While non-REM (NREM) sleep has been strongly implicated in the reactivation and consolidation of memory traces, the role of rapid-eye movement (REM) sleep remains unclear. A growing body of research on humans and animals provide behavioral evidence for a role of REM sleep in the strengthening and modulation of emotional memories. Theta activity—which describes low frequency oscillations in the local field potential within the hippocampus, amygdala and neocortex—is a prominent feature of both wake and REM sleep in humans and rodents. Theta coherence between the hippocampus and amygdala drives large-scale pontine-geniculo-occipital (PGO) waves, the density of which predicts increases in plasticity-related gene expression. This could potentially facilitate the processing of emotional memory traces within the hippocampus during REM sleep. Further, the timing of hippocampal activity in relation to theta phase is vital in determining subsequent potentiation of neuronal activity. This could allow the emotionally modulated strengthening of novel and gradual weakening of consolidated hippocampal memory traces during REM sleep. Hippocampal theta activity is also correlated with REM sleep levels of achetylcholine - which is thought to reduce hippocampal inputs in the neocortex. The additional low levels of noradrenaline during REM sleep, which facilitate feedback within the neocortex, could allow the integration of novel memory traces previously consolidated during NREM sleep. We therefore propose that REM sleep mediates the prioritized processing of emotional memories within the hippocampus, the integration of previously consolidated memory traces within the neocortex, as well as the disengagement of consolidated neocortical memory traces from the hippocampus. Though the body may seem inert during sleep, the brain most definitely is not. Mammalian sleep cycles through multiple electrophysiologically and neurochemically distinct sleep stages. These stages are generally split into two categories, based on the occurrence of rapid-eye movements (REMs), i.e., REM and non-REM (NREM) sleep. While evidence strongly supports a pivotal role of NREM sleep in memory consolidation, the function of REM sleep remains elusive.In this review, we propose that REM sleep represents a unique brain state that allows the emotionally modulated integration and recombination of neocortical memory traces previously consolidated during NREM sleep. In addition, we suggest that REM sleep is involved in the gradual disengagement of successfully consolidated memory traces from the hippocampus—thus mediating the decontextualization of novel memories, allowing generalization, abstraction, etc. To support this, we initially review behavioral evidence linking REM sleep and emotional memory processing in both rodents and humans. We then discuss how this relationship may be mediated by electrophysiological (in particular theta) activity within the hippocampus, amygdala and neocortex during REM sleep.REM and NREM sleep differ remarkably in several ways: while NREM sleep is characterized by high amplitude, low frequency (0.3–4 Hz) electroencephalographic (EEG) activity reflecting synchronization across large neuronal populations, the low-amplitude, mixed frequency EEG activity observed during REM sleep more closely resembles that of quiet wake . NeuromoIn humans, NREM sleep is divided into light sleep and slow-wave (or deep) sleep , while iThe idea that sleep is important in the consolidation and processing of both recent and remote memories is well established , or else it is used as an incentive to perform a memory task . Similarly to the work in humans, there is a large body of evidence supporting a role of amygdala activity in emotional memory  in rodents  on memory consolidation and encoding in rodents. Post-training REMSD was consistently found to impair avoidance learning , avoidanA recent study using REMSD provides more striking evidence for a role of REM sleep in emotional memory processing: In humans, sleep in general has been shown to benefit fear extinction . The amoPhysiological evidence also supports a role of REM sleep in memory. Areas implicated in memory processing during wake, in particular limbic circuits within the medial temporal lobe, are highly active during REM sleep . At a ceA distinct role for REM sleep in memory is also supported by the striking similarities and contrasts between neuromodulator states in REM sleep and wakefulness. The wake-like levels of acetylcholine in the limbic system suppress excitatory feedback potentials within the hippocampus and in the cortex . During Based on these neuromodulator states, it has been proposed that during REM sleep, memories within the neocortex—free from interference from the hippocampus—recombine and potentially integrate into existing memory networks between periods of NREM sleep-dependent memory consolidation . It is iRapid-eye movement sleep possesses a unique physiology which appears particularly amenable to the processing of emotional memories . FunctioIn addition to a general role of REM sleep in emotional memory processing, a separate line of research has emerged concentrating on a more specific link between REM sleep and the modulation of emotional responses. Whether this role is part of the same mechanism, or relies on distinct processes is unclear and needs to be investigated more systematically. This section reviews evidence specifically linking emotional response modulation and REM sleep.Though many psychological disorders are comorbid with sleep disorders, it is particularly of note that mood disorders tend to be associated with unusual REM sleep: in depression, REM sleep is pathologically increased ; a hallmSome evidence suggests a sleep-dependent decrease in both subjective emotional arousal and autonomic response to negative stimuli compared to an equally long period of wake in humans . In lineIn contrast, Theta activity describes synchronized oscillating local field potentials of neuronal populations within the range of 4–10 Hz initially observed in rodents . It is cHippocampal theta activity appears to originate from nuclei within the brain stem which project via the hypothalamus to the septal complex comprising of the medial septum and a subregion of the Broca area . The sepGiven the strong link between acetylcholine and theta activity, the role of acetylcholine in memory processes within the hippocampus is highly indicative of the function of theta activity. It appears that high levels of acetylcholine enhance memory encoding during wakefulness, yet do not affect retrieval in a range of learning tasks in both rats and humans  activity reliably predicts encoding, working memory and navigation  locations appear to be preferentially activated during wake SWRs . SequencCritically, coordinated sequential activity of hippocampal place cells also occurs during REM sleep  and thusThough there are no human studies investigating sequential reactivation of place cells during REM sleep, the phenomenon of cued-memory reactivation during sleep (both during SWS and REM sleep) supports the idea that memories are reactivated during both sleep stages. Thus, sound cues associated with Morse code presented during REM sleep resulted in improved performance following sleep, though only if cueing occurred during phasic—not tonic (the distinction is elaborated further on)—REM sleep . In a seSequential reactivations of cell assemblies representing experienced space are a clear candidate for stored memories. The fact that these occur during REM sleep – in coordination with ongoing theta activity – and that these reactivations are biased by exposure to memory cues during REM sleep – provides a strong case for REM sleep having a role in the processing of these memories.Rapid-eye movement sleep theta activity appears to share a common generation mechanism with other prominent REM sleep features. Thus, the generation of REM sleep theta, REMs, REM sleep atonia and pontine-geniculo-occipital (PGO) waves all depend on the ventral part of the oral pontine reticular nucleus vRPO; . PGO wavBesides having an overlapping generation mechanism, PGO waves and REMs tend to be phase-locked to theta waves , 2007. IThough readily detectible in rats and cats, scalp EEG does not suffice in revealing PGO activity in humans. However, based on fMRI evidence, the pontine tegmentum, thalamus, primary visual cortex, putamen, and limbic areas activate in synchrony with the occurrence of REMs, which strongly suggests a similar activity in humans . Due to Taken together, it appears that in rodents, theta activity during REM sleep—and specifically the theta synchronization between the amygdala and hippocampus—drive large scale synchronized activation in the form of PGO waves, which in turn enhance synaptic plasticity within the hippocampus and amygdala. This, in combination with the evidence linking PGO wave density with emotional memory processing, strongly supports a specific role of REM sleep theta activity in the selective processing of emotional memories. It is possible that a similar relationship between theta coherence within the limbic system and PGO waves exists in humans, however evidence for this is lacking.In addition to evidence linking the occurrence of REM sleep with emotional memory consolidation, there is some evidence directly linking REM sleep theta with emotional memory processing. Following training in an avoidance task, compared to a preceding control night, rats displayed an increased amount of REM sleep periods 17–20 h following training, as well as increased theta power during these REM sleep periods . InteresA more compelling link between REM sleep theta and fear memory consolidation was shown in a study on rats by Due to the lack of an adequate non-emotional control task in rodent studies, we cannot exclude the possibility that non-emotional memory consolidation is equally dependent on REM sleep theta activity. Human studies allow a clear separation of emotional and non-emotional memories—though human intracranial studies investigating the role of hippocampal theta activity during REM sleep on emotional memory processing are limited. However, given the coherence of theta activity between the hippocampus, amygdala and neocortex in rats both during wake , 2013 anRapid-eye movement sleep itself is not a homogenous state. It is often described as either phasic or tonic—with tonic REM sleep taking up approximately 95% of REM sleep in rats . During The existence of phasic REM sleep in humans is assumed based on a similar occurrence of phasic bursts of REMs accompanied by muscle twitches and cardiorespiratory irregularities . InteresIn rats, theta and gamma synchrony within the hippocampus are increased during phasic vs. tonic REM sleep . During In contrast, phasic REM sees an increase in theta and gamma coherence in DG together with an increase in gamma coherence in CA3/CA1, thus facilitating greater information exchange between the hippocampus and neocortex during REM sleep , in whicBased on the evidence reviewed above, it appears that theta activity during REM sleep drives the emotionally modulated processing of novel memory traces within the hippocampus, possibly in preparation for consolidation during NREM sleep. This is supported by the observation that theta activity—in particular theta coherence between the hippocampus