory infections ), any isolate not classified as susceptible was categorized as being resistant. The outcome for logistic models was the phenotypic resistance or non-resistance of each NTSEC or M. haemolytica isolate, and separate models were evaluated in parallel for each antimicrobial drug tested. Analysis of the variance structure relative to the hierarchy of the data  revealed that almost all of the clustering (correlation in resistance outcomes) occurred at the isolate level . As such, lack of independence among isolates was controlled in models using generalized estimating equations (GEE) using an exchangeable correlation structure. Separate models for each antimicrobial drug and for each methodological comparison were built using a priori understandings of the hierarchical data structure using commercially available software . Odds ratios, 95% confidence intervals (95%CI), and the associated P-values were obtained for each model. Due to lack of precision in the estimates and the impact on model stability, models were not evaluated if resistance to a particular antimicrobial was less than 2%.To facilitate analysis involving logistic regression, isolate susceptibility was dichotomized as resistant and non-resistant (which included both intermediate and susceptible classifications). For AMDs that did not have a published intermediate susceptibility classification  were included for the comparison of detected differences in resistance between two susceptibility tests. Conditional logistic regression with matching on the isolate level was used to evaluate the association between resistance to each AMD and susceptibility test. The type of susceptibility test was the only predictor included in these models (automated disk diffusion vs. broth microdilution). There were 7 AMDs that could be analyzed in these models as these drugs were evaluated on both susceptibility panels . Separate models were analyzed for NTSEC recovered from individuals and all M. haemolytica that were recovered from the same individuals were evaluated in conditional logistic regression models (matching on isolate) to investigate systematic differences in resistance for the two organisms. Only susceptibility results from testing with broth microdilution were included in these analyses. Separate models were evaluated for each resistance outcome  and organism was the only predictor included in these models (NTSEC vs. M. haemolytica).Isolates of NTSEC and To investigate the potential that there may be differences in resistance prevalences among NTSEC recovered from individual fecal samples when compared to those recovered from composite (pen-floor) samples, isolates recovered from these two sample types were compared using logistic regression. Only susceptibility results from testing with broth microdilution were included in these analyses. Lack of independence among isolates was controlled in models using generalized estimating equations (GEE) using an exchangeable correlation structure. Separate models were evaluated for each AMD and the sample type was the only predictor included in these models . Since pens were occasionally stocked over a number of days, only the isolates that were recovered from composite and individual samples that had been collected on the same day were included in these analyses.M. haemolytica versus those that were culture-negative. Only susceptibility results from testing with broth microdilution were included in these analyses. Only isolates collected from culture-positive and culture-negative cattle that were housed in the same pens and sampled within the same time frame were included in these analyses. Lack of independence among isolates recovered from individuals housed within the same pens was controlled using GEE using an exchangeable correlation structure, and separate models were investigated for each AMD. The M. haemolytica culture status of individuals (culture-positive vs. culture-negative) was included as the fixed effect of interest, and additional predictor variables were controlled in the analyses to account for potential differences in resistance that might be associated with feedlot, sampling time-frame , and a single interaction term between sample time-frame and M. haemolytica culture status.Logistic regression was used to compare the likelihood for detecting resistance prevalences among NTSEC recovered from cattle that were culture-positive for M. haemolytica nasopharyngeal isolates were evaluated for susceptibility using both broth microdilution and disk diffusion. Results indicated that there were strong differences in the likelihood of resistance classification for NTSEC isolates from individual fecal samples for 5 of 6 AMDs .There were also strong differences in the likelihood of detecting resistance for M. haemolytica that were both recovered from the same 2190 individuals were identified for this comparison and evaluated by broth microdilution. Results indicate that there was a strong statistically detectable difference in the likelihood of detecting resistance in NTSEC for all drugs except nalidixic acid, when compared to M. haemolytica .Isolates of NTSEC and ca Table . Non-typP>0.30; Table A total of 412 NTSEC recovered from 137 individual fecal samples collected per rectum on the second sampling day were used in this analysis, along with 198 NTSEC isolates collected from 40 composite pen-floor samples that were collected from the same pens on the same days. Controlling for the lack of independence created by analyzing multiple isolates from the same sample and obtaining multiple samples from cattle housed in the same pen, the proportion of resistant isolates was not different for any of the drugs evaluated . Modeling as described, results indicated that there was no detectable difference in the proportion of resistant isolates when comparing NTSEC isolates recovered from these 2 groups  that were culture-positive for M. haemolytica culture status affect results related to resistance in NTSEC. Thus surveillance programs could be flexible relative to these factors.This study was performed because of the practical questions that naturally arose when attempting to design a large-scale surveillance program for AMR in beef feedlot cattle. Specifically these questions were: Does it matter which susceptibility testing methods are used? Is resistance different for enteric surveillance targets compared to testing of a respiratory pathogen? How should fecal samples be collected? And, does selective sampling of cattle bias results? These results suggest the method for evaluating susceptibility and the target bacteria chosen for surveillance programs must be considered carefully relative to the purpose for conducting surveillance, as these choices will systematically affect the results of the investigations. However, there was no difference in resistance estimates obtained from fecal samples collected per rectum or as composite pen floor samples, nor did in vitro test results for antimicrobial resistance, analyses were heavily dependent upon interpretive criteria used in classifying susceptibility of isolates. Ideally, breakpoints specific to animal species, disease, pathogen, drug, and the administration regimen  should be used for the most clinically valid interpretations. However, approved guidelines have not been defined for all of the antimicrobial drugs tested in this study regarding feedlot cattle. Therefore, each antimicrobial drug was carefully investigated relative to NTSEC and M. haemolytica to identify appropriate CLSI interpretive criteria when possible and published acceptable alternatives when CLSI criteria were not defined , as neither can be considered a perfect reference. Therefore, while these results showed that for most of the drugs evaluated there were strong differences in the likelihood classifying organisms as being resistant, the superiority of one test over the other for correct classification of resistance cannot be asserted. Again, the interpretive criteria used in this study could have affected the estimates for resistance classification. While the interpretive criteria established by CLSI are intended to provide the same classification results for broth microdilution and disk diffusion when comparing test results for the same isolates and extreme care was used to ensure that appropriate breakpoints were used throughout this investigation, results could have been affected by the absence of standardized criteria for all bacteria-drug combinations and specifically defined for cattle. Other studies have evaluated the MICs estimated by algorithms based on the zone diameters and compared them to the MICs determined by broth microdilution to show both close agreement as well as widely divergent results ,28. InteM. haemolytica would not be recoverable from all cattle, while NTSEC is ubiquitous [M. haemolytica. The ability to recover M. haemolytica might be related to the treatment history of an individual as well as the sample time . Further, the ability to recover this bacterium could also be related to its resistance status. Therefore, a direct comparison between M. haemolytica positive and M. haemolytica negative NTSEC isolates was warranted to verify if results might be biased by the design consideration to only recover NTSEC from individuals culture positive for M. haemoltyica. Other factors were also controlled in these analyses which may have affected the likelihood of detecting resistant NTSEC. Even though the interaction (sample time*M. haemolytica status) was not found to be statistically significant in any of the models, it was still forced into models due to its biological relevance.Investigations of AMR in animal populations have much greater relevance to producers if important bacterial pathogens are incorporated in surveillance efforts alongside monitoring an indicator of AMR for the purposes of public health surveillance. However, based on previous research, it was known that the iquitous . TherefoM. haemolytica, the distributions of sampling days (DOF) were similar and sampling time was included as a variable in the analysis.There was wide variability in the timing of when the second samples were collected because this sampling was coordinated with the need to restrain and handle individual cattle for routine management procedures  This routine handling occurs at different times because of variability in cattle weights/ages and sex, in addition to the practical logistics of managing large feedlots. Regardless, this variability should not have systematically altered results of this study as comparisons of multiple bacterial isolates recovered from individual cattle were limited to those recovered on the same day . Also, for comparisons of NSTEC isolates recovered from cattle that were culture-positive or culture-negative for M. haemolytica recovered from feedlot cattle is appropriate in order to improve the relevance by inclusion of an animal health pathogen, especially since AMR in NTSEC does not predict results for M. haemolytica. Sampling strategies used in AMR surveillance for NTSEC recovered from feedlot cattle could possibly employ composite pen floor fecal sampling interchangeably with sampling feces from individual cattle per rectum. Feces could be sampled from individuals to recover NTSEC without regard for M. haemolytica culture status as an efficiency when both agents are targets for surveillance.We found that for use in AMR surveillance, the susceptibility test must be carefully chosen considering the purpose of the surveillance since the ability to correctly classify target organisms apparently can vary between these tests. This study was not designed to evaluate the accuracy of either test, but this investigation demonstrates that results for the two tests varied among the different drugs. Surveillance of AMR in E. coli; Amox-Clav: Amoxicillin-clavulanic acid; Trimeth-Sulfa: Trimethoprim-sulfamethoxazole.AMR: Antimicrobial resistance; AMDs: Antimicrobial drugs; NTSEC: Non-type specific The authors declare that they have no competing interests related to this manuscript.KMB participated in study design, conducted all statistical analyses, participated in interpretation of analyses, and drafted the manuscript. SPG and TAM participated in study design, provided oversight of laboratory analysis, and participated in interpretation of analyses. SC participated in study design and assisted with interpretation of analyses. CWB participated in study design, provided oversight for sample collection, and assisted with interpretation of analyses. PSM conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors have read and approved the final manuscript.


