Taken together, this article aims to provide an overview of the current status of the cellular and pathological mechanisms involved in blast overpressure injury and argues for the urgent need to identify potential biomarkers that can hint at the different mechanisms involved.Among the U.S. military personnel, blast injury is among the leading causes of brain injury. During the past decade, it has become apparent that even blast injury as a form of mild traumatic brain injury (mTBI) may lead to multiple different adverse outcomes, such as neuropsychiatric symptoms and long-term cognitive disability. Blast injury is characterized by blast overpressure, blast duration, and blast impulse. While the blast injuries of a victim close to the explosion will be severe, majority of victims are usually at a distance leading to milder form described as mild blast TBI (mbTBI). A major feature of mbTBI is its complex manifestation occurring in concert at different organ levels involving systemic, cerebral, neuronal, and neuropsychiatric responses; some of which are shared with other forms of brain trauma such as acute brain injury and other neuropsychiatric disorders such as post-traumatic stress disorder. The pathophysiology of blast injury exposure involves complex cascades of chronic psychological stress, autonomic dysfunction, and neuro/systemic inflammation. These factors render blast injury as an arduous challenge in terms of diagnosis and treatment as well as identification of sensitive and specific biomarkers distinguishing mTBI from other non-TBI pathologies and from neuropsychiatric disorders with similar symptoms. This is due to the “distinct”  Traumatic Brain Injury (TBI) represents a major public health problem with an over 150,000 military personnel diagnosed with form of mild traumatic brain injury (mTBI), due to the exposure to blast resulting in a wide range of neurological and psychological symptoms . Blast-rBetween 2000 and 2010, the Department of Defense (DoD) reported ~200,000 head injuries as a consequence of combat-related incidents as well as events occurred in a non-deployed environment (civilian injuries) . Howeverprimary, secondary, tertiary, and quaternary injuries) is often accompanied by hemorrhagic blood loss, multiple fractures, burns, and systemic injury coupled with TBI  What is the experimental and human evidence that blast is associated with progressive alterations in the brain and via what mechanism(s) they are mediated? (ii) What is the relation between blast-induced brain injury and the development of neuropsychological disorders such as post-traumatic stress disorder (PTSD)? (iii) What are the biochemical markers that can identify, track and predict the injury and symptoms observed in patients exposed to blast injury?Blast overpressure-induced injury results from an explosion characterized by an abrupt release of energy in such a short period of time within a small volume creating a non-linear shock and pressure wave . The blaprimary injury resulting from the BOP waves due to the shock-wave overpressure or/and under pressure. This event is usually associated with contusion, edema, hemorrhage, and diffuse axonal injury (DAI) (secondary injury that is due to shrapnel or hard objects propelled at the body. (iii) The tertiary insult involves head translation/rotation coupled with acceleration/deceleration due to blunt impact arising from blast wind and finally (iv) the quaternary insult resulting from thermal burns or the probable use of toxic gases or chemicals.Blast can cause four different types of insults: (i) the ry (DAI) , 21, 22.Compared to previous past conflicts, the majority of war zone wounds have been attributed to secondary blast injury (shrapnel propelled by explosions), while tertiary and quaternary blast injuries were related to terrorist-linked acts involving structural collapse and the use of toxic material. Previous studies on primary injury (BOP) have traditionally focused on gas-containing hollow organs such as the lungs and gastrointestinal tract , 23.In one study by Clemedson discussing blast injury, the term “blast injury” has been used to describe the biophysical and pathophysiological events post exposure to high explosion or the shock wave associated with it . The greFurthermore, BOP can induce a mild form of brain injury with significant neurological conditions involving cerebral edema, neuroinflammation, and vasospasm along with DAI and neuronal death. This neuronal injury phase is followed by a series of complex neuropsychiatric symptoms which may include memory loss and behavioral changes , 31–33. Blast wind passage to the skull causes acceleration/rotation to the brain comprising the direct injury. Indirect injury involves the compression of the abdomen and chest transferring kinetic energy to the body’s biofluid. This rippling effect