and amygdala—drives high-amplitude PGO waves, the density of which predicts the expression of plasticity-related genes within the hippocampus and amygdala. As amygdala-hippocampal coherence during wake predicts future recall of emotional events, it is possible that this coherence in REM sleep reflects the selective processing of emotional memory traces.The timing of spike activity within the hippocampus with respect to theta phase is vital in determining whether long term potentiation (LTP) or depression (LTD) occurs. While hippocampal activity associated with a novel experience occurs at a similar phase during REM sleep compared to wake—thus promoting LTP—activity associated with a familiar experience occurs at a phase promoting LTD. In addition, evidence suggests theta activity during REM sleep increases unit synchrony and decreases firing rate variability. Taken together, this implies REM sleep in processes of separation and offloading of traces previously consolidated during NREM sleep, thus freeing capacity to encode and process novel memories.In addition to emotionally modulated memory processing within the hippocampus, theta activity during REM sleep could also mediate the integration of novel memory traces within the neocortex. Acetylcholine—which reduces hippocampal interference in the neocortex—is raised during REM sleep and directly correlated with theta activity within the hippocampus. In addition to the rise in acetylcholine, noradrenaline levels are extremely low during REM sleep. As noradrenaline suppresses recurrent activation within the neocortex, this drop in noradrenaline during REM sleep could allow encoding-like processes of a more elaborate nature than wake, i.e., allowing recombination and integration of novel memory traces into pre-existing memory networks. This neocortical integration may give rise to the vivid dreaming experienced during REM sleep.Those who refute a role of REM sleep in memory  refer toThis emotionally modulated processing of memories within the hippocampus may prioritize the consolidation of emotional memories during NREM sleep. The following integration of these novel memories could allow emotional experiences to guide future behavior in similar situations. This is backed by behavioral evidence, which associates REM sleep with the selective strengthening of emotional memories and the modulation of emotional responses. The REM sleep dependent bidirectional changes in arousal reported in the literature could also be explained by integratory processes during REM sleep: if an emotional response associated with a given experience is of no direct use to the memory itself, this may be lost in this process. In contrast, an emotional response which is integral to the memory , the emotional response may be strengthened along with the memory. Thus the main function of REM sleep may be the emotionally modulated optimization of behavior.This proposed function of REM sleep would explain why the suppression of REM sleep does not cause noticeable cognitive deficits in patients using REM-suppressing antidepressants. If not faced with emotionally trying experiences or significant changes to the environment—which would require rapid adaptation—the patients can rely on “outdated” behavioral responses to similar situations. Therefore, we hypothesize that REM sleep suppression during early childhood development would result in more striking behavioral deficits. In addition we hypothesize that parametrically varying emotional weight in the form of rewards/punishments in behavioral tasks should change the degree of theta synchronization between limbic structures, distribution of theta-phase firing of individual cells and also manifest itself as a differential number of sequence reactivations during REM sleep. Despite behavioral evidence predominantly pointing toward a role of REM sleep in predominantly emotional memory, it remains unclear whether non-emotional memories are processed independently of REM sleep—it is possible that they are also processed during REM sleep though with a lower priority. Further, it would be interesting to consider how the previously alluded to evidence linking REM sleep and procedural memory ties in with the proposed role of REM in emotional memory processing.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.


Just as there is a range of paediatric imaging techniques available during life, a similar repertoire is available as part of the foetal and perinatal postmortem examination. In this article, we review the literature regarding the diagnostic utility of postmortem radiography, US, CT and MRI in this clinical setting. There is limited direct evidence on the diagnostic utility of any of these techniques, apart from postmortem MRI, which when combined with other noninvasive investigations, has been shown to be highly sensitive and specific for many foetal postmortem diagnoses. The main disadvantages of postmortem MRI include the longer duration of imaging, the need for appropriate training in the interpretation of normal postmortem changes, and possible non-diagnostic imaging examinations in early gestation foetuses. As less-invasive autopsy becomes increasingly available, the true utility of these techniques will evolve, and clinical guidelines for maximal diagnostic yield can be developed. In many developed countries, foetal and paediatric autopsy acceptance rates remain at historically low levels, with overall acceptance rates of about 12% in the United States and 15% in the United Kingdom . A combiThe ultimate role of autopsy is to determine the underlying cause and mechanisms of death, and in cases of stillbirth or foetal demise, to provide a unifying diagnosis that may have significance for the management of future pregnancies or implications for other family members. Obstetric US screening programs for early antenatal diagnosis have increased the frequency of terminations in early pregnancy during the last decades, and improvements in antenatal US imaging mean that there is now generally good agreement between prenatal US and autopsy findings, with 90% concordance in specialist centres . This suPostmortem imaging adds value in three main ways. First, it provides a direct diagnosis, such as the radiographic phenotype in skeletal dysplasia. Second, it provides additional value to guide the autopsy, such as image-guided biopsy or identification of an unsuspected lesion. Third, in cases that parents do not agree to an invasive postmortem examination, postmortem imaging can be offered as at least some form of acceptable investigation after death, with the limitations of such an approach clearly defined. In discussing the perinatal postmortem imaging options in this article, we have drawn on the paediatric and adult literature where no perinatal evidence is available to discuss their relevance to perinatal postmortem imaging.Several types of imaging can be employed in the investigation of foetal demise or termination of pregnancy including conventional postmortem radiography, US, CT and MRI. As might be expected, there are large differences in how these techniques are employed between institutions, across European countries and worldwide. A recent survey of European Society of Paediatric Radiology members reported that not only was there a lack of consistent approach regarding which sub-population of postmortem cases to image  Fig. , there wSkeletal radiography provides an overview of bone structure and development, and bone biometry, and it allows identification of focal or generalised bone abnormalities; a specific diagnostic approach to the postmortem radiography is provided elsewhere in this mini-symposium . RoutineBefore the widespread introduction of routine antenatal sonography, foetal gestational age estimation and accurate skeletal imaging was difficult. Postmortem radiography therefore developed as a standard tool for these indices from assessments of ossification centres and long-bone length measurements . Postmorroutine postmortem radiographic examinations is low with regard to providing significant additional information in apparently anatomically normal foetuses and stillbirths. A particular challenge for radiologists in this setting is differentiating skeletal dysplasias from normal skeletal development at increasingly early gestation (less than 20 weeks), where knowledge of normal foetal appearances is crucial [Modern antenatal sonography now provides highly accurate determination of gestational age and reliable detection of a wide variety of foetal abnormalities, including skeletal dysplasias. As antenatal diagnostic sonography continues to improve , combine crucial . Other iSkeletal abnormalities or dysplasias are extremely rare in infants who do not have external stigmata. This has been shown by studies in the 1970s and 1980s in which routine postmortem radiography was diagnostic in about 15% and of no value in >50% –13. A moadditional value of postmortem radiography over an autopsy, or vice versa, because in all cases both investigations (autopsy and postmortem radiography) were performed and a composite final report issued. In studies where the autopsy rate was high, the additional yield of routine radiographs is likely to be minimal, with the main exception being specific clinical settings identified prior to autopsy, such as suspected skeletal dysplasias or complex genetic syndromes, in which postmortem radiography often provides a specific diagnosis. Two more recent studies of the objective usefulness of postmortem radiography have concluded that it is useful in only a minority of cases [It is often difficult to extract from published data the objective of cases , 16. TheInitial cost estimates suggest that a directed approach could also represent a cost saving: while postmortem radiography is fairly cheap as a diagnostic tool, the workload volume in routine imaging may not justify its overall service cost. An evidence-based selective postmortem radiography imaging protocol could yield significant cost savings, without a reduction in diagnostic yield. We conclude that routine postmortem radiography in all cases has a low diagnostic yield in clinical practice and is therefore neither diagnostically useful nor cost-effective, and therefore we suggest that postmortem radiography remain indicated for specific cases in which an appropriate abnormality is suspected from the antenatal findings, or in cases in which a specialist paediatric pathologist determines that there is a clinical indication to exclude or include specific features from the clinical history or initial external examination. Limiting skeletal radiography to selected cases would significantly reduce the radiology case workload. This would support the use of additional cross-sectional examinations such as postmortem CT in selected cases, yet still represent an overall cost-saving.Foetal sonography, although used widely in both antenatal and neonatal contexts, has, somewhat surprisingly, not been used or researched extensively in the postmortem setting. Postmortem US has been used in adults to demonstrate predominantly abdominal findings such as ascites, gallbladder stones, kidney and liver disease, and the presence of an intrauterine device. However normal postmortem changes in the body can cause difficulties at US, including hyperechoic abdominal and thoracic walls, gas distension of the