Carnivores, such as the dolphin and the domestic cat, have numerous adaptations that befit consumption of diets with high protein and fat content, with little carbohydrate content. Consequently, nutrient metabolism in carnivorous species differs substantially from that of non-carnivores. Important metabolic pathways known to differ between carnivores and non-carnivores are implicated in the development of diabetes and insulin resistance in non-carnivores: (1) the hepatic glucokinase (GCK) pathway is absent in healthy carnivores yet GCK deficiency may result in diabetes in rodents and humans, (2) healthy dolphins and cats are prone to periods of fasting hyperglycemia and exhibit insulin resistance, both of which are risk factors for diabetes in non-carnivores. Similarly, carnivores develop naturally occurring diseases such as hemochromatosis, fatty liver, obesity, and diabetes that have strong parallels with the same disorders in humans. Understanding how evolution, environment, diet, and domestication may play a role with nutrient metabolism in the dolphin and cat may also be relevant to human diabetes. Tursiops truncatus) exhibit metabolic changes after fasting that are similar to those observed in humans with diabetes, including an elevation in fasting plasma glucose concentration (Felis catus), another obligate carnivore, exhibits metabolic characteristics during fasting that are similar to those observed in dolphins. A clinical syndrome of diabetes mellitus described in pet cats has been proposed as a model for human diabetes  review adaptations in nutrient metabolism that have appeared during evolution in mammals that consume a meat-based diet that is naturally low in carbohydrate; (2) examine parallels between select metabolic features of normal carnivores and metabolic disturbances observed in humans with diabetes; (3) discuss the usefulness of carnivore species as diabetic models.For purposes of the following discussion, “carnivore” is used to describe any animal that consumes a diet composed of at least 70% animal tissue . While mThe domestic cat is among the most extensively studied carnivores and serves as a representative model for metabolic adaptation to carnivory. Numerous anatomic, behavioral, and physiologic adaptations have appeared during feline evolution as a result of pressures imposed by the nutritional needs and feeding ecology of cats . Underlytas1R2 gene, which encodes one subunit of a heterodimeric sweet receptor (tas1r gene isoforms from six carnivores (one felid and five non-felids) showed that only the felid (Asiatic lion) showed no taste preferences when offered water or water with added sweetener  in protein-rich foods . Lacking substantial dietary carbohydrate, cats use dietary protein to supply brain energy needs via the latter mechanisms  for the presence of erythrocyte adaptations the support transport of large amounts of glucose in the blood . In mostprimates , 32. Theprimates . The verKm hexokinase (HK) activity  activity and the predominance of low activity , 35. Theactivity ]. For exactivity , 38. It activity , 40, butMustela vison), an obligate carnivore, also exhibits active hepatic gluconeogenesis. In fact, activities of G6Pase and PEPCK are higher than measured in cat liver  to study adaption to obesity and diet yielded unexpected results. Despite insulin resistance and impaired glucose tolerance, chronically obese cats exhibit lower hepatic glucose production during fasting than lean counterparts  and liver are major organs involved in blood glucose sensing, but important roles are hypothesized for specialized neurons in the brain and enteroendocrine cells in the gut. Precise minute-to-minute regulation of insulin secretion from the pancreatic beta cells requires a glucose-sensing mechanism to modulate beta cell function according to the prevailing glucose concentration, while glucose-sensing pathways in hepatocytes ensure proper balance between hepatic glucose usage and production . CentralGCK gene, is found only in glucose-responsive tissues where it is the de facto “glucose sensor” , which is localized in the hepatocyte nucleus. GCK undergoes a well-described nuclear-cytosolic translocation cycle and its subcellular localization varies with the nutritional state . In the activity .GCK and GCKR (the gene that encodes GKRP) mRNAs are absent from feline liver and activities of GCK and GKRP proteins are not detectable . However, like the cat, GCKR knockout mice are susceptible to stress-induced hyperglycemia and exhibit mild glucose intolerance compared with wild-type that express GCKR  of hexokinase I means that enzyme activity will be maximal at glucose concentrations below the basal blood glucose concentration and cannot increase its rate of glucose phosphorylation in the face of increasing blood glucose levels . However, GKRP has effects on GCK gene and protein levels distinct from its action as a regulator of GCK activity. Thus, the lack of GCKR expression in cats may contribute to reduced GCK mRNA expression and low enzyme activity observed in feline liver as was shown for mice with GCKR knockout . The kinetic features , which metabolizes fructose and serves as an upstream regulator of GK:GKRP binding, is preserved in the cat. Although fructose can be digested and absorbed by cat intestine . Each MODY subtype (there are five subtypes) results from mutation of a gene involved in glucose metabolism; GCK mutations cause MODY type 2 (MODY2), which account for as many as 60% of all MODY cases    lends suGCK and GCKR expression in liver of the domestic cat has been convincingly demonstrated. But is the lack of expression of these genes unique to the cat or is it a universal feature of carnivore metabolism? Direct determination of GCKR expression or activity in vertebrate liver has not been widely explored. A study by Vandercammen and van Schaftingen  and a splice mutation in exon 2. GCKR in Tursiops had predicted frame shift mutations in seven exons  and a splice mutation in exon 1  in all of the mammals examined , separate genetic events leading to the evolutionary loss of GCKR likely represents independent adaptations to carnivory. Like GCKR, other hepatic genes with metabolic roles appear to have adapted to serve carnivore metabolism during evolution of carnivory  obtained from Atlantic bottlenose dolphins that revealed significant differences between non-fasted  or fasted  conditions. In fasted dolphins, a slight but significant increase in glucose was detected. Fasted samples also had elevated platelet count, chloride and creatinine concentrations, and increased activities of serum alkaline phosphatase, gamma-glutamyl transferase, and creatine kinase. Fasting decreased serum concentrations of blood urea nitrogen, potassium, carbon dioxide, triglycerides, and uric acid. Changes in glucose, uric acid, GGT, ALP, platelet count, and acid-base indicators observed in fasted samples paralleled changes observed in humans with diabetes. A follow-up study by the same investigators examined dynamic glucose responses in dolphins fed either a dextrose solution or a fish meal . Such comparisons are useful and can inform understanding of normal carbohydrate metabolism for a particular species but differences should not be interpreted to imply pathology in one or the other species. The value in gaining and understanding of the mechanisms that govern glucose and insulin responses in healthy carnivores is the insight provided into the possible pathologic mechanisms that produce similar changes leading to glucose intolerance in non-carnivore species.In the diabetic liver, failure of insulin to induce a switch from glucose production to glucose utilization reflects hepatic insulin resistance –110. TheIn carnivores consuming a natural diet, the low carbohydrate content minimizes the possibility that large changes in glucose will occur during the absorptive phase following a meal. When the diet contains low amounts of glucose, hepatic gluconeogenesis is predicted to be the major pathway to maintain blood glucose. Consistent with the latter expectation, the activities of key gluconeogenic enzymes  are increased in the liver of normal cats , 35. WhoInsulin resistance occupies a prominent pathophysiologic role in the development and propagation of the diabetic state. Insulin resistance can be conceptually defined as a resistance (impaired sensitivity) to insulin action at the tissue level. Insulin resistance is mediated at the cellular level with muscle being the most important affected tissue, but adipose tissue and liver also affected. Early insulin resistance is characterized by hyperinsulinemia but not hyperglycemia. Hyperglycemia does not develop because the reduced effect of insulin at target tissues is offset by an increase in insulin secretion. Later, when beta cell function is impaired and the total insulin secretory capacity falls, hyperglycemia develops because insulin resistance is now accompanied by a relative or absolute insulin deficiency.In animal studies, insulin resistance is difficult to measure directly except under carefully controlled conditions using euglycemic clamp methods and is not routinely performed outside of the research setting. The euglycemic clamp is the gold standard used to measure insulin resistance in humans, too, but algorithms have been developed that use simple clinical measures to identify insulin resistant humans . The algDolphins develop two hepatic disorders, hemochromatosis and fatty liver, that have been associated with acquired insulin resistant states in humans. Hemochromatosis, also known as iron overload, is a disorder associated with