generates oscillating waves from blood to the brain distant from the contact point. In turn, this kinetic energy transfer will induce functional and morphological changes in brain structures which represent a distinct complex feature of blast-induced brain injury not present in other traditional brain injury models , 31, 35.c-Myc, c-Fos, and c-Jun and impaired axonal transport along with oxidative stress with elevated nitric oxide generation  have shown evidence of altered cellular, molecular and biochemical processes, and behavioral outcomes. For instance, different studies have shown a heterogeneous profile of brain-associated cellular impairments including: elevation in β-amyloid precursor protein, altered expression of protooncogenes neration , 36–44. neration , 44–46. neration , 43, 47.Furthermore, such heterogeneous neural profile has been attributed to several factors including the suitable experimental model systems that can closely mimic “composite” primary, secondary, tertiary, and quaternary components of blast exposure, the lack of standardized blast wave instruments, different body localization and body armor, and the use of different animal species , 41, 48 Several studies have been performed to assess neuropathological effect of BOP coupled with other comorbid factors , 47–51. One representative study is that of Kamnaksh et al. where they assessed different stressors and their contribution to blast injury. These stressors included transportation and blast sound with or without blast injury. Of interest, all groups exhibited increased anxiety, while injured and blast noise-exposed rats showed elevated corticosterone, interferon-c (IFN-c), and interleukin-6 (IL-6) in the amygdala and hippocampus. Injured animals showed elevated Iba1, GFAP, and apoptotic immunoreactivity . These dPun et al. using a rat model, assessed the effects of a single sublethal blast over pressure (BOP) exposure (48.9–77.3 kPa) in an open-field set up. Histopathological analysis of inflicted brains revealed “darkened” and shrunken cortical neurons with narrowed vasculature at day 1 post-injury. Signs of recovery were demonstrated at days 4 and 7 post-blast exposure. Oligodendrocytes and astrocytes showed TUNEL-positivity in the white matter at day 1. Acute axonal damage was observed in the white matter as indicated by elevated amyloid precursor protein immunoreactivity with no sign of macrophages/microglia change. Major gene changes were observed at day 1 and 4 post-blast pointing toward signs of repair at day 4 and 7. These findings suggest that the BOP levels in the study resulted in mild cellular injury and white matter perturbations . In anotIn a different study, de Lanerolle et al. used a swine model to assess different scenarios of blast exposure including: simulated free field (blast tube), high-mobility multipurpose wheeled vehicle surrogate, and building 4-walled structure. Of interest, histological changes in the three blast scenarios showed minimal neuronal injury with fiber tract demyelination and intra-cranial hemorrhage. Neuropathological changes involving increased astrocyte activation coupled with proliferation and periventricular axonal injury detected were observed with β-amyloid precursor protein .Long et al. assessed blast-induced physiological, neuropathological, and neurobehavioral changes coupled with Kevlar protective vest encasing the thorax and part of the abdomen using a compression-driven shock tube (at 126- and 147-kPa). Kevlar vest effect reduced air blast mortality and also ameliorated the widespread fiber degeneration in rat brains. BOP was shown to induce abnormal neurologic and neurobehavioral performance along with cardiovascular disruptions involving hemorrhagic hypotension with disruption in cardio-compensatory resilience  . SimilarIn an interesting study, Garman et al. characterized the neuropathological changes produced by a single blast exposure in rats with body shielding using a helium-driven shock tube (exposure of 35 Psi with left side-head-only exposure) . NeuropaIn conclusion, these different blast studies presented distinguished heterogeneous results  cross talk involved in blast-induced trauma related to the activation of autonomous nervous system and the neuroendocrine–immune system which contributes significantly to the mechanism of blast injury. Inflammation has been proposed to play an important role in the pathogenesis of long-term neurological deficits due to blast . ExperimIn vivo real time imaging of myeloperoxidase (MPO) inflammatory enzyme activity of activated phagocytes was conducted on three groups of rats: (1) whole-body blast; (2) blast with “body armor,” (chest and abdomen) with the head exposed; or (3) blast with “helmet” as head protection (neck and skull) while the rest of the body exposed. One day post-blast exposure, MPO activity