digestive tract, and putrefaction of the subcutaneous tissues in infants . PostmorA skilled sonographer can obtain excellent imaging of the cranial contents, spine, limbs, chest and abdomen, and at our institution sonography has been found to be particularly useful in two settings: when the foetus is too small to be reliably imaged with routine postmortem MRI , and to address a specific issue raised at postmortem radiography or MRI. Examples include suspected ray abnormalities in the hand that cannot be resolved using plain radiographs, or abnormalities such as cystic hygroma in a small foetus Fig. .Fig. 2CyCompared to antenatal US examination, postmortem US has several significant potential advantages for achieving optimal imaging: the body can be positioned in an optimal orientation with no movement, and the high-frequency probes can be used in close proximity to the target tissue. However, both pliability and postmortem changes within tissues can pose a problem in accurate localisation of lesions in postmortem US in foetuses, and rigor mortis can present further adaptation problems. Postmortem US also requires the operator to be in prolonged direct contact with the body, which may inhibit the widespread uptake of this technique by practitioners. With relatively inexpensive and easy-to-acquire hardware, postmortem US has the potential to contribute greatly to postmortem imaging in this setting but further research is required to determine optimal methods and approaches.Although percutaneous organ biopsy using surface landmarks has been attempted , 19, US-The main advantages of postmortem CT over postmortem MRI are speed, availability and the increased bone detail that is achieved in CT. Although postmortem CT angiography is now the mainstay of adult postmortem imaging , 22 to dIn the context of foetal and childhood pathology, unenhanced postmortem CT gives excellent bony detail and provides good diagnostic-quality images in suspected skeletal dysplasias, with possible additional benefits from 3-D reconstructions Fig. , and in One study has suggested that postmortem CT can be useful in selected cases; in a small cohort of 47 deaths in infancy, good concordance between postmortem CT and autopsy findings was reported  although deaths remained unexplained in 29 of their 47 cases (62%) . Other pThe main disadvantages of postmortem CT compared to postmortem MR in foetuses and children include (1) markedly inferior soft-tissue contrast from reduced abdominal and subcutaneous fat, and (2) the lack of intravenous contrast agent, which makes assessment of the thoracic and abdominal cavity organs almost impossible Fig. . AlthougMicro-CT is a potential alternative diagnostic modality for imaging small bodies, using CT but at improved resolution down to micrometers rather than millimetres. Micro-CT is becoming more widely used in postmortem forensic work  and has Foetal postmortem MRI shows the greatest promise as an adjunct and possible alternative to conventional perinatal and paediatric autopsy. Many advances have been made since several early postmortem MR studies were published in the late 1990s suggesting reasonable sensitivity and specificity for brain and spinal cord abnormalities –34. MoreThe largest recent prospective trial of postmortem MRI versus standard traditional autopsy in foetuses, stillbirths and children showed that postmortem MRI had the highest diagnostic accuracy in the foetal age group (Magnetic Resonance Imaging in Autopsy: MARIAS study) . This stPostmortem MRI alone did not detect about one-quarter of all major foetal diagnoses, cases in which additional genetic or placental examination was required, or detect cases of sepsis . This coHowever, foetal postmortem MR cannot be approached and introduced without appropriate safeguards. An understanding of the imaging correlates of normal postmortem changes such as fluid redistribution  remains elusive and the subject of ongoing research. Similarly, reliably discriminating pathological processes from normal postmortem changes on imaging alone is particularly difficult in the lungs and the abdomen. Foetal imaging is further complicated by the potential superimposed effects of maceration following in utero death, and subsequent postmortem interval-related changes, which vary with gestational age. A comprehensive review of the normal findings on postmortem MR is also included elsewhere in this issue .With the increasing introduction of routine first-trimester antenatal US screening, foetuses may be submitted for autopsy examination at early gestational ages. These represent a challenge, both for traditional autopsy examination and imaging approaches, and in certain instances postmortem imaging is useful where the autopsy is non-diagnostic. However, in the MARIAS study  postmortIn some circumstances postmortem MR might be superior to traditional autopsy and might provide diagnostic information over and above that achieved at standard open autopsy. In the MARIAS study , postmorPostmortem imaging may provide specific information to allow targeted tissue sampling or direct endoscopic assisted sampling in cases in whom parents do not agree to traditional autopsy. In cases where the parents do not consent to any form of invasive investigation, postmortem MR can, of course, provide more information than no examination at all. The varied clinical circumstances in which postmortem MR may be requested must be appreciated when attempting to determine its value, because this differs according to the specific clinical question to be addressed and the availability or unavailability of other forms of postmortem investigation in any individual case.Just as MRI now offers an additional step in the antenatal diagnostic imaging pathway, perinatal postmortem imaging has two significant indications: (1) to confirm that antenatal findings were correct , and (2) to confirm the absence of any other abnormality missed on antenatal scanning. For instance, a foetus terminated for apparently isolated ventriculomegaly on antenatal scanning in whom no other abnormality is identified on postmortem imaging or autopsy represents a different process compared to one in whom additional findings are present to suggest a specific underlying syndromic diagnosis. In the former, where the findings are confirmed, the parents may be reassured regarding the antenatal findings and this provides an important governance tool. It is difficult to quantify the effect that a normal finding may have on grieving parents during a future pregnancy, but this role should not be underestimated.By virtue of the population being assessed, and the evidence from the large cohort trials already performed, the majority of imaging and autopsy investigations by whatever means are likely to be normal. Can we quantify how useful a normal postmortem imaging study could be? The majority of postmortem radiographic and postmortem MR studies have a high negative predictive value, which suggests that when imaging is normal few pathologies are missed. This can be highly reassuring to both the pathologist and parents, particularly when further invasive autopsy is declined. However, there are two other implications to a normal study in which no significant abnormality was identified.First, a failure to find an abnormality in the foetus following intrauterine death, stillbirth or miscarriage potentially implicates a greater likelihood of a maternal–placental unit pathology as the underlying cause. For example, an unexpected late stillbirth with normal antenatal history and normal antenatal US examinations, who then has a normal postmortem MR, could help to direct the pathologist to assess the placenta and the obstetrician to investigate other maternal abnormalities. The approach to the autopsy may also be significantly modified if a specific abnormality is suspected on postmortem imaging, such as a brain or cardiac abnormality. This can therefore be useful to direct resource allocation.Second, a normal postmortem imaging investigation represents a cohort of foetuses in whom no specific anatomical diagnosis is present, in whom future studies investigating other specific ancillary investigations can be targeted, for example cardiac arrhythmias or genetic studies , 43, repThere is one specific indication for which postmortem MRI, with or without other imaging modalities, represents an essential and integral part of the process, namely the minimally invasive autopsy. A minimally invasive autopsy is based on postmortem imaging followed by targeted tissue examination using a variety of techniques including endoscopic guidance. This process allows similar clinically relevant diagnostic information to be obtained compared to standard traditional autopsy for selected indications in whom the parents do not consent to a standard traditional autopsy approach. Because neither selected tissue biopsy nor endoscopic examination and sampling allow adequate anatomical information to be obtained, it is essential that postmortem MR is carried out beforehand. Finding either a normal or specifically abnormal anatomical examination is extremely useful to direct further investigation, to allow the maximum diagnostic yield with the least invasive approach. It should also be recognised that in some cases parents will not agree to any form of autopsy tissue sampling, but in these cases postmortem imaging combined with ancillary investigations, such as placental histological examination, can still provide useful clinical information. Initial studies indicate that postmortem imaging using MRI is a highly acceptable approach even to those who do not agree to a standard autopsy and, furthermore, the minimally invasive autopsy appears more acceptable than the standard open procedure; it is therefore highly likely that these approaches will become more widespread –47.The integrated foetal postmortem imaging service will include a multidisciplinary group of obstetricians, foetal medicine specialists, paediatric radiologists, perinatal pathologists, and geneticists, to name but a few, who might be involved in the ongoing care and counselling of bereaved parents. By combining these clinical skill sets and recognising the contribution of each imaging and other modality to the final diagnosis, the optimal approach to the investigation after death can be determined for each case. We consider that following foetal death, a stepwise approach is the most logical and efficient, using, where available, a full clinical history and examination that take into account gestation, presentation and likely diagnosis Fig. . SkeletaThe use of postmortem imaging in foetal and paediatric diagnosis will continue to evolve and improve. We suggest that a targeted, rather than routine, radiographic approach and more routine postmortem MRI approach will give the highest diagnostic yield, particularly in cases in which a formal autopsy is declined. By understanding the advantages and limitations of each imaging technique, we can employ each technique to its maximal advantage and counsel parents appropriately as to what a normal scan means in the appropriate context. As minimally invasive autopsy becomes increasingly available, the true utility of these techniques will become clear and help to develop clinical guidelines for maximal diagnostic yield and parental acceptability of the investigation after death.