hepatic iron accumulation and severe insulin resistance . HemochrFatty liver of dolphins resembles human non-alcoholic fatty liver syndrome and hepatic lipidosis described in cats and other veterinary species. A recent investigation that examined archived pathology samples from 18 captive dolphins that had died over a 25-year period reported finding histological evidence of fatty liver in nearly 40% of dolphins . LipidosFeline obesity has many parallels with obesity in humans and may serve as a model for the disorder in humans . ObesityEuglycemic clamp studies in lean and obese cats consistently show insulin sensitivity is decreased in obese cats compared with lean cats –45. BodyIn studies with carnivores, it is important to consider possible ways that captivity or domestication might impact “normal” carbohydrate metabolism. Factors such as environment, husbandry, diet, and ability to adapt to captivity are potential confounding factors that must be considered when investigating carbohydrate metabolism using captive populations of bottlenose dolphins. Likewise, the effects of domestication must be taken into account when using the cat as a model for carnivore metabolism. Domestic cats live in a research colony or as pets, consume processed diets, and have a relatively sedentary life style compared with wild felids. Further selective breeding, which has occurred in cats for thousands of years, may have intentionally or unintentionally introduced traits that alter nutrient metabolism. For example, it is possible that the high prevalence of diabetes in pure bred Burmese cats from catteries in Europe and Australia is an unintended outcome of selective breeding since Burmese breed has not been identified as a diabetes risk factor in the U.S. feline population , 133. AlVariability in fasting glucose is one example of a possible effect of captivity or confinement on metabolic measurements in carnivores. In three separate studies, fasting glucose in captive bottlenose dolphins was determined to be between 108 and 194 mg/dL , 103. ThSimilarly, the blood glucose of cats may vary unpredictably under some experimental conditions. The propensity for rapid development of hyperglycemia when handled or during interventions is a confounding factor that must be considered when interpreting studies of glucose homeostasis in cats. Investigators have addressed potential “stress hyperglycemia” by using behavioral desensitization techniques to adapt subject cats to the experimental situation prior to obtaining baseline sample , 136. NeDiet is another factor that might be expected to influence carbohydrate metabolism in captive and free-living populations. A captive or domestic carnivore may not consume a natural prey-based diet and the substitute diet provided may not replicate exactly the natural diet. The importance of diet for glucose homeostasis is illustrated by the results from a study of captive cheetahs. Cheetahs fed whole prey had different colonic short chain fatty acid profiles and lower colonic propionate concentrations than when they were fed meat alone . ColonicIn cats, the wide variety of available commercial diets formulated using different protein, fat, and carbohydrate compositions, means that diet effects must be carefully controlled for in studies of feline nutrient metabolism. Complete discussion of the complex effect of diet composition on glycemia and metabolism in cats exceeds the scope of this article but the subject has been summarized in detail by others . HoweverIn the domestic cat, substantial elevations in blood glucose are achieved during challenge tests, such as the intravenous glucose tolerance test, due to the rapid injection of large amounts of glucose, a simple carbohydrate –147. HowDiabetes mellitus in the domestic cat is a frequently encountered endocrinopathy in veterinary medicine but is probably rare in free-living felids and other carnivores. Overt diabetes mellitus – with hyperglycemia and severe insulin deficiency as prominent features – can be considered a disorder of domestication in that affected cats would die without treatment. Thus, among pet cats and the myriad of other domestic and captive species in which diabetes has been reported, the diabetes prevalence is a reflection of the willingness and ability of veterinarians and pet owners to provide care and treatment –154. SimHemochromatosis is well documented in dolphins maintained in captivity but is poorly documented in free-living dolphins. Similarities between hemochromatosis of dolphins and humans has already been discussed but there is evidence that hemochromatosis is a disorder of captivity. A study comparing iron indices reported that serum iron, ferritin, and transferrin saturation levels were lower in free-living dolphins than in captive dolphins, suggesting an association between iron disorders and captivity .Knowledge of the genetic, molecular, and physiologic mechanisms that govern carnivore metabolism is incomplete, yet elucidation of their unique metabolism is essential for understanding the physiology, ecology, and husbandry of carnivore species and their evolutionary adaptation to a specialized ecological niche. Likewise, the potential for carnivores to serve as comparative models for human diabetes has yet to be fully realized. The similarities between normal metabolic processes in carnivores and diabetes pathology in humans suggest carnivores may be useful models for human type 2 diabetes. While no single animal model completely reproduces the human disease, the dolphin and cat models represent a unique opportunity to study important features of human diabetes and offer several advantages as research subjects. First, a major advantage is that diabetes does not have to be induced because the metabolism of interest is present in healthy animals. This avoids a drawback of many traditional animal diabetes models in which the disease must be induced, the animals become clinically ill or require extensive treatment. Second, compared with humans, normal carnivores appear to have mild glucose intolerance and reduced insulin sensitivity, which offers opportunity to study the metabolic pathways that underlie glucose intolerance and insulin resistance, two hallmarks of diabetes pathology in humans. Third, the animals are long lived, which permits study of individual animals over many years, easy to maintain in a controlled environment, are cooperative, and accessible for study.Carnivores are remarkably adapted to their ecological niche as predators , 159–161Thomas Schermerhorn is the sole author and conceived, researched, organized, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Emotional experiences leave vivid memories that can last a lifetime. The emotional facilitation of memory has been attributed to the engagement of diffusely projecting neuromodulatory systems that enhance the consolidation of synaptic plasticity in regions activated by the experience. This process requires the propagation of signals between brain regions, and for those signals to induce long-lasting synaptic plasticity. Both of these demands are met by gamma oscillations, which reflect synchronous population activity on a fast timescale (35–120 Hz). Regions known to participate in the formation of emotional memories, such as the basolateral amygdala, also promote gamma-band activation throughout cortical and subcortical circuits. Recent studies have demonstrated that gamma oscillations are enhanced during emotional situations, coherent between regions engaged by salient stimuli, and predict subsequent memory for cues associated with aversive stimuli. Furthermore, neutral stimuli that come to predict emotional events develop enhanced gamma oscillations, reflecting altered processing in the brain, which may underpin how past emotional experiences color future learning and memory. Emotions are layered upon the stream of sensations that we experience throughout our lives. Beyond being merely another event in an experience, they shape the quality, vividness, and persistence of what is remembered. The mechanisms underlying this likely encompasses processes during encoding of the experience, its subsequent consolidation, and eventually retrieval.In principle, the neural processes that support emotional memory should exhibit similar properties to emotional memory itself. Such an isomorphism underpins virtually every attempt to construct neural explanations for psychological processes Boring, . This rede facto psychological paradigm in neuroscience, memory, like all psychological processes, is inferred by observing the actions of an organism. The other defining characteristic is the presence of affective events. What constitutes affective is deceptively simple. Affective stimuli often have a direct impact on an organism's homeostasis, either by providing nourishment or inflicting physical distress. But for many situations, the emotional stressor is just perceived or anticipated. For instance, emotional memory can arise from viewing a gruesome picture, or the delivery of bad news. Thus, an emotional experience does not require an overt biologically salient stimulus. And even if the stimulus is overtly affective, its salience is influenced by a subject's goals and circumstances; a food reward will not be arousing to a satiated subject.Before addressing the neural basis of emotional memories, we must outline their properties and how they are studied. Readers familiar with these concepts can skip to the subsequent sections. Emotional memories are typified by their endurance, vividness, and ease of recall  activation, that received extensive investigation as a