was observed in the gastrointestinal tract and the diaphragmal mediastinal parts of the lungs  induces the activation of autonomous nervous system in rabbit exposed to BOP. Distinct pathological components in the brain including impaired energy metabolism, and increase in the sodium–potassium ATPase measured in the brainstem and erythrocyte membranes were coupled with edema formation , 60. In he lungs .In the brain, this activity was observed at 7, 14, and 30 days post-blast injury. Of interest, MPO increase in the brain was independent of head protection at 14 and 30 days post-injury suggesting chronic inflammation and highlighting the role of systemic origin of the inflammatory activation mediating brain injury which highly reflects on the role of the vagal afferent neurons mediating gut–brain communication. Taken together, the results of this study clearly demonstrate the importance of the indirect, i.e., blast–body interaction as well as the decisive role of autonomous nervous–neuroendocrine–immune systems interaction in the pathogenesis of blast-induced brain trauma .Similarly, Chavko et al. assessed the theory of the indirect effect of kinetic energy transfer via the blood vessels and the surrounding cerebrospinal fluid (CSF) to the CNS . In theiAnother significant aspect of blast injury is psychological health which is highly affected. Many injured troops returning from war zones are afflicted with blast-induced BI experiencing post concussive symptoms (PCS), characterized by memory and cognitive disruption, irritability, anxiety, and fatigue . Among tThe nomenclature of mTBI has been a challenge for both civilian and military settings as described by Rosenfeld et al. . mTBI, aMild traumatic brain injury is the most frequent form of brain trauma among deployed military populations . It has Primary blast component of blast injury is among the main contributors in developing neuropsychiatric impairments associated with the primary phase profile , 73. ThePost-traumatic stress disorder, a psychiatric condition that arises after exposure to a life threatening experience such as conditions experienced in combat war zone with or without blast exposure as a form of mTBI . This, bPsychological stress resulting from exposure to blast wave leads to an altered psychological health status which contribute significantly to the development of PTSD , 70. HowPost-traumatic stress disorder is deemed an effect of psychological and emotional determinants/trauma , while mild bTBI is a result of destructive biomechanical forces acting on the brain . There iClinical evaluation of a blast-exposed personnel can be challenging as symptoms may range from neurologic problems, psychiatric, or emotional difficulties which may be attributed to blast or due to other psychiatric disorder where in several instances the occurrence of TBI and PTSD may be suggested , 87. ForBased on the above, it is of high interest that an accurate detailed knowledge of blast injury biophysics and injury threshold may assist clinicians in better diagnosis . This inOver the last several decades, a number of experimental animal models have been implemented to study the mechanisms of blast wave impact which included rats, mice, ferrets, rabbits, and larger animals involving sheep and swine , 90–97. There are limited available basic and translational studies relevant to the mechanisms of primary blast-induced brain injury. A better understanding of injury mechanisms is required for the development of protection and treatment options and biomarker identification for prognosis.Several animal models have been proposed at translating intra-cranial biophysics and pathophysiology experienced in human blast exposure . These mTraumatic brain injury leads to progressive pathophysiological changes resulting in a reduction in cerebral blood flow and a decrease in tissue oxygen levels leading to ischemia, BBB disruption with brain edema . Death oAt the cellular level, the biphasic nature of secondary injury is mediated by numerous disturbed pathways which include: (a) excitotoxicity caused by an excess of the neurotransmitter glutamate; (b) free radical generation by mitochondrial dysfunction, causing damage to proteins and phospholipid membranes of neurons and glia; and (c) the neuroinflammatory response which takes place due to both CNS and systemic immunoactivation. Thus, diffuse brain injury mediated immune responses, BBB alterations, and neuroinflammation seem to play an important role in the pathology of BOP. The increase in BBB permeability was shown to recover by the third day after the blast exposure , 102. Fo+, K+-ATPase, a membrane-bound enzyme required for cellular transport. Dysfunction of this pump is a common feature in CNS pathologies related to ischemic conditions and TBI. The activity of Na+, K+-ATPase pump is very sensitive