Exposure to stress or traumatic events can lead to the development of depression and anxiety disorders. In addition to the debilitating consequences on mental health, patients with psychiatric disorders also suffer from autonomic imbalance, making them susceptible to a variety of medical disorders. Emerging evidence utilizing spectral analysis of heart rate variability (HRV), a reliable non-invasive measure of cardiovascular autonomic regulation, indicates that patients with depression and various anxiety disorders  are characterized by decreased HRV. Social stressors in rodents are ethologically relevant experimental stressors that recapitulate many of the dysfunctional behavioral and physiological changes that occur in psychological disorders. In this review, evidence from clinical studies and preclinical stress models identify putative biomarkers capable of precipitating the comorbidity between disorders of the mind and autonomic dysfunction. Specifically, the role of corticotropin releasing factor, neuropeptide Y and inflammation are investigated. The impetus for this review is to highlight stress-related biomarkers that may prove critical in the development of autonomic imbalance in stress -related psychiatric disorders. Stress that stems from one’s social environment is the most common form of stress encountered by people and is perceived as more intense than other types of stressors . SociallOne physiological change observed in patients with psychiatric disorders thought to contribute to increased CVD risk is dysfunction within the autonomic nervous system as evidenced by changes in heart rate variability (HRV). HRV reveals beat-to-beat changes in heart rate measured by the electrocardiogram. Variability within the heart rate is regulated by the sympathetic nerves, which accelerate heart rate and the parasympathetic (vagus) nerve, which slows it. Therefore, HRV provides a non-invasive measure of the balance between the sympathetic and parasympathetic nervous system. Healthy cardiac activity is characterized by a high degree of parasympathetic input and thus increased variability, protecting the heart against adverse cardiac events such as heart failure and myocardial infarction . AlternaStress is a common risk factor for both CVD and psychiatric disorders. Therefore, identifying stress-sensitive systems that mediate cardiovascular and behavioral/emotional responses could shed light on the shared pathophysiology that links these comorbid disorders. Two reliable ethologically relevant animal model of social stress have proven particularly useful for studying this link. One is the resident-intruder paradigm of social defeat . This moAcute stress responses are adaptive in helping the individual cope with the stressor, however, if unabated in the face of chronic stress this can lead to pathological changes. Therefore, social defeat in rodents and social isolation in prairie voles have behavioral and physiological consequences that are relevant to human pathologies. Cardiovascular telemetry allows for detailed 24 h cardiac monitoring in unrestrained animals and has been critical in establishing cardiovascular repercussions to stress, allowing for direct comparison to humans. From a physiological perspective, social defeat renders rats with disruption of the circadian rhythm for heart rate and core temperature  and malaThere are several stress-sensitive biological molecules that have both pro-depressive or anxiogenic effects and impact the autonomic nervous system. As such, these molecules are classified as putative biomarkers driving the comorbidity between psychiatric disorders and autonomic dysfunction. One potential biomarker is corticotropin-releasing factor (CRF). This neuropeptide is considered the “hallmark” of the stress response . In stre1 receptor distribution and quantity in brain and pituitary  is yet another neuroendocrine peptide that has demonstrated central control over both behavioral and cardiovascular responses to stress. NPY is widely distributed in the brain and expressed in regions implicated in psychiatric disorders. NPY is often co-expressed with the neuropeptide CRF and as such, it is poised to impact central regulation of stress-related behavior, neuroendocrine and cardiovascular responses. For example, the LC , the amyeceptors . Becauseeceptors  this NPYeceptors . Furthereceptors . Elevateeceptors . Moreoveeceptors . Importaeceptors . Combat-eceptors  and NPY eceptors . Along teceptors . Therefo1 receptor revealed that NPY serves to potentiate NE-evoked vasoconstriction (In addition to its cognitive effects, NPY has prominent cardiovascular impact. Like NPY’s effect on anxiety, its hemodynamic effects are also in contrast to CRF; central administration of NPY lowers blood pressure and heart rate at rest and in response to social defeat in rats . The martriction . As suchtriction . Nonethetriction  and in atriction , 2014. SProinflammatory cytokines are another such mediator that can be persistently up regulated as a result of stress in vulnerable individuals and has roots in the pathogenesis of both psychiatric disorders and CVD . In factProinflammatory cytokines also possess potent cardiovascular effects. For example, both central and peripheral infusion of the proinflammatory cytokine interleukin-1β induces a pressor response and tachycardia in rats . In humaThere is a large body of evidence to suggest that psychosocial stress plays a prominent role in the etiology and progression of certain CVDs and psychiatric disorders. As such, there is a strong association between psychiatric disorders and increased cardiac morbidity and mortality. This review highlights proinflammatory cytokines, CRF, and NPY systems amongst those capable of generating both depressive or anxiety-like behaviors and reductions in HRV, thereby increasing CVD risk. Targeting these systems in preclinical social stress models and in psychiatric disorder patients with evidence of decreased HRV may prove beneficial and warrant further evaluation.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.


Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries—physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, we begin by summarizing the technical challenges of constructing metagenomic libraries and emphasize their value as resources. We then discuss libraries constructed using the popular cloning vector, pCC1FOS, and highlight the strengths and shortcomings of this system, alongside possible strategies to maximize existing pCC1FOS-based libraries by screening in diverse hosts. Finally, we discuss the known bias of libraries constructed from human gut and marine water samples, present results that suggest bias may also occur for soil libraries, and consider factors that bias metagenomic libraries in general. We anticipate that discussion of current resources and limitations will advance tools and technologies for functional metagenomics research. Functional metagenomics involves isolating DNA from microbial communities to study the functions of encoded proteins. It involves cloning DNA fragments, expressing genes in a surrogate host, and screening for enzymatic activities. Using this function-based approach allows for discovery of novel enzymes whose functions would not be predicted based on DNA sequence alone. Information from function-based analyses can then be used to annotate genomes and metagenomes derived solely from sequence-based analyses. Thus, functional metagenomics complements sequence-based metagenomics, analogous to how molecular genetics of model organisms has provided knowledge of gene function that is widely applicable in genomics.cos-based vector, allowing packaging by lambda phage for subsequent transduction of Escherichia coli , it is not easy to determine the cause.Other challenges include the sensitivity of packaging extracts and preparation of purified digested and dephosphorylated vector DNA for ligation. Although excellent commercial products are available for both, in-house vector preparation may still be required when specific expression hosts are to be used in functional screening outside the host range of available commercial vectors . In recent years, as functional metagenomics has gained traction, metagenomic libraries from remarkably diverse environments have been constructed using pCC1FOS (Table cat) marker that is superior to the common ampicillin resistance (bla) marker, obviating the occurrence of satellite colonies associated with beta-lactamase secretion that can be problematic for the dense platings often required for library construction. In addition to an F plasmid oriV for single-copy maintenance, pCC1FOS also carries an oriV from the RK2 plasmid. The RK2 oriV is broad-host-range, conferring replication ability in diverse members of the Proteobacteria  carries trfA under the control of an inducible promoter that is advertised to increase copy number from 1 copy per cell to 10–200 copies. The strain likely possesses a trfA copy-up mutant allele under control of araC-PBAD, which is induced by L-arabinose , as have others . The importance of devising systems that allow for functional screening in diverse expression hosts has been reviewed by others  for integrase-mediated site-specific recombination of cloned insert DNA into the genomes of landing pad strains, including S. meliloti and Agrobacterium tumefaciens .Beyond the practical questions of how to optimize vectors for library construction and how to maximize valuable existing libraries, there is a technical question that we find particularly interesting: how much of the sequence diversity present in original DNA extracts is captured in constructed libraries, and what affects this? Though not so much a concern for functional screens, it is interesting to consider the factors that influence library representativeness; elucidating these factors may lead to development of better strategies for accessing the full potential of environmental metagenomes. We previously used shotgun sequencing to examine bias in a human fecal library . The high level of host contamination could be due to preferential amplification of template during PCR based on differences in DNA conformation: though present in very small quantities, linear DNA may be more efficiently amplified over supercoiled or closed circular plasmid DNA  from the Natural Sciences and Engineering Research Council of Canada, by Genome Canada for the project “Microbial Genomics for Biofuels and Co-Products from Biorefining Processes,” and by a University of Waterloo CIHR Research Incentive Fund. KL was supported by a CGS-D scholarship from the Canadian Institutes of Health Research.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.