signature of emotional processing. Since at least the 1940s, it has been known that emotionally salient stimuli drive the nervous system differently. EEG studies were the first to identify the stimulus-driven neural correlates of arousal. Cortical EEG activation was one such response, referred to variously as “activation,” “desynchronization,” “low-voltage fast,” or “alpha-blocking,” because it replaced the slow EEG rhythms that accompany a relaxed state with a fast low amplitude chatter. What made the activation response compelling was that it increased with the arousing properties of the triggering stimuli and could be acquired by neutral stimuli that gained behavioral relevance Magoun, . The actSoon, it was found that cortical activation and behavioral arousal reactions could be elicited by electrical stimulation of brain stem nuclei  and EEG, have recently received particular attention because they co-occur with numerous psychological processes, and are attractive candidates for coordinating activity within local circuits and between distant brain regions Fries, . Their revoked, defined by their consistent phase-locking with stimulus onset. This consistency leads to the evoked gamma oscillation being readily observable by averaging the neural response (EEG or LFP) across repeated stimulus presentations. They usually occur at a relatively short-latency from stimulus onset, with a laminar profile similar to potentials evoked by thalamic stimulation , therefore gamma-band responses to single stimuli must be understood. After this, we will cover combinations of stimuli, where subjects learn that a neutral stimulus precedes an affective event. Each of the phases supporting this learning will be covered separately, namely acquisition, consolidation, and retrieval.conditioned and unconditioned. Unconditioned stimuli have affective qualities that typically drive behavioral responses without prior training. Depending on their intensity they can elicit emotional responses. In contrast, conditioned stimuli generally do not evoke a behavioral response, except when they are novel or out of place  signals , they have also been extensively investigated in the hippocampus  predicts an aversive or rewarding event (US), leading to the development of a conditioned behavioral response. Acquisition is generally defined as the time period during which subjects develop a reliable conditioned response to the CSPerhaps the first investigation focusing on gamma activation during the acquisition of classical conditioning was Miltner et al. . For thiA more recent study found that enhanced gamma power develops to a visual grating stimulus that is repeatedly paired with negative affective pictures (Keil et al., Overall, the studies reviewed above, and others (e.g., Jeschke et al., To overcome this, and to examine the relationship between gamma and emotional memory in particular, Headley and Weinberger  recordedemotional tag, perhaps in addition to its role in facilitating memory. Indeed, if gamma just supported memory for events as they happened irrespective of emotionality, then enhanced gamma to the CS− should have resulted in improved discrimination. Since this was not the case, the memory facilitation tied to gamma was related to the subjective emotional content of the experience.To test this, the same subjects underwent several sessions of discrimination conditioning; a novel tone was introduced that did not predict shock, the CS−. Subjects discriminated between the CSs; they maintained their fear responses to the CS+, and decreased responding to the CS−. However, not all subjects acquired this discrimination immediately. There was a range of performance, with some subjects still treating the CS− as if it predicted shock on the second discrimination training session. Curiously, these subjects exhibited stronger CS− induced gamma during the first discrimination training session. These results suggest that gamma serves as an This study also found that gamma-band activation modulated the magnitude of CS+ related plasticity in the auditory cortex. Previous work had established that fear conditioning with auditory stimuli shifts frequency tuning curves in auditory cortex so that the frequency of the CS+ elicits increased responses (Weinberger, Presumably the gamma oscillations generated in auditory cortex are facilitated by the activation of the cholinergic afferents projecting from the NB. Thus, with respect to the auditory cortex, direct NB stimulation should emulate an unconditioned stimulus (McLin et al., Despite an abundant literature on the consolidation of emotional memories McGaugh, , no studOvercoming this, Laitman et al.  analyzedComplicating this picture is a study on gamma coherence during post-training slow wave sleep and memory for neutral word pair associations (Molle et al., Instead of correlating gamma oscillations during the consolidation period with subsequent memory, an alternative is to induce them in brain regions involved in consolidation of emotional memories. A recent study has done just that, using optogenetic tools to selectively excite neurons in the amygdala immediately after training rats on an inhibitory avoidance task (Huff et al., Following up on these early findings, Huff et al.  delivereThe basolateral nucleus of the amygdala plays a critical role in the post-training consolidation of emotional memory McGaugh, . If suchIn contrast with the paucity of studies on the role of gamma on consolidation, their presence during the expression of emotional memories is well documented. An interesting observation to emerge from this work is that, just as affective stimuli enhance gamma oscillations, so do neutral stimuli that have come to signal affective events.One of the first studies to show a relationship between gamma and performance on a task that could be construed as emotional was done in hungry dogs that acquired a cued instrumental response for food Dumenko, . It was However, perhaps more important than just demonstrating a correlation between gamma and emotional memory is to uncover its consequences for the circuits that support later performance and retrieval. Bauer et al.  showed tA similar pattern of results was also found when comparing activity in the amygdala and striatum (Popescu et al., Besides studies relying on appetitive training tasks, negative emotional learning has also been explored. Using four daily sessions of tone/shock classical fear conditioning, Headley and Weinberger  tracked The synchrony arising from gamma oscillations is thought to support the binding of neurons into coactivated cell assemblies, allowing them to drive each other and their mutual downstream targets more effectively (Womelsdorf et al., evoked, having a short-latency and time-locked to stimulus onset, they exhibit modulation by emotional content that can be unconscious (Keil et al., Induced gamma on the other hand, which occurs several hundred milliseconds after stimulus onset and without phase-locking, reflects a subject's awareness of emotional content (Keil et al., Bringing the reviewed studies together, several patterns emerge. The first is that affective stimuli, particularly those with negative valence, elicit enhanced gamma oscillations in both the neocortex and amygdala, the two structures that were the focus of this review. When these responses are Second, induced gamma is the form that appears most affected by learning. This is especially true for neutral events that have been paired with affective ones. The initially neutral stimuli acquire the ability to elicit stronger induced gamma oscillations. This increase typically occurs near the end of the stimulus, close to when the reinforcer would normally occur.Third, the enhanced gamma that accompanies emotional learning appears to promote coordinated spiking between neurons. For example, this was the case for neurons in auditory cortex that responded to a fear conditioned stimulus (Headley and Weinberger, Finally, there are presently four under-addressed issues in the study of gamma oscillations and emotional memory. The first is the dearth of studies investigating gamma's role in consolidation. Apart from the few reviewed above, to the best of our knowledge no other studies have probed this issue. The second issue is whether the function of gamma oscillations in emotional memory can be dissociated from their role in attention. Studies of gamma oscillations during emotional memory consolidation, when conducted during the post-training consolidation period, lack the discrete attention grabbing stimuli that are often reinforced. Thus, studies of gamma during this period may provide insights into their ability to coordinate activity without the ongoing influence of attentional processes. The third issue is the long-term consequences of the changes in gamma coordination that occur for stimuli that come to predict affective events. How does this enhanced gamma-band activation affect future learning about these stimuli, and are these changes in any way unique to emotional learning? Lastly, are gamma oscillations recruited in a qualitatively different manner during emotional experiences compared with non-emotional ones, or are these differences only quantitative? Perhaps emotional stimuli fall along a continuum that includes stimuli that are novel, goal-related, or devoid of emotional inflection, with each of these engaging circuits that mediate gamma oscillations to different degrees. Answering these questions could go a long way toward explaining the salience, vividness, and accessibility of emotional memories.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.