to free radical reactions and lipid peroxidation. Reductions in this activity can indicate membrane damage indirectly. Thus, Na+, K+-ATPase is clearly down regulated under low O2 conditions which in turn triggers brain edema, enhances the loosening of tight junctions and causes BBB breakdown. MPO activity, an index for neutrophil infiltration, also increases as an evidence of inflammation  of injury occurring in the brain after BOP exposure. Due to the biochemical nature of BOP compared to physical nature of TBI (impact or penetrating injury), subtle molecular changes such as free radical-mediated oxidative stress occur and contribute to the manifestation of BOP-induced brain injury , 44, 110The widespread recognition of the brain vulnerability to blast exposure and inadequate approaches to diagnose blast-related TBI led to design an mTBI Diagnostics Workshop  and the Current available imaging modalities, such as computed tomography (CT) and magnetic resonance imaging (MRI), primarily detect major structural changes in the brain ; howeverTo date, several biomarkers such as GFAP , UCH-L1 More recently, Balakathiresan et al. proposed microRNAs as novel serum diagnostic biomarkers of bTBI. They investigated microRNA signatures in CSF and serum of rats exposed to BOP injury. Specifically, microRNA let-7i was elevated in both CSF and serum post-blast wave exposure and was considered as an ideal candidate biomarker of brain injury . ImportaStudies investigating biomarkers of mTBI in humans continue to be limited as illustrated in one study by Ingebrigtsen and Romner . In theiInterestingly, recent experimental and human studies are suggesting a link between blast exposure and chronic traumatic encephalopathy (CTE), a tau protein-linked neurodegenerative disease –131. To For long, TBI has been considered one of the “signature injuries” of current conflicts in Iraq and Afghanistan which attracted concern from the DoD, Department of Veteran Affairs, and National Institutes of Health, encouraging combined efforts to understand brain injury pathophysiology and identify therapeutics and assess different approaches for rehabilitation platforms as well as deciphering novel blast specific biomarkers , 11. BetA non-trivial and urgent issue in biomarker-panel design will be determining an appropriate instrument platform that is suited to measure these biomarker changes. At present, biomarkers are analyzed in clinical laboratories using closed, high throughput immunoassay analyzers allowing for high performance in terms of accuracy and precision which are suitable for major hospitals. Future recommendation is to focus research on the development of a miniaturized point-of-care (POC) system, which can be transported in the “field” (military and civilian) providing accurate measurements at a reasonable cost with short turnaround time .Drs. Prima and Svetlov are employees and receive salaries from Banyan Biomarkers, Inc. The other co-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.


Somatostatin (SST), cortistatin (CORT), and its receptors (sst1–5), and ghrelin and its receptors (GHS-R) are two highly interrelated neuropeptide systems with a broad range of overlapping biological actions at central, cardiovascular, and immune levels among others. Besides their potent regulatory role on GH release, its endocrine actions are highlighted by SST/CORT and ghrelin influence on insulin secretion, glucose homeostasis, and insulin resistance. Interestingly, most components of these systems are expressed at the endocrine pancreas and are actively involved in the modulation of pancreatic islet function and, consequently influence glucose homeostasis. In addition, some of them also participate in islet survival and regeneration. Furthermore, under severe metabolic condition as well as in endocrine pathologies, their expression profile is severely deregulated. These findings suggest that SST/CORT and ghrelin systems could play a relevant role in pancreatic function under metabolic and endocrine pathologies. Accordingly, these systems have been therapeutically targeted for the prevention or amelioration of certain metabolic conditions (obesity) as well as for tumor growth inhibition and/or hormonal regulation in endocrine pathologies (neuroendocrine tumors). This review focuses on the interrelationship between SST/CORT and ghrelin systems and their role in severe metabolic conditions and some endocrine disorders. The pancreas is a physiologically and biologically complex organ organized in exocrine and endocrine compartments which are modulated by a wide variety of neuronal and hormonal signals in an integrated manner. The exocrine function of the pancreas is performed by more than 90% of the whole pancreatic tissue and is essentially composed of acinar and ductal cells that respectively, synthesize and transport