With the significant advancement of highthroughput technologies and diagnostic techniques throughout the past decades, molecular underpinnings of many disorders have been identified. However, translation of patient-specific molecular mechanisms into tailored clinical applications remains a challenging task, which requires integration of multi-dimensional molecular and clinical data into patient-centric models. This task becomes even more challenging when dealing with complex diseases such as neurodegenerative disorders. Integrative disease modeling is an emerging knowledge-based paradigm in translational research that exploits the power of computational methods to collect, store, integrate, model and interpret accumulated disease information across different biological scales from molecules to phenotypes. We argue that integrative disease modeling will be an indispensable part of any P4 medicine research and development in the near future and that it supports the shift from descriptive to causal mechanistic diagnosis and treatment of complex diseases. For each ‘P’ in P4 medicine, we demonstrate how integrative disease modeling can contribute to addressing the real-world issues in development of new predictive, preventive, personalized and participatory measures. With the increasing recognition that application of integrative systems modeling is key to all activities in P4 medicine, we envision that translational bioinformatics in general and integrative modeling in particular will continue to open up new avenues of scientific research for current challenges in P4 medicine as outlined in the concluding part of this paper.


Sc). Prion diseases affect humans and animals, the latter including the food-producing ruminant species cattle, sheep, goats and deer. Importantly, it has been shown that the disease agent of bovine spongiform encephalopathy (BSE) is zoonotic, causing variant Creutzfeldt Jakob disease (vCJD) in humans. Current diagnostic tests can distinguish different prion types and in food-producing animals these focus on the differentiation of BSE from the non-zoonotic agents. Whilst BSE cases are now rare, atypical forms of both scrapie and BSE have been reported, as well as two types of chronic wasting disease (CWD) in cervids. Typing of animal prion isolates remains an important aspect of prion diagnosis and is now becoming more focused on identifying the range of prion types that are present in food-producing animals and also developing tests that can screen for emerging, novel prion diseases. Here, we review prion typing methodologies in light of current and emerging prion types in food-producing animals.Prions are an enigma amongst infectious disease agents as they lack a genome yet confer specific pathologies thought to be dictated mainly, if not solely, by the conformation of the disease form of the prion protein (PrP Sc conformer of a benign host encoded membrane glycoprotein (PrPC) in the absence of any coding nucleic acid [C to PrPSc result in increased insolubility in detergents [C has a wide tissue distribution and is present as di-, mono- and un-glycosylated forms. It is thought that PrPSc is responsible for the conversion of additional molecules of PrPC into PrPSc and that this is the central molecular event that occurs during TSE disease. This conversion event is largely restricted to those cells of the lymphoreticular tissues (LRS) and central nervous system (CNS), and the end product has a propensity to aggregate and form amyloid. It is unclear as to the effect of the loss of function of PrPC that occurs after conversion to PrPSc as the physiological function of PrPC remains a controversial matter. However, it is thought that either this PrPSc end product, or a conformer of this protein formed along the folding pathway is toxic to cells of the CNS and is responsible for the characteristic brain pathology including the spongiosis, astrocytosis and neuronal loss that are commonly associated with these diseases.The transmissible spongiform encephalopathies (TSEs), commonly referred to as prion diseases, are a group of infectious, fatal neurodegenerative disorders that affect a number of mammalian species, including man. The prion hypothesis proposes that the infectious agent is largely comprised of the PrPeic acid . The contergents  and prottergents . PrPC haPrion diseases occur in humans, for example Creutzfeldt-Jakob disease (CJD) and kuru. They also occur in food-producing animals , namely TSEs often present as a spectrum of different clinical signs in the infected animal. In scrapie-affected sheep and goats, infection may show as pruritus, weight loss, wool pulling, the swaying of hips and hind limbs, and animals can develop a body tremor and have an increased sensitivity to noise and movement. As disease progresses animals can develop an inability to stand, and exhibit other behavioural changes. In BSE affected cattle clinical observations may be subtle or obvious and can include changes in temperament such as nervousness or aggression, animals can also be seen to develop an abnormal posture, demonstrate a lack of coordination and have difficulty in rising. There is decreased milk production, and animals often show weight loss. With CWD, clinical signs may include emaciation, an excessive salivation, ataxia, the drooping of head and ears, weakness and behavioural changes. C and PrPSc in post mortem samples by their distinct resistances to proteinase K (PK): under defined conditions, PrPC is completely degraded and PrPSc is cleaved at the N-terminus leaving a protease resistant core of 27 to 30 kDa  staining techniques that define foci of PrPSc aggregation in the absence of protease treatment. For all ruminant prion diseases, clinical signs are not specific enough for diagnosis and often these signs may be subtle or go unnoticed. The most common methods for diagnosing prion diseases rely on differentiating between PrPo 30 kDa . PrPSc iSc [Sc can adopt different conformations within the secondary, tertiary or quaternary structure of the protein. Experimental evidence illustrating that TSE strains are likely to be made up of different conformational forms of PrPSc has been repeatedly demonstrated by the different strain-to-strain properties of the PrPSc protein with regard to protease digestion with PK [Sc from a particular isolate, however the true identification of a TSE strain can only be confirmed by determining a stable disease phenotype in specific lines of inbred rodent strains. Prion strains are defined TSE isolates that when transmitted to a specific host species  exhibit phenotypic disease traits that are distinct from one another. These traits include differences in disease incubation time, the lesion profile and IHC staining pattern within the CNS, and the biochemical characteristics of the PrPSc . A chara with PK  and trea with PK . Within C is expressed in the host and the infecting inoculum, as is the case when using transgenic rodents for bioassay [C primary structures [The ability of a TSE to infect a distinct host species is governed by a phenomenon commonly referred to as the species barrier . When prbioassay . The natructures  and in nructures , presumaSc conformers are possible within a given strain, but that within each prion ‘strain’ only a subset of those conformers are able to interact with the host PrPC that is presented within any given infection. Thus there will be a range of PrPSc infectious conformers that are able to become the dominant species upon transmission within a new host. For an efficient infection to proceed the PrPC from the host has to be capable of forming a conformation that is compatible with the infecting PrPSc strain. Thus the selection of the new dominant species is dependent on the appropriate conditions being present for its selection. The selection of a minor conformer within a newly infected host and its preferential replication over the previous dominant ‘strain’ would go some way to explaining how prion strains can apparently mutate  fibrils [in vitro prion replication method  to amplify prions from a scrapie-infected brain sample, another study showed that when RNA was removed from and then returned to the replication environment, a novel prion strain developed which was not present in the original brain sample, further supporting the deformed template theory [Studies have suggested that novel synthetic prions can be created  fibrils ,20,21. Ue theory .in vitro and in vivo selection of novel prions in both cell culture and in mice, using biologically cloned murine adapted prions. In these experiments prions could be seen to adapt to the selection pressures of either brain (infection of mice) or cell culture replication where the action of the anti-prion compound quinacrine could select for resistant prions. In addition, Li and co-workers [Prion strains can appear to undergo Darwinian selection given the right selective pressures. Ghaemmaghami and co-workers  have dem-workers  demonstrSc biological/biochemical properties. This data indicates that environmental reservoirs of ruminant prions may be dynamic and the dominant “strain” may change over time potentially even leading to the emergence of novel prions. Understanding how TSE strains are maintained within populations and how they might have the ability to cause new infections within new hosts or after prolonged incubation in environmental reservoirs given the correct selective pressures is of importance if we are to minimise the potential for these agents to affect human health.A recent study used transgenic mouse bioassay to examine a mixture of ovine scrapie isolates before and after incubation on soil for 13 months to determine whether the dominant types were consistent during this incubation period within an environmental matrix . The datSc as compared to classical BSE [Sc with a lower molecular weight than classical BSE together with a lower proportion of the diglycosylated form [BSE was first discovered in 1986 in UK cattle  and led ical BSE ,28,30, ated form  . Masujin and co-workers [Mice transgenic for bovine PrP-workers  describe-workers . Togethe-workers ,49,51, pC [Transmission of CWD prions to wild type mice lines is inefficient. However, mice expressing cervid PrPC  have higSc deposition within the CNS and periphery.Alternate rodent models have been developed for the monitoring for prion infectivity and prion type. In recent years the bank vole has been demonstrated to have a high susceptibility to different prion types from a variety of sources ,54. The PRNP genotype [Sc distribution patterns that were distinct from those seen with classical scrapie: vacuolation is primarily in the cortex of the cerebellum and cerebrum and not