In vitro toxicity results do not easily translate into in vivo toxicity. ▶ Current research lacks a unifying protocol for the toxicological profiling of NPs.▶ NP toxicity depends on NP characteristics, administered dose and route. ▶  in vitro and in vivo results are described along with the need for a unifying protocol for reliable and realistic toxicity reports.In recent years, nanoparticles (NPs) have increasingly found practical applications in technology, research and medicine. The small particle size coupled to their unique chemical and physical properties is thought to underlie their exploitable biomedical activities. Here, we review current toxicity studies of NPs with clinical potential. Mechanisms of cytotoxicity are discussed and the problem of extrapolating knowledge gained from cell-based studies into a human scenario is highlighted. The so-called ‘proof-of-principle’ approach, whereby ultra-high NP concentrations are used to ensure cytotoxicity, is evaluated on the basis of two considerations; firstly, from a scientific perspective, the concentrations used are in no way related to the actual doses required which, in many instances, discourages further vital investigations. Secondly, these inaccurate results cast doubt on the science of nanomedicine and thus, quite dangerously, encourage unnecessary alarm in the public. In this context, the discrepancies between  Despite the theoretically brilliant logic, animal and human studies have taught us differently and highlighted the issue of the feasibility of correlating organ toxicity with the pre-determined dose; there exists a widely acknowledged problem of extrapolating in vitro concentrations into in vivo scenarios which can be subdivided into two points; firstly, it has yet to be determined how efficiently any administered NP dose is reaching the target tissue and secondly, NPs can induce biochemical changes in vivo which may have gone unnoticed in isolated cell-based studies. With the potentially disastrous consequences in mind, new ways of predicting as yet unpredictable, non-dosage-dependent actions of NPs in vivo must be sought. Apart from the dosing issue, another, so far underexposed area of nanotoxicity relates to the route of particle administration which may also, quite independently from the dose, influence toxicity in an adverse fashion. It is sensible to assume that biodistribution, accumulation, metabolism and excretion of NPs will differ depending on the route of administration as will its toxicity. So far, no reviews have focused on the association between different routes of administration and NP toxicity.Nanoparticles have a large surface area to volume ratio which leads to an alteration in biological activity compared to the parent bulk materials. In the past two decades, the use of nanoparticles (NPs) in experimental and clinical settings has risen exponentially due to their wide range of biomedical applications, for example in drug delivery, imaging and cell tracking Substances may enter the body via oral ingestion, inhalation, dermal penetration and intravascular injection and subsequently distribute to any organ system. In this review, we aim to identify clinically relevant NPs and critically appraise organ-based toxicological studies which have been carried out on a cellular and pre-clinical level as well as on human volunteers. Furthermore, emphasis will be placed upon the importance of particle size and the route of administration with respect to toxicity as the authors believe that the latter has not received adequate evaluation despite its fundamental importance with respect to the clinical setting.in vivo imaging and diagnostics NPs have a vast potential in the medical arena as drug and gene delivery vehicles, fluorescent labels and contrast agents in vitro and in vivo absorption, distribution and biocompatibility of NPs have been reviewed and are critically appraised in this article. This widespread use of different types of NPs in the biomedical field raises concerns over their increasing access to tissues and organs of the human body and, consequently, the potential toxic effects. Various studies evaluating Due to their small size and physical resemblance to physiological molecules such as proteins, NPs possess the capacity to revolutionise medical imaging, diagnostics, therapeutics as well as carry out functional biological processes. But these features may underlie their toxicity. Also, depending on the mode of administration and sites of deposition, toxicity may vary in severity. Therefore, to maintain clinical relevance, information on toxicity is presented using a system-based approach focusing on experimental lung, dermal, liver and brain targets see .The lung is an attractive target for drug delivery due to the non-invasive nature of inhalation therapy, the lung's large surface area, localization/accumulation of drugs within the pulmonary tissue and avoidance of first-pass metabolism, thus reducing systemic side effects co-glycolic acid) (PLGA) for use as drug carrier devices Polymeric NPs are biocompatible, surface modifiable and are capable of sustained drug release. They show potential for applications in the treatment of various pulmonary conditions such as asthma, chronic obstructive pulmonary disease (COPD), tuberculosis (TB) and lung cancer as well as extra-pulmonary conditions such as diabetes in vivo lung cancer model While such drug loaded nano-configurations demonstrate promising alternatives to current cancer treatment, cytotoxicity needs to be evaluated. PLGA NP successfully improve therapeutic outcome and reduce adverse effects via sustained and targeted drug delivery. Additionally, the use of biological capping materials such as chitosan or BSA further reduce toxicity while their biocompatibility and biodegradative capacity making them an intuitive choice for nanoparticulate surface modification. Romero et al. demonstrated a reduction in cytotoxicity of PLGA NPs stabilized with BSA compared to synthetic coating materials in cultured lung cancer cells Biological capping materials reduce cytotoxicity by mimicking the physiological environment, thus ‘hiding’ from the immune system. However, the possibility of enzymatic degradation due to biophysical resemblance needs further investigation.in vivo inhalation models and can be subdivided into SWCNTs and MWCNTs with the former being considered more cytotoxic. This difference in toxicity has been attributed to the larger surface area of SWCNTs compared to the multi-layered alternative CNTs are frequently used for in vitro and dose-dependent cytotoxicity at a critical concentration of 50 μg/mL as demonstrated on A549 cells Silica NPs are already in wide-spread use in the non-medical field as additives to chemical polishing, cosmetics, varnishes and food stuffs 3. In order to evaluate potentially acute and delayed adverse pulmonary effects of AgNP, Sung et al. have carried out a series of inhalation studies focusing on the acute, subacute (28 days) and subchronic (90 days) toxicity of AgNP in rats 3; 7.5 times higher than the limit), no significant body weight changes or clinical changes were observed. Furthermore, lung function tests revealed no statistical differences between exposed and control groups. Repeated administration of AgNP for 4 weeks showed similar results. In contrast, subchronic inhalation for 13 weeks at a maximum concentration of 515 μg/m3 (5 times the limit) revealed time- and dose-dependent alveolar inflammatory and granulomatous changes as well as decreased lung function The most common route of pulmonary exposure to silver NPs (AgNP) is via the occupational inhalation of airborne particles during manufacturing The skin is the largest organ of the body and functions as the first-line barrier between the external environment and the internal organs of the human body. Consequently, it is exposed to a plethora of non-specific environmental assaults within the air as well as to distinct and potentially toxic substances within creams, sprays or clothing. Topically applied NPs can potentially penetrate the skin and access the systemic circulation and exert adverse effects on a systemic scale.Ag is one of the most consistently studied NPs in terms of toxicity. This arises from the fact that AgNP possess proven anti-microbial effects which are currently used in many products ranging from wound dressings to clothing. However, the specificity of AgNP toxicity towards micro-organisms must be elucidated in order to exclude potentially adverse effects mediated by such particles on other exposed cell types within the body. Ag ingestion and topical application can induce the benign condition known as argyria, a grey–blue discoloration of the skin and liver caused by deposition of Ag particles in the basal laminae of such tissues 2 NPs have several properties which make them an advantageous ingredient for commercial sunscreens and cosmetics. They exhibit UV-light blocking properties and confer better transparency and aesthetics to creams. In vitro studies demonstrated cell type-dependent TiO2 toxicity affecting cellular functions such as cell proliferation, differentiation, mobility and apoptosis in vivo. In order to assess penetrative capacities, dermal infiltration studies have been carried out on human volunteers using different investigative techniques. Lademan et al. investigated the penetrative effect of repeated administration of TiO2-containing sunscreen on the skin of volunteers 2 penetrated into the open part of a hair follicle as opposed to the viable layers of the epidermis or dermis. Furthermore, the titanium amount in any given follicle was less than 1% of the applied total amount of sunscreen. Surface penetration via hair follicles or pores was also suggested by a study conducted by Bennat and Muller-Goymann where skin permeation was greater when sunscreen was applied to relatively hairy skins 2 deposition in hair follicles or skin layers 2 NPs as well as with the number of follicular pores within the skin facilitating particle uptake.TiOin vivo rabbit model in vivo rabbit skin model. These results highlight the superiority of HSEM compared to culture-based systems in terms of evaluating relative dermal toxicities of NPs and emphasize the discrepancies encountered if one tries to extrapolate