enzymes crucial for nutrient digestion at the gastrointestinal tract.The endocrine function of the pancreas is in turn achieved by distinct cell types organized in major structures called Islet of Langerhans, which are scattered throughout the organ and are in close contact with the vascular environment. At least five major different endocrine cell types have been described: glucagon-secreting alpha-cells, insulin-secreting beta-cells, somatostatin (SST)-secreting delta-cells, ghrelin-producing epsilon-cells, and pancreatic polypeptide-producing cells. The distribution and proportion of endocrine cells within the pancreatic islets varies between species . The cooSomatostatin was originally isolated from ovine hypothalamus based on its potent inhibitory action on pituitary growth hormone (GH) secretion . At circA large amount of this wide biological capacity of SST is mediated via binding and activation of SST receptors, a family of five specific transmembrane proteins (named sst1–5) belonging to the superfamily of G proteins coupled receptors (GPCRs), and encoded by five distinct intronless genes . All fivThe pleiotropic activity featured by SST fits well with both its ample tissue distribution and its multiple receptors. Moreover, it can also be likely related to the existence of a highly similar peptide of the same family, CORT, which was originally discovered in frogs and subsequently in rodents and humans . Like SSAlthough CORT was initially discovered in the brain and it is especially abundant in the cortex (where its name comes from), further reports showed that this neuropeptide is also widely distributed at peripheral tissues including gastrointestinal tract and pancreas. Based on its ability to activate ssts, CORT exhibits almost endocrine and most non-endocrine actions of SST . Actuallin vitro by a GHS-R1a antagonist, a biological action that is not elicited by SST , a former orphan receptor originally suggested as specific for CORT. However, this receptor appears to be a quite promiscuous GPCR that also shows some specificity for proadrenomedullin and related peptides . Interesd by SST .GHRL, encloses a 20-bp non-translated exon (Ex0), four coding exons (Ex1–4) and three introns, being a 28-aa native ghrelin peptide the predominant product of the 117-aa precursor pre-proghrelin  crucial for its biological activity. This post-translational modification is catalyzed by the ghrelin O-acyltransferase , protein kinase C (PKC), protein kinase A  intracel kinases .Different components of the ghrelin system have been found to be ubiquitously represented in the organism. Specifically, ghrelin was originally described to be predominantly produced by endocrine cells of the stomach submucosa . In addiOn the other hand, GHS-R1a expression has been also widely detected in tissues or organs including pancreas, liver, stomach, adipose tissue, small and large intestine, immune system, and others . In striAccording to the wide tissue distribution of the different ghrelin system components, many physiological actions have been documented for this system. Based on the high conservation degree among species of ghrelin and its receptor sequences, it has been suggested that ghrelin system plays essential biological actions. In fact, and as mentioned earlier, acylated-ghrelin was initially identified based on its ability to stimulate GH release upon GHS-R1a coupling in a similar manner to that described for GH secretagogues . Also inOn the other hand, unacylated-ghrelin, plays both equal or opposite biological actions to that above described for acyl-ghrelin, most probably through its coupling to a still unknown receptor different from GHS-R1a, as it has been recently suggested and documented . MoreoveRegarding obestatin, this 23-aa peptide is mainly produced in the stomach and at lower level in the pancreas, spleen, testis, duodenum, jejunum, colon, and mammary gland . To dateSomatostatin has long been known to potently modulate pancreatic function by playing a regulatory role on insulin and glucagon secretion. This tight regulation is accomplished by the coordinated action of SST biologically active forms. In this sense, SST-14 is the major product released by adult pancreatic delta-cells, whose contribution to total circulating SST accounts for nearly 5%. SST-14 immediate actions imply the paracrine regulation of other pancreatic endocrine cells. In addition, endocrine pancreatic function is also under the control of the major circulating SST form, SST-28. In this context, it has been documented that SST-14 and SST-28 differently target pancreatic alpha- or beta-cells. Accordingly, SST-14 has been associated with the inhibition of glucagon secretion while SST-28 seems preferentially to inhibit insulin secretion . In eithThe SST action on pancreatic hormones is mediated by its interaction with