the medulla oblongata [Sc accumulation in caudal brainstem in clinically affected compared to asymptomatic BSE-infected cattle [Vacuolation patterns are routinely used in mouse bioassay (often following repeat sub-passage) as a diagnostic feature of a prion strain/type. Different prion types can influence lesion profiles in sheep scrapie as well, for example when compared to natural classical scrapie, SSBP1 experimental scrapie produces relatively few lesions in the medulla oblongata ,58. Howeblongata ,59. Amylblongata  and is eblongata . Intraneblongata . The lesblongata ,64. Howed cattle .Sc molecular phenotype very similar to BSE PrPSc; [In goats, BSE and CH1641  produceE PrPSc; ,67. For CWD infections of deer or elk, again lesions are routinely found in various brain regions although within elk the lesions are generally less severe. In deer, pathology is usually found in the medulla oblongata, olfactory bulb, cortex and hypothalamus (reviewed in ). Overall, whilst vacuolation patterns are a diagnostic feature of a prion strain/type in rodent bioassays, they have very limited application in the definition of prion types in natural infections due to high variability ,68,69.Sc is largely consistent between individuals when analysing distinct neuroanatomical sites and distinct patterns are observed for different BSE types [Sc compared to C-BSE: L-BSE PrPSc is present at comparatively lower levels in brainstem and higher levels within more rostral sites of the brain such as the olfactory bulb and olfactory cortex as well as the hippocampus [Sc deposition [With BSE in cattle the accumulation of PrPSE types ,64. L-BSpocampus . At presposition . Sc type that can diagnose BSE from classical scrapie [Sc morphological types, cell-type associations as well as levels of PrPSc accumulation across multiple neuroanatomical sites allows the discrimination of ovine BSE, CH1641, atypical/Nor98 scrapie and classical scrapie. This technique has been termed PrPSc profiling and has been pioneered by Jeffrey and co-workers [Sc accumulation have been described, each associated with distinct cell types: four associated with cell surface or extracellular PrPSc and three with intracellular PrPSc  is a distinct technique that also displays a high level of discrimination [Sc containing the 84-102 amino acid region in dark zone tingible body macrophages (TBMs) of the LRS [Sc were consistently higher for ovine BSE than scrapie across multiple brain regions when detected with a range of antibodies [Sc for ovine BSE appeared to be determined by the cell type with three distinct sites being identified, all of which result in smaller PrP fragments compared to classical scrapie PrPSc [Sc that is distinct from both classical scrapie and ovine BSE as it is truncated even further from the N-terminus than BSE [The analysis of both intra- and extracellular PrPmination . Using t the LRS ,75 and itibodies . Interesthan BSE . Howeverthan BSE . Sc accumulation was distinct from classical scrapie with increased accumulation in the cerebellar cortex and no accumulation in the medulla oblongata [Sc staining was of a diffuse granular type that has not been seen with classical scrapie [Sc in the LRS [With atypical/Nor98 scrapie the pattern of PrPblongata . Such ce scrapie . In addi the LRS . Sc staining was seen with SSBP1 or CH1641 infections, whereas goats infected with BSE show clear PrPSc accumulation particularly in the thalamic, hypothalamic nuclei and in the basal ganglia [Sc when comparing goat and sheep infections, with intraneuronal PrPSc for BSE being truncated further from the N-terminus compared to classical scrapie [With goat scrapie, very little PrP ganglia . Almost  scrapie . Interes scrapie . Sc accumulation in the medulla oblongata at the level of the obex make this region important for the diagnoses of prion disease. The caveat to this is that this region is less involved in pathology in atypical/Nor98 diseases and therefore other brain regions require examination, such as the cerebellum in sheep [For classical BSE, classical scrapie and CWD the presence of spongiform lesions and PrPin sheep . Sc profiling and PrPSc epitope mapping techniques provide highly discriminatory descriptions of PrPSc phenotypes capable of differentiating BSE, CH1641 and classical scrapie infections in small ruminants. It has also been suggested that these techniques may even be able to define distinct classical scrapie types in the natural host [Sc staining patterns and require the availability of high quality tissue covering an extensive range of neuroanatomical sites. For PrPSc profiling, some of these issues have been addressed by the simplification of the technique by analysing more restricted neuroanatomical areas  [Whilst vacuolation profiles are consistent for BSE infections in ruminants, other prion diseases of these species produce highly variable profiles indicating the very limited application of this technique for prion type identification. However, this technique may have an application in distinguishing atypical/Nor98 and classical scrapie infections. In contrast, IHC analysis shows great promise in defining prion types in the natural host. L-BSE and C-BSE can be distinguished by IHC examination as can atypical/Nor98 and classical scrapie infections. The PrPral host ,77. Poteephalon) ,77.Sc from PrPC is the high proteolytic resistance of PrPSc. Of course, this trait is not absolute and it is likely that for all prion diseases PrPSc will exist as both protease-resistant (PrPSc-res) and protease-sensitive (PrPSc-sen) conformers. At one extreme are 263K scrapie infection in hamsters and human Gerstmann–Sträussler–Scheinker syndrome P102L infection of transgenic 101LL mice, where both infection models accumulate high infectivity titres in the absence of PrPSc-res [Sc-res that are detectable by conventional immunoassays. As such, immunoassay detection of PrPSc-res has become the mainstay of prion diagnostics including defining prion types in ruminant diseases. When detected in western blots, the pattern of the PrPSc-res fragments can be used to differentiate prion types as it is thought that their distinct PrPSc conformers will present distinct cleavage sites to the protease  but retained during scrapie PrPSc digestion. When compared to an antibody binding further C-terminally in the protease resistant core, ratios of P4: “core antibody” are much higher for classical scrapie that for BSE or CH1641. By far the most common protease used in prion typing is PK, which digests PrPreported  that BSEhe PrPSc  and the he PrPSc  can inflhe PrPSc ,82,83. The PrPSc  who usedSc-res triplet bands could be indicative of prion type, for instance with bovine and ovine BSE displaying a dominant diglycan compared to classical scrapie [Sc can be influenced by host factors such as the brain region and PRNP genotype [PRNP genotypes [It was also noted that on western blots the glycoform pattern of the PrP scrapie . The robgenotype ,86. Howegenotype ,87,88 anenotypes ,88. It henotypes ,89. Whilenotypes . Sc, the ratio of P4:‘core antibody’ binding and glycoform profiling to search for BSE infections. Samples displaying one or more traits of BSE are usually subsequently analysed by mouse bioassay for confirmatory prion typing [Screening of small ruminant prion diseases has mainly been designed to distinguish the zoonotic agent BSE from the non-zoonotic agents of scrapie. Most studies use a combination of molecular weight of PK-resistant PrPn typing ,89,91. Gn typing ,83,91. Sc for ovine BSE compared to classical scrapie that was not influenced by host genotype or scrapie type [In addition to PK cleavage, an alternative protease thermolysin can also be applied to distinguish BSE from classical scrapie in small ruminants. Digestion resulted in distinct cleavage patterns of PrPpie type .Sc-res with three distinct antibodies that bind to different regions on PrP. Due to distinct PK-cleavage sites for PrPSc from different TSE types, all three antibodies capture PrPSc-res from classical scrapie, two bind PrPSc-res from atypical scrapie and a different combination of two antibodies bind to PrPSc-res from BSE and CH1641. BSE and CH1641 can then be differentiated due to the distinct binding ratios of these two antibodies.Recently, there have been further developments in the differentiation of BSE from natural scrapie with particular emphasis on differentiating BSE from CH1641 which is not possible using any of the criteria described above . PirisinSc fragments that may also be indicative of prion type in ruminant, such as the so-called C2 fragment in scrapie cases but not in experimental BSE or CH1641 cases [Endogenous proteolysis also results in PrP41 cases  and the 41 cases .Sc-res with a lower protease resistance than classical scrapie and PK digestion patterns for atypical/Nor98 scrapie often  also co always, .Sc has led to the description of variants of BSE in cattle. Biacabe and co-workers [Sc further N-terminally than in classical BSE, resulting in the efficient retention of the 12B2 epitope and therefore relatively high levels of binding to this antibody [Sc-res, termed BASE or L-BSE. In addition to the differences in PK cleavage sites, L-BSE can be distinguished from C-BSE and H-BSE by a distinct glycoform pattern [Sc-res is sufficient to diagnose such infections, the difference in molecular weight between L-BSE and C-BSE PrPSc-res is just 0.3kDa for the unglycosylated band and is not suitable for routine prion typing [The analysis of PK cleavage patterns of PrP-workers  reportedantibody . Casalonantibody  reported pattern ,28 altho pattern ,85,86,88 pattern  that can pattern . Whilst n typing . Howevern typing  demonstrSc molecules led to the development of tests to characterise the stability of these conformers. These methods were developed into two complementary assays that quantified the disruption of prion conformations with GndHCl [Sc molecules upon denaturation and the conformational stability assay (CSA) measured the loss of PK-resistance of the PrPSc molecules upon denaturation. The underlying theory that prion characteristics are defined by the conformational make-up of the PrPh GndHCl ,99. The The CDI was developed and applied to rodent passaged strains and could distinguish multiple prion strains in hamsters . DespiteSc-res . With this CSSA assay, different ovine classical scrapie isolates with a range of PRNP genotypes could be differentiated from a similarly diverse set of atypical/Nor98 scrapie isolates.Pirisinu and co-workers  also adaSc from PrPC. Such analyses show significant promise in defining prion types and correlating structural properties of the prion with disease pathologies such as the duration of asymptomatic or