results gained from cell-based studies into human scenarios.Silica NPs are frequently incorporated in drug additives and cosmetics as well as being used as nano-vehicles for drug delivery Due to facile means of synthesis and the potential for bio-functionalization, gold NPs (AuNP) are being investigated for clinical applications including dermal drug-delivery Being the site for first-pass metabolism, the liver is particularly vulnerable to NP toxicity and has consistently been shown to accumulate administered substances, even long after cessation of exposure. Thorough evaluation of NP-mediated hepatocellular toxicity thus remains of prevailing importance.AuNP play an interesting role in biomedicine and have multiple possible applications in the fields of imaging, drug and gene delivery Well known for their anti-microbial effects, AgNP may gain access into the circulation via various routes. Once within the systemic circulation, first-pass metabolism via the liver is likely, if not probable, thus potentially rendering hepatocytes vulnerable to toxic insults. Many studies have indicated the propensity for AgNP to accumulate within the liver and induce oxidative stress-related toxicity Biomedical applications for silica NPs are widespread and include diagnosis and drug delivery 2) or poly(ethylene glycol) (PEG) coatings. Cytotoxicity was tested on exposure to each type separately by measurement of macrophage cell viability and LDH release. All QDs were shown to induce significant cytotoxicity after 48 h and coating materials as well as liberated Cd ions were suggested to be the causative agents. It is likely that a breakdown of physically labile surface material resulted in ion liberation and subsequent toxicity. Recently, Seifalian and colleagues have demonstrated that the novel synthetic nanomaterial polymeric oligohedral silsesquioxane (POSS), when incorporated onto CdTe cored QDs, shows significantly enhanced cytocompatibility than conventionally used materials, even without ZnS shelling (unpublished data). POSS was shown to be non-toxic by preventing ion leakage from the core. These results underline the importance of the type of coating material used and suggest that the most important factor influencing QD toxicity remains heavy metal ion leakage from the core due to inadequate surface coverage.Semiconductor nanocrystals, or QDs may be used in a variety of biomedical applications. The general structure of QDs comprises an inorganic core–shell and an organic coating to which biomolecules may be conjugated to enable targeting to specific areas within the body. Such close proximity and interaction with the physiological environment necessitates toxicological evaluation of these particles. Cell based studies focusing on QD-induced adverse effects found toxicity most likely arises from the liberation of metal ions released from the heavy metal core The brain, unlike the liver, has very limited regenerative capacity and must therefore be particularly protected from exogenous insults. This is achieved by the protective BBB which separates the cerebrospinal fluid (CSF) surrounding the brain from the systemic blood circulation via tight junctions around the capillaries and is of undoubted benefit in preventing blood-borne pathogens from gaining entry and causing potentially irreversible damage while allowing the diffusion of smaller lipophilic molecules such as oxygen. However, bypassing the BBB may be beneficial and potentially life-saving in the management of acute conditions such as cerebral meningitis as well as chronic illnesses like dementia or Parkinson's disease. Targeted drug delivery allows the use of smaller drug doses and hence reduces systemic adverse effects. Nevertheless, the fate of NPs after translocation into the structures of the central nervous system (CNS) requires toxicological analyses. External insult to the BBB could potentially activate cerebral epithelial cells thus inducing oxidative stress-mediated injury. Additionally, the integrity and biostability of the particle's coating materials require rigorous assessment Recently, Oberdorster et al. demonstrated the uptake of NPs by sensory nerve endings within the pulmonary epithelium and subsequent axonal translocation into the CNS which presents a further route of exposure to potentially harmful NPs in vitro and in vivo cell tracking. Studies suggesting AuNP as imaging agents of the CNS are manifold and could prove to be useful in the diagnoses and therapeutics of CNS pathologies in vivo models AuNP are valuable imaging modalities for In contrast to Au, AgNP have been shown to exhibit neurodegenerative abilities +) resulted in a marked increase in turnover of the neurotransmitters dopamine (DA) and serotonin (5-HT) which are key components in the reward, anxiety and sensorimotor pathways. Ag+-induced neurotransmitter hyperactivity resulted in behavioural changes consistent with a lower anxiety-threshold.This apparent potential for AgNP to accumulate and cause harm requires further investigation with particular attention assigned to the influence of NP size on the distribution and toxicity. Powers et al. demonstrated the potential for AgNP to behave as a developmental neurotoxin in zebrafish embryos and cause long-term changes in synaptic functioning SPIONs and ultra-small SPIO nanoparticles (USPIONs) consist of an iron oxide core and a variable carbohydrate coating which determines cellular uptake and biological half life. The degree of surface coverage has been postulated to be the main parameter in cellular uptake as incomplete surface coverage was shown to promote opsonization and rapid endocytosis whereas fully coated SPION escaped opsonization which, as a result, prolonged plasma half-life in vitro studies have shown the capacity for SPION to induce ROS generation, impair mitochondrial function and cause leakage of LDH – all of which could incite neurotoxicity as well as potentially aggravate pre-existing neuronal damage in vitro toxicity of plain, uncoated magnetic iron oxide NPs (P particles) with either dextran derivatized (DD) or albumin derivatized (AD) NPs. P particles as well as DD particles exhibited similar toxicities, whereas AD particles managed to induce cell proliferation The unique physio-chemical properties shared by all NPs, such as nanometer size and a large surface area to volume ratio, makes SPION particularly valuable for novel therapeutic and diagnostic applications. However, such dimensional reductions may potentially induce cytotoxicity and interfere with the normal components and functions of the cell In a study by Muldoon et al., the distribution, cellular uptake and toxicity of three FDA approved SPION of different sizes and surface coatings were compared to each other and to a laboratory reagent in vivoin vivo toxicity studies calls for the need for considerable attention to the systematic evaluation of QDs intended for brain-specific purposes.QDs may be functionalized to serve both diagnostic and therapeutic purposes. Presently, QDs find invaluable experimental applications in visualizing neural stem and progenitor cells in developing mouse embryos NPs have certain unique characteristics which can be and have been exploited in many biomedical applications. However, these unique features are postulated to be the grounds for NP-induced biotoxicity which arises from the complex interplay between particle characteristics , administered dose and host immunological integrity. Recently, more emphasis has been placed onto understanding the role of the route of particle administration as a potential source for toxicity. Current research focuses on elucidating the mechanistics underlying NP toxicity which are postulated to range from inflammatory cell infiltration and cellular necrosis to ROS-induced apoptosis. Despite the wealth of toxicity studies available, the authors have identified several points of criticism which currently hinder the progression into clinical settings. Firstly, the application of the so-called ‘proof of principle’ approach where cell cultures or experimental animals are exposed to ultra-high NP concentrations to ensure cytotoxicity leads to unrealistic results which cannot be extrapolated into the human scenario since diagnostic and therapeutic interventions usually only require the administration of minimal concentrations. Thus, not only are we faced with scientifically unreliable data, such practices may, quite dangerously cause unnecessary alarm in the public. Additionally, the authors have identified two further limitations of current toxicity studies; firstly, the chronicity of NPs exposure in the case of therapeutic applications needs more thorough long-term evaluation; secondly, different studies apply different particle formulations leading to conflicting and unreliable results. Consequently and for the future, more emphasis should be placed on defining the dose of NPs in relation to the route of administration. As mentioned at the beginning, end-organ accumulation and distribution as well as metabolism and excretion are variable depending on the routes of administration, such that intravenous NP administration may have more implications with regards to systemic adverse effects than dermal application of NPs. However, one must treat and compare toxicity results from different studies with caution, as current toxicity protocols lack uniformity with respect to NP formulations and application protocols. It has become apparent that a unifying protocol for the toxicological profiling of NPs may be required in order to achieve reliable outcomes that have realistic implications for the human usage of NPs. In summary, current difficulties in evaluating NP toxicity originate in the inherent discrepancies found amongst toxicity study protocols such that is has become apparent that a unifying protocol for the toxicological profiling of NPs may be required in order to achieve reliable outcomes that have realistic implications for the human usage of NPs. Not only is there a pressing need for long-term studies, the future of nanotoxicology must also more heavily rely on realistic particle dosages and composition principles as well as differentiating more carefully between various routes of administrations if nanotechnology is to fully unfold its clinical potential.