the different receptor isoforms, however, conflicting data about receptor expression at the pancreatic level have been published which, might be related with the different method used and/or species analyzed. In general, it is likely that endocrine pancreas expresses all five sst isoforms, being probably sst1, sst2, and sst5 those expressed in a predominant manner. Specifically, it has been demonstrated by RT-PCR that rodent pancreas expresses all sst isoforms except sst4. In turn, by double immunohistochemistry methods, it has been confirmed the expression of sst2 and sst5 in both rodent beta- and delta-cells. In humans, it has been reported a predominant expression of sst2 in alpha-cells, sst1 and sst5 in beta-cells, and sst5 in delta-cells while there is not consistent data on the expression of the rest of ssts .knock out mouse models with deletion of the different ssts isoforms have been employed. These issues have been amply revised elsewhere . Furthermore, it has been reported that insulin and SST inhibit ghrelin release while glucagon inhibits its secretion in rodent although stimulate ghrelin release in humans  as well as its inhibitory action on intestinal glucose absorption, predicted its use as a key tool to potentially regulate glucose homeostasis and insulin sensitivity in diabetes and obesity  on hormones that mediate glucose homeostasis has been described in healthy volunteers . AccordiIn different series of acromegalic patients, a significant prevalence of impaired glucose metabolism and diabetes mellitus has been described, a consequence that is believed to be originated by the excess GH . In suchSomatostatin analogs have also been applied in the management of NET suffering patients in which, similarly to acromegalic individuals, an altered glucose tolerance has been documented . Such alHypercortisolism condition, a distinctive characteristic of Cushing’s disease, also leads to hyperglycemia and reduced glucose tolerance and as a result, in an increase of the prevalence of diabetes in this pathology . FurtherIn sum, hyperglycemia conditions occur in an elevated proportion of individuals suffering of acromegaly, Cushing’s disease or NET and accordingly, it has been proposed that regular blood glucose testing and insulin analogs will be required, particularly when SST analogs are therapeutically used in these pathologies .in vivo and in vitro and its influence on glucose tolerance. Accordingly, it has been proposed that the antagonism of ghrelin system components could improve glucose homeostasis and/or beta-cell function under certain metabolic disorders as T2DM, a complex disease with a strong genetic, behavioral, and environmental background that is characterized by two distinctively conditions: insulin resistance and progressive beta-cell dysfunction. In T2DM, beta-cells become unable to adequately increase insulin release to compensate insulin resistance and consequently leading to a situation of hyperglycemia. It is well known the close association between T2DM and obesity in terms of metabolic imbalance and their common features, insulin resistance, in which ghrelin system could be of relevance based on its ability to modulate both glucose homeostasis and weight loss  were adjusted, probably as a direct effect of insulin on ghrelin-producing cells . Based oOn the other hand, obesity conditions have been associated with some ghrelin and GHS-R gene variations although some discrepancies exist depending on studies and population considered . In thisHowever, it has been well documented that under obesity conditions plasma ghrelin levels negatively correlate with BMI and consequently with factors or parameters that are elevated in obesity such as insulin, leptin, and fat mass . In thisTargeting ghrelin system components by different pharmacological, immunological, and genetic approaches have been addressed in order to promote weight loss and to improve obesity conditions . Thus, some studies evaluated pharmacological approaches to block or neutralize either ghrelin or its receptor under diet-induced obesity and how these methods ameliorate obesity condition by reducing appetite or food intake, and ultimately inducing weight loss . AnotherIn addition, it has also been proposed that GOAT by a specific inhibitor could be a potential treatment against obesity by inhibiting ghrelin acylation and consequently avoid weight gain .On the other hand, diet-induced weight loss elicits an increase in circulating ghrelin levels thus normalizing them until near optimal concentration, rise that probably may hamper the sustained weight loss . In caseUnfortunately, there is not yet a ghrelin system based therapy that ensures a sustained weight loss, although ghrelin antagonist and/or GOAT inhibitors may be considerate good therapeutic candidates for the treatment of T2DM and obesity.The