clinical phases of disease. However, relatively little data is available to date on the application of such assays to ruminant samples.Overall, the analysis of the stability of prion conformers can be monitored through changes in epitope accessibility or protease-resistance, and total PrP can be analysed directly or following separation of PrPC gene was permissive to ovine scrapie and was found to accumulate high levels of PrPSc [A number of cell lines permissive to prion multiplication have been developed including established cell lines, neuronal stem cells, and primary neuronal cells. Whilst initial cell lines were only permissive to rodent-adapted prion strains, the rabbit epithelial RK13 cell line expressing an ovine PrPof PrPSc . Since tof PrPSc , and celof PrPSc . Cell moof PrPSc .Sc containing cells determined by an ELISA using ELISPOT plates with automated counting. [The standard scrapie cell assay (SSCA) involves the use of susceptible N2a cells which are exposed to prion containing samples for 3 days, grown to confluence, split 1:10 three times, then the proportion of PrPounting. . The SSCounting. . The Rovounting. . Howeverin vitro prion replication assay that relies on iterative rounds of sonication and incubation [C into PrPSc during the incubation period. The technique allows the amplification of minute amounts of PrPSc when spiked into normal brain homogenate substrate where the prion is amplified over multiple (24–48) cycles of sonication/incubation (a round) and the substrate is then replenished by diluting the amplification products into fresh substrate. Any number of rounds can then be carried out. When analysing a range of rodent passaged scrapie strains or synthetic prions, Gonzales-Montalban and co-workers [Sc particles and also the total yield of PrPSc produced in the assay [C substrate [A non-radioactive cell free conversion assay that used recombinant mouse PrP as the substrate to examine the replication of murine passaged scrapie strains and BSE demonstrated that the conversion efficiency differed between some strains . More recubation  (Figure -workers  demonstrhe assay . Other she assay , as wellubstrate . PRNP genotypes. These observations have been developed into a sensitive assay using a combination of the VRQ/VRQ and AHQ/AHQ brain homogenate substrates [C substrate with distinct PRNP genotypes. In terms of the analysis of prions from their ruminant hosts, PMCA has been applied to a range of ovine scrapie types as well as ovine passaged BSE. Taema and co-workers  demonstrbstrates . These tbstrates ,114. Thebstrates . These aTogether, the data supports the hypothesis that a range of substrate/co-factor conditions in tandem with a range of amplification conditions could provide a panel of defined PMCA conditions capable of defining prion types in the natural host; effectively producing an “amplification profile” for each ruminant prion type. in vitro replication assay known as quaking induced conversion or “Quic” has been described [in vitro amplification and prion typing of unknown samples may be possible using this rapid and sensitive technique.Recently, the use of recombinant bank vole PrP as a universal substrate for a similar escribed  (Figure Sc type or in vivo pathology will be best suited for diagnosing new prion types. Whilst the cases of BSE in ruminants is now very low and the associated concern for the contamination of the human food chain with the zoonotic BSE agent has eased, there are still concerns surrounding the exposure of humans to prions from food-producing animals. The more recent description of atypical/Nor98 scrapie in goats/sheep and atypical bovine BSE as well as the discovery of two distinct types of CWD all raise the possibility that further types of prions are circulating in ruminants that are not detected and/or defined by current assay methods. An additional concern is that novel types may emerge in these animals. One diagnostic challenge in prion biology is to develop and apply prion typing tests to fully elucidate the range of existing prion types in ruminants and to monitor for the emergence of novel types. This is a significant challenge as it is unknown what molecular and pathological differences any novel type will have compared to those already described. Therefore, assays that have a wide range of distinct measurements that describe a PrPin vitro prion typing assays. The development of PrPSc profiling and PrPSc epitope mapping provide IHC methods that can differentiate classical, atypical/Nor98 and CH1641 scrapie from BSE in sheep and has the potential to further discriminate between classical scrapie types. The methods examine multiple anatomically distinct brain regions and cellular prion locations and/or map multiple epitopes of PrPSc. The methods therefore have a wide range of possible test outcomes that are examined and have great diagnostic application. The drawbacks of these methods are that they are labour intensive and require extensive good quality brain material. Prion typing tests are almost certainly going to be most applicable to post-mortem derived tissues from routine sampling sites in animals confirmed as having a prion disease. Therefore, the application of these IHC methods using routine surveillance samples to test for novel or existing types is perhaps limited. The examination of proteolytic fragments of PrPSc by western blotting has formed the mainstay of high throughput prion typing assays for ruminants. Recent advances using multiple antibody probes for single western blots along with the partial denaturation of samples prior to proteolysis have improved the diagnostic capability of such tests. However, these assays have each been specifically designed to differentiate defined existing prion types by looking for a specific fragmentation pattern and therefore may be limited in their application to detecting novel prion types. In contrast, the CSA measures the stability of a prion type over a range of denaturation conditions and therefore may well define novel types with distinct stabilities. It could be envisaged that the use of multiple denaturation conditions within CSA combined with epitope mapping of the resulting PrPSc fragments by western blot could provide a range of test outputs that measure the conformation of PrPSc to define types. A limitation of PrPSc analysis in all of these methods may be that PrPSc can display variation in its neuroanatomical location dictated by the prion type pathology and sufficient PrPSc may not be present at the routine sampling sites to allow analysis by these methods. A methodology that could detect prion types at very low concentration would therefore be advantageous. PMCA may provide such a method. It has been reported that PMCA usually maintains the PrPSc molecular profile of the seed and is exquisitely sensitive in detecting PrPSc. A combination of PMCA amplification followed by analysis of the PrPSc product by an assay such as CSA may allow a robust prion typing assay to detect novel types even when PrPSc is present at low levels in the tissue sample. Alternatively, PMCA methods have now also been directly applied to prion typing in ruminants demonstrating that different prion types display different replication efficiencies under defined conditions. The data supports the hypothesis that a range of substrate/co-factor conditions in tandem with a range of amplification conditions could provide a panel of defined PMCA conditions capable of defining prion types from the natural host; effectively producing an ‘amplification profile’ for each type. There have been significant advances in recent years in in vitro prion typing assays the definition of a novel prion type would need to be assessed as being representative of an inheritable in vivo strain phenotype rather than of a quasispecies that was selected/identified during the in vitro analyses. Therefore, any novel type would require analysis within rodent bioassay to define a novel and consistent pathology that is stable upon sub-passage and can therefore be described as a novel prion strain. The recent advent of transgenic murine models and a range of rodent host species have extended the use of rodent bioassay to a relatively wide range of natural ruminant isolates making this a much more realistic possibility.For all


Although in anurans the predominant mode of intra- and intersexual communication is vocalization, modalities used in addition to or instead of acoustic signals range from seismic and visual to chemical. In some cases, signals of more than one modality are produced through or by the anuran vocal sac. However, its role beyond acoustics has been neglected for some time and nonacoustic cues such as vocal sac movement have traditionally been seen as an epiphenomenon of sound production. The diversity in vocal sac coloration and shape found in different species is striking and recently its visual properties have been given a more important role in signalling. Chemosignals seem to be the dominant communication mode in newts, salamanders and caecilians and certainly play a role in the aquatic life phase of anurans, but airborne chemical signalling has received less attention. There is, however, increasing evidence that at least some terrestrial anuran species integrate acoustic, visual and chemical cues in species recognition and mate choice and a few secondarily mute anuran species seem to fully rely on volatile chemical cues produced in glands on the vocal sac. Within vertebrates, frogs in particular are suitable organisms for investigating multimodal communication by means of experiments, since they are tolerant of disturbance by observers and can be easily manipulated under natural conditions. Thus, the anuran vocal sac might be of great interest not only to herpetologists, but also to behavioural biologists studying communication systems. •Our view of anuran communication has changed drastically during the last few decades.•Anurans use diverse communication strategies often directly related to the vocal sac.•Distinctive vocal sac shapes, sizes and colours suggest functional diversity.•Evidence increases that the vocal sac also plays a role in chemical signalling.