The enteric nervous system (ENS) is a network of neurons and glia within the wall of the gastrointestinal tract that is able to control many aspects of digestive function independently from the central nervous system. Enteric glial cells share several features with astrocytes and are closely associated with enteric neurons and their processes both within enteric ganglia, and along interconnecting fiber bundles. Similar to other parts of the nervous system, there is communication between enteric neurons and glia; enteric glial cells can detect neuronal activity and have the machinery to intermediate neurotransmission. However, due to the close contact between these two cell types and the particular characteristics of the gut wall, the recording of enteric glial cell activity in live imaging experiments, especially in the context of their interaction with neurons, is not straightforward. Most studies have used calcium imaging approaches to examine enteric glial cell activity but in many cases, it is difficult to distinguish whether observed transients arise from glial cells, or neuronal processes or varicosities in their vicinity. In this technical report, we describe a number of approaches to unravel the complex neuron-glia crosstalk in the ENS, focusing on the challenges and possibilities of live microscopic imaging in both animal models and human tissue samples. Many aspects of gastrointestinal function are controlled without major inputs from the brain. Instead, the enteric nervous system (ENS), a ganglionated neuronal network that resides within the gut wall, autonomously controls gastrointestinal motility, secretion, and blood flow Furness, . The ENSEnteric glial cells are considered to be active partners in ENS function  imaging and present some novel analysis tools that serve this purpose.In this technical report, we describe a number of approaches to disentangle the complex neuron-glia crosstalk in the ENS, in both animal models and human tissue. We discuss the techniques that have been used to examine neuron-glia interactions in the animal and human ENS with an emphasis on live intracellular calcium concentration   were loaded with the Can Figure . Simple n Figure . Thus, aex vivo preparations, is very attractive to investigate with imaging techniques. However, the assumption of 2D structure is only valid when entire cells are considered. Once synaptic contacts and cellular processes are of interest, the analysis faces all the technical problems that are associated with 3D organization and ROIs will easily incorporate scattered light emanating from structures close by. Hence, the intimate relationship between enteric glia, neurons and their processes . To correct for residual movements, we use translation stabilization routines  and the nicotinic agonist dimethylphenylpiperazinium (DMPP). Enteric glial cells, on the other hand, do not respond to these stimuli directly, but show delayed responses that can be modulated by intervening with several components of purinergic signaling -exponential decay, while in glia a secondary close-to-Gaussian shaped response is observed. This typical fingerprint can thus be used also in tissue to identify different cell types , a difference that is more pronounced in ex-vivo tissue preparations in comparison to primary culture. Using the ratiometric dye Fura2, we tested whether this difference was due to higher resting level of intracellular Ca2+. We found the differences  to be only very small and definitely not sufficient to explain the large differences in resting Fluo4 fluorescence. This was further confirmed by using Rhod2, a non-ratiometric Ca2+ indicator with an even higher Kd, which is classically used in astrocyte research . Taken together, this suggests that glial cells take up and/or metabolize the AM ester more easily, which is probably a reflection of a higher metabolism.It is of note that enteric glial cells display higher baseline fluorescence after Fluo4 loading compared to neurons  derivatives, have been placed under the direct control of GFAP or S100β regulatory elements to study astrocytic function can also be used to visualize enteric glial cells in live imaging experiments. Furthermore, the conditional expression of fluorescent reporters by Cre-Lox recombination technology enables identification of enteric glia as illustrated by Joseph et al.  imaging studies , such as GCaMPs, allow imaging of Ca2+ signaling in genetically defined cell populations, thus providing a powerful means to study neuron-glia interactions in the ENS. Recently, a reporter mouse was developed that expresses GCaMP3 (Tian et al., Rosa26 locus (Zariwala et al., Wnt1-Cre transgene (Danielian et al., 2+ imaging in enteric neurons and glia (Figure Another application of transgenic methods lies in the recent development of several optogenetic tools, an opportunity that has yet to be exploited in ENS research. The core instruments of these novel techniques are genetically-encoded optical indicators Knopfel,  and actua Figure . Becausea Figure , 2013. Tin vitro and ex vivo animal models, and given the difficulty to obtain healthy human gut tissues for experimental purposes, our current knowledge about enteric neuron–glia interactions in the human gut is rather poor. The limited information about human enteric glia function originates from in vitro studies using enteric glia isolated from surgical resection specimens. These studies indicated that human enteric glia actively participate in inflammatory responses (Cirillo et al., Because most investigations have been carried out using For these reasons we have recently developed a method to culture human enteric glial cells isolated from routine intestinal biopsies (Boesmans et al., ex vivo preparations. Although the interplay between enteric neurons and glia was not specifically envisaged, Mueller and colleagues were able to record enteric glial cell activity in resection specimens obtained from patients undergoing surgery (Mueller et al., 2+ imaging in submucosal ganglia dissected from duodenal biopsies obtained from healthy volunteers. This technique also allows exploring human enteric glia function (Figure Of course, in order to fully understand neuron-glia interactions in the human gut, also these should ideally be studied in intact n Figure . Again, n Figure . Moreoven Figure . Here agDespite significant progress in understanding enteric glia function, the exact signaling mechanisms and possible “gliotransmitters𠄍 that act in a physiological context such as during the different gastrointestinal motility patterns remain elusive. Nonetheless, several studies indicate that enteric glial cells are active partners in enteric neurotransmission. In particular, the aforementioned live imaging studies have provided invaluable information about how enteric glial cells can detect neuronal activity (Gomes et al., Concept and design, interpretation of data, statistical analysis, drafting and editing of the manuscript were done by Werend Boesmans and Pieter Vanden Berghe; Werend Boesmans, Michiel A. Martens, Nathalie Weltens, and Marlene M. Hao performed experiments, image analysis and contributed to paper writing; Carla Cirillo performed and analyzed experiments on human enteric glia and was involved in paper writing; Jan Tack was responsible for mucosal biopsy collection; Pieter Vanden Berghe and Michiel A. Martens wrote analysis software; and Jan Tack and Pieter Vanden Berghe obtained funding.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.


Land plants secure a constant supply of water and nutrients by developing a deep and densely branched root system. Nutrients are often distributed unevenly in the soil; formation of lateral roots must therefore be responsive to environmental conditions. Over time, lateral root initiation has become an accessible model system for studying the basic regulatory mechanisms that control postembryonic organ development. While there has been much interest in the phenomenology of root branching, it is only recently that the underlying molecular processes have started to be understood .Arabidopsis thaliana has a relatively simple root anatomy when compared to other plants such as grasses. Arabidopsis roots are composed of single concentric layers of epidermal, cortical, endodermal and pericycle cells, which surround a double-stranded vascular cylinder  is being cleared from the tissue by its specific cellular uptake carrier? mentally , suggestmentally . Indeed,in vivo at cellular and subcellular resolutions. The continuous emergence of novel technologies, such as light sheet microscopy and sensors for quantitative ratiometric time-resolved analysis of auxin dynamics (The study by dynamics , may all


To investigate longitudinally the effect of alcohol consumption and related acute alcohol-related dysfunction on employment status.A total of 1143 men aged 25–55 years in regular paid employment and resident in the city of Izhevsk, Russia were interviewed between 2003–06 and then re-interviewed (2008–09) and their employment status ascertained.-beverage alcohols) and alcohol-related dysfunction, measured by a latent variable defined in terms of frequency of alcohol-related dysfunctional behaviours and by one or more episodes of zapoi (a period of continuous drunkenness lasting 2 or more days). The outcome of interest was whether or not men were still in regular paid employment at follow-up. The inter-relationship between these variables was investigated using structural equation modelling.Exposures of interest were baseline alcohol intake . Acute alcohol-related dysfunction was an important mediator of the relationship between non-beverage alcohol use and employment status.Total volume of ethanol consumed had no substantive effect on future employment status; however, taking into account education and other socio-demographic factors, there was strong evidence that loss of regular paid employment at follow-up was influenced by non-beverage alcohol consumption , latent acute alcohol-related dysfunction  and Acute alcohol-related dysfunction is an important factor in determining whether men remain in employment and an important mediator of the effects of alcohol intake. As well as having a negative impact on health , heavy aPathways from alcohol consumption to unemployment may be through chronic effects on health which make it difficult or impossible to remain in work, but also through acute dysfunctional consequences of alcohol such as hangover, which can directly affect an individual's ability to function in the work-place. Whether or not alcohol consumption results in acute dysfunctional behaviour could be particularly important for any effect of alcohol use on employment status. Acute dysfunction may be an important mediator between alcohol intake and loss of employment, because even when consuming the same volume of ethanol individuals can differ in their vulnerability to dysfunction and therefore the impact of their drinking on their work life.et al. found that self-reported drunkenness once a week or more predicted employment status in Scandinavian construction workers, but not forest workers  for every standard deviation unit increase in dysfunction score. Additional adjustment for health problems (model 3) had very little impact on the association between alcohol-related dysfunction and employment, suggesting that the association between alcohol and employment was not mediated importantly through any negative effect on chronic health problems. There remained strong evidence of an association between dysfunction and employment status when men whose proxies reported ‘serious work-related or employment problems’ at IFS-1 were excluded (data not shown). There was no evidence of interaction between fifth of dysfunction score and occupation type (zapoi) with employment are shown in Fig. zapoi and routine acute alcohol-related dysfunction, but there was no evidence that non-beverage alcohol use had a direct effect on employment status once zapoi and routine alcohol-related dysfunction were included in the model. Volume of ethanol had no indirect effect via zapoi, but there was strong statistical evidence of a small indirect effect via routine alcohol-related dysfunction. There was strong statistical evidence that both zapoi and the latent factor of routine alcohol-related dysfunction directly influenced employment status at IFS-2.The relationships between alcohol intake  and acute alcohol-related dysfunction  and routine alcohol-related dysfunction at baseline were associated with higher odds of no longer being in regular paid employment at follow-up. Acute alcohol-related dysfunction was also an important mediator of the effects of alcohol intake  on employment. Once alcohol-related dysfunction was included in the model there was no evidence that either measure of alcohol intake directly increased the probability of no longer being in regular employment. No evidence was found of interaction by occupation type.To our knowledge, this is the first study to consider the effects of acute alcohol-related dysfunction on employment status. We were able to assess this longitudinally and found that high levels of both sporadic  than IFS-2, but the effect size found for average daily consumption of alcohol was also very small  [The findings for total volume of beverage alcohol are in contrast to findings from previous studies ,3, incluzapoi) and routine dysfunction was higher in non-beverage alcohol drinkers compared to those in the highest category of beverage alcohol consumption  which may explain why non-beverage alcohol consumption predicted employment status in the logistic regression model while volume of beverage alcohol did not. This is supported by the finding that the effects of non-beverage alcohol use were completely explained by alcohol-related dysfunction in the structural equation model.All data on alcohol use were obtained from self-or proxy report and therefore subject to measurement error. Using proxy reports of drinking behaviour may be more accurate than self-reported data, as proxies have less reason to under-report socially unacceptable behaviours. However, proxy report is not reliable for certain aspects of alcohol use such as volume of ethanol consumed per occasion, and therefore could not be used for measuring alcohol intake. Self-reported alcohol intake is very likely to be affected by measurement error, because when asked about usual frequency and volume of consumption participants often report their mode rather than mean consumption, ignoring less frequent heavy drinking episodes . Dysfunczapoi. Both were associated strongly with transition out of regular paid employment. The findings of this study are particularly important given the high levels of hazardous drinking found in Russia [In addition to the more general findings with respect to acute alcohol-related dysfunction, this is the first study to investigate the effects of two distinctive features of Russian drinking on employment: non-beverage alcohol consumption and n Russia ,25.There are some limitations in terms of generalizability of these findings. First, the need for a proxy respondent at baseline meant that men who were living alone were excluded. These men are likely to have been different to those included and therefore results are not applicable to all men in Izhvesk. The age distribution of our study population was also skewed towards older men. Furthermore, men who were lost to follow-up were less likely to have been married at baseline; however, with the exception of frequency of hangover, drinking behaviour at baseline was not associated with whether or not they were followed-up, suggesting that these men do not represent a heavier drinking population. Despite the longitudinal study design there may have been some problems with reverse causality, as men may start to drink more hazardously in response to work-place problems even though they are still in employment at that time. Nevertheless, there remained strong evidence of an association between alcohol-related dysfunction and employment status even when men who were perceived as having ‘serious work or employment related problems’ at baseline were excluded. There was no evidence of interaction between occupation type and alcohol use on employment status at IFS-2, although the relatively small number of men who became unemployed between the studies meant that in order to increase power in detecting interaction we used a binary categorization of occupational type, which may not have been sensitive enough at identifying occupational groups at particular risk. Very few men with higher education became unemployed and therefore it was not possible to investigate interaction by education. There may also have been other effect modifiers related to employment, such as income, which were not measured in these studies. Therefore, these results should be interpreted with caution as they may not apply equally to men of all occupational types or educational level. Although we adjusted for chronic health problems, the measure used was relatively simple and so may not have accounted for all the effects of chronic ill health. However, adjusting for health problems made very little difference to the estimated effects of alcohol intake and dysfunction on employment, suggesting that this was not an important pathway. Finally, this study assessed quantitatively the relationship between alcohol use at baseline and employment status at follow-up (3–5 years later), but qualitative work is needed alongside this to understand fully the role of alcohol in employment transitions.In conclusion, non-beverage alcohol use and both sporadic and routine alcohol-related dysfunction were related prospectively to remaining in employment. Acute alcohol-related dysfunction was an important mediator of the relationship between alcohol intake and employment and should be considered in addition to conventional measures of alcohol consumption when investigating the impact of alcohol consumption on work. If further studies support our findings, dysfunctional behaviour could be used for identifying those who would benefit from interventions to reduce alcohol consumption. Reducing dysfunctional behaviour should be considered an important treatment aim for hazardous drinkers alongside reducing overall consumption.


Hepatitis C virus (HCV) infects an estimated 3% of humanity It is now recognized that effective prevention and treatment of HCV infection requires an appreciation of the virus' evolutionary behaviour. HCV evolves very rapidly during infection within a host, resulting in a genetically diverse viral population (often termed a “quasispecies”) whose composition is determined by a combination of evolutionary processes that include mutation, replication rate, natural selection, and random genetic drift. As a consequence, patterns of HCV genetic diversity within infected patients  should reveal information about the evolutionary dynamics of infection and could uncover links between viral evolution and the progression of clinical disease.diversity of sequences sampled at any one time and the divergence among sequences sampled at different times. No clear-cut patterns in these statistics among patients with HCV have emerged. For example, with respect to patient outcomes, high diversity in the HCV envelope region is associated with progression from acute to chronic infection Studies that have followed this logic are too numerous to review here and vary widely in the number and health status of patients investigated, the timespan over which viruses are sampled, and the quality of viral genetic information obtained . In most studies, HCV genomic sequences are obtained from peripheral blood and are summarized using two measures: the model is always required to infer the behaviour of a viral population from sequences sampled from it It is helpful to interpret these results in the context of evolutionary theory. First, it is well-established that much of the evolutionary information inherent in sequence data is lost when they are compressed into summary statistics such as diversity or divergence whole viral population, which is comparatively constant through time  will be of limited utility; we instead recommend that analyses begin with a phylogenetic approach. Second, our model suggests that viruses sampled from peripheral blood give an incomplete picture of HCV infection dynamics. Where possible, future studies should incorporate additional sites, including liver biopsies, explant livers, and other cell types. Third, population structure can maintain high viral diversity within a patient even when serum viraemia and diversity are low, perhaps contributing to the evolution of drug resistance and to treatment failure. The emergence of new anti-HCV drugs therefore makes the understanding of HCV evolutionary dynamics a research priority.