•The anuran vocal sac may be of great interest for studies on communication systems. Anolis lizards rely almost entirely on visual signals; their colourful throat display advertises position, repels males and attracts female mates and habitat light conditions are suggested to promote signal diversity , a so-called foot-flagging species that performs leg waves during agonistic male interactions, only displays this behaviour in response to conspecific calls accompanied by vocal sac inflation. The visual cue was suggested to improve detection and discrimination of acoustic signals by making them more salient to receivers amid complex biotic background noise  which might facilitate floating in water and/or better airborne sound transmission  the proposed signal cocktails consist of 65 different compounds, whereas specific combinations of sesquiterpenes, alcohols and macrolides are correlated with species identity . In Canebrake frogs, Aplastodiscus perviridis, males rest their vocal sac on females suggesting pheromone transmission during courtship  there is substantial variation in body coloration, morphology and reproductive modes, but males of all reed frog species share a common feature: a prominent gular patch on the vocal sac, which is particularly conspicuous once the vocal sac is inflated . Althougidentity . Additioidentity , and mulourtship .Figure 2Apart from the use of the anuran vocal sac, unimodal chemical signalling is widespread in amphibians but has received relatively little attention in the vast field of anuran communication . The folMost amphibians have a biphasic life cycle, hence spend part of their life in water and because of their often thin and permeable skin  it seemsAscaphus truei, live alongside noisy stream habitats, similar to foot-flagging species. Tailed frogs that were exposed to water previously containing reproductive males or females showed a preference for chemosignals of the opposite sex which suggests chemical mate recognition  females tested in Y-maze experiments showed a clear preference for water containing homogenized male postaxillary breeding glands or water previously containing live males  denote the ability to receive airborne as well as waterborne odours  related to certain environmental conditions or habitat features . To shed light on signal function, more across-species comparisons of how single and combined signal components influence receivers are needed. Furthermore, detailed descriptions of the conditions in which signals are produced are essential to draw more general conclusions on vocal sac signal function. Finally, we should question the constraints of different modalities and therefore the costs imposed by sexual or natural selection, to explain differences and similarities of this multimodal communication tool. Moreover, the majority of descriptions of amphibian glands involved in chemical courtship and mating signals lack information on the structure of the glands. This information would allow for more specific hypotheses on the origin and function of the glands described.Within vertebrates, amphibians possess a rather basal nervous system leading to a mostly stereotypic behaviour (but see for example Amphibians show a vast diversity in reproductive modes e.g.  and owinTo date, a number of amphibian pheromones have already been identified , but in The vast diversity of anuran vocal sacs suggests a multitude of different functions in intra- and intersexual communication shaped over evolutionary time by natural and/ or sexual selection. The modalities used in addition to or instead of acoustic signals range from seismic and visual to chemical. The vocal sac, a single morphological feature, has the potential to generate multimodal signals simultaneously or sequentially. Signals of the same modality might be directed towards and perceived by one or more conspecifics and heterospecifics. Furthermore, even signals shaped under similar evolutionary constraints may influence receivers differentially. Thus, the anuran vocal sac might be of great interest not only to herpetologists, but also to behavioural biologists investigating unimodal or multimodal communication in the acoustic, visual or chemical domains. Amphibians are highly suitable organisms to study chemical unimodal and multimodal signalling in a basal system. The focus of chemical signal research in amphibians, however, used to be mainly on urodeles. In anurans the salient acoustic and visual signals might to a certain extent have deflected our attention from equally fascinating chemical signalling strategies. Possibly, terrestrial anurans might have lost once-present chemical signals owing to the evolutionary success of acoustic communication. In some species acoustics might not have been sufficient, and thus chemical signals were ‘rediscovered’ and incorporated into a multimodal signal or in some species even fully replaced acoustic signals.In this review we hope to have stimulated researchers to investigate potential chemical signals in anuran communication systems and to recognize the vocal sac as a unique multimodal signalling tool.


Piwi-interacting RNAs (piRNAs) originate from genomic regions dubbed piRNA clusters. How cluster transcripts are selected for processing into piRNAs is not understood. We discuss evidence for the involvement of chromatin structure and maternally inherited piRNAs in determining their fate. Drosophila, piRNAs are derived from long precursor transcripts that originate from distinct genomic regions dubbed piRNA clusters . ,22. DrosIt is essential that cells generate a proper pool of piRNAs that can target potentially dangerous transposons yet inhibit processing of transcripts from normal coding and non-coding genes. Next we discuss possible mechanisms that might be responsible for differentiation between sequences that are meant to be processed into piRNAs and other cellular transcripts.The fact that only a very specific subset of transcripts is processed into piRNAs indicates that some features of either the genomic locus or the transcript itself must differentiate piRNA precursors from other cellular RNAs. Three hypotheses can be proposed on how piRNA-producing loci are identified. First, distinct sequence or structure motifs in precursor transcripts or DNA inside or around clusters could signal the biogenesis machinery to process the transcript into piRNAs. Second, the chromatin structure of piRNA clusters could be marking these genomic regions for differential processing of their transcripts. Finally, piRNAs that are inherited from the previous generation could be responsible for selection of regions for piRNA processing in the progeny.Caenorhabditis elegans piRNAs, called 21U-RNAs, are individually encoded by separate genes, all of which contain an octamer motif roughly 40 bp upstream of the piRNA sequence that is recognized by the Forkhead family of transcription factors [ factors . Transcr factors . AlthougIn flies and mice, no unique sequence motifs have so far been identified in or around piRNA clusters. Although A-myb has binding motifs in the promoters of pachytene clusters in mouse, this motif is also present at promoters of several protein-coding genes whose transcripts are not processed into piRNAs, indicating that binding of A-myb cannot be a signal that discriminates piRNA loci. Similarly, no distinct structural motifs have been identified within piRNA clusters. Unlike in the case of siRNAs, there is no phasing of piRNAs: although they have a strong preference for uridine at their 5′ base position, piRNAs can start at any nucleotide within the cluster. Insertion of a heterologous sequence into a cluster leads to piRNA production from this sequence, indicating that any sequence can be processed into piRNAs . These dpiRNA-producing loci could be specified by a unique chromatin structure. Chromatin structure has been mostly linked to transcriptional regulation of the underlying DNA. However, chromatin might also have an impact on the post-transcriptional fate of transcripts. How is this possible? Either by regulating processes that happen co-transcriptionally, such as splicing and polyadenylation , or by mDrosophila do have a unique chromatin environment. Most piRNA clusters are found at the boundary of euchromatic and heterochromatic regions in the pericentromeric and subtelomeric regions of the genome [Increasing evidence indicates that piRNA clusters in e genome . piRNA ce genome ,28. Moree genome . Rhino de genome . Nucleare genome  that RhiAlthough it seems clear that chromatin has an important role in regulating the expression of piRNA clusters, currently it is not clear whether the chromatin state simply provides a permissive environment for piRNA cluster activity, or whether, at least in some cases, it is by itself sufficient to specify such regions.Drosophila might be defined by a molecular memory provided by the pool of piRNAs inherited from the previous generation. Piwi proteins and the associated piRNAs expressed in the maternal germline during oogenesis are deposited into the developing egg and are present in the early embryo before the start of zygotic transcription [Drosophila, such maternally inherited piRNAs are essential for effective piRNA-mediated silencing and fertility of the progeny. Indeed, recent studies of a long-known phenomenon called hybrid dysgenesis showed that the absence of piRNAs inherited from the previous generation causes a failure to produce cognate piRNAs in the progeny [Instead of intrinsic features such as specific sequence signals or a unique chromatin signature, the loci that generate germline piRNAs in cription . In Dros progeny .trans-generationally inherited piRNAs in specifying the activity of piRNA loci in the next generation has recently been revealed by studies of transgenes that generate piRNAs. It was shown that a transgene that generates piRNAs is able to induce piRNA production from another locus that was originally incompetent for piRNA generation [The role of neration . After ineration  revealedtrans-generationally inherited piRNAs can activate piRNA generation both by inducing chromatin changes and by initiating precursor processing through the Ping-Pong cycle. It should be noted that these mechanisms can only regulate piRNA production in germ cells but not in follicular cells of the Drosophila ovary: piRNAs specific to follicular cells are not deposited into the embryo and thus cannot serve as a template for either mechanism. In addition, follicular cells also lack components necessary for the Ping-Pong amplification loop.Two possible mechanisms can be envisioned for how piRNAs inherited from the mother might act to start generation of new piRNAs in the progeny Figure b. They mtrans-generationally inherited piRNAs as two components that are required for activity of regions that generate piRNAs. Chromatin and inherited piRNAs can act in concert to identify genomic loci for piRNA generation, with chromatin state licensing certain genomic regions and inherited piRNAs providing further direction using both the Ping-Pong cycle to initiate processing and a feedback loop to ensure that the compatible chromatin state is preserved.Taken together, recent studies implicate chromatin state and