complex relationship of ligand–receptor architecture of SST/CORT and ghrelin systems is complemented by the functional relevance of their common tissue targets. Indeed, besides their opposite influence on GH release at the pituitary level, SST/CORT and ghrelin systems act on the same cellular target to influence common and relevant biological actions as it is the case of their interactions on beta cell function and survival as well as on glucose homeostasis and insulin resistance. However, the underlying molecular mechanisms of these actions have not been fully elucidated yet. Interestingly, different and severe metabolic dysfunctions as T2DM and obesity have been described to modulate their circulating levels and the expression of some components of both systems at hypothalamic, pituitary, or pancreatic level. In sum, there are an increasing number of evidences that support a potential contribution of SST/CORT and ghrelin system components in the endocrine pancreas dysfunction in prevalent neuroendocrine-metabolic pathologies (T2DM and obesity) which suggest that these systems could be considered as future valuable therapeutic targets for the prevention or treatment of such metabolic disorders. In this sense, future research will be of particular importance in order to ascertain whether SST/CORT and ghrelin systems and its receptors will act at pancreatic islets under physiological and pathological conditions. Likewise, the underlying molecular mechanisms as well as the precise role and the contribution of islet derived-SST, -CORT, and -ghrelin in the pancreatic endocrine deregulation and/or the resulting insulin resistance under severe metabolic conditions should be investigated.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.


One area in which the influence of miRNAs is most prominent is cancer. Numerous expression surveys and more focused studies have revealed miRNA involvement in carcinogenesis, cellular pathology, cell behavior and prognosis. Large-scale comparisons of miRNA expression in varioius types of cancer have not been previously possible. However, The Cancer Genome Atlas (TCGA), an extensive multi-centered effort to characterize the genomes of hundreds of types of cancer, has enabled such comparisons. In the present study, the expression patterns of hundreds of miRNAs in thousands of tumors covering seven types of cancer: uterine corpus adenocarcinoma, ovarian serous adenocarcinoma, breast adenocarcinoma, prostate adenocarcinoma, pancreatic adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma were analyzed. The results showed that miRNA expression patterns among these cancer types are highly correlated (0.874>ρ>0.974) and that miRNA expression in all seven cancer types is dominated by miRNAs belonging to the most evolutionarily ancient miRNA families. This raises the possibility that more ancient miRNAs are involved in the fundamental cell processes that are central to tumor evolution. AmongIn the present study, we extracted miRNA expression data on seven types of cancer for the purpose of examining shared and unique expression patterns. The cancer types we selected to extract are uterine corpus adenocarcinoma (designated as ENDOCA), ovarian serous adenocarcinoma (OVARCA), breast adenocarcinoma (BRSTCA), prostate adenocarcinoma (PROSCA), pancreatic adenocarcinoma (PANCCA), colorectal adenocarcinoma (COLNCA), and lung adenocarcinoma (LUNGCA). Our rank order correlation analyses of miRNA expression in these seven types of cancer reveal a high degree of homogeneity. Closer examination of individual miRNAs shows that evolutionarily ancient miRNA families are significantly over-represented among the most highly expressed miRNAs. We hypothesized that the two observations suggest that relatively minor variations in miRNA expression are sufficient to play a role in establishing and maintaining tumorigenesis and the regulatory targets of many of the most highly expressed miRNAs are likely to be involved in fundamental cellular processes that are re-programmed in cancer.The data used in this study and the analyses reported herein have been approved by the The Cancer Genome Atlas Program Office at the National Cancer Institute of the United States National Institutes of Health (25/10/13).6 bases). Standardization was performed to avoid bias potentially introduced by closely related miRNAs or by miRNAs occurring in clusters.TCGA miRNA expression in each cancer was determined via deep sequencing tumor-derived RNAs on the IlluminaHiSeq_miRNASeq platform (?). Potential microRNA sequences were verified against primary miRNA (pri-miRNA) and precursor miRNA (pre-miRNA) sequences contained in the miRBase microRNA database. Molecule counts for human miRNA sequences were standardized within each miRNA and reported as ‘hits’ per megabase (10www.wessa.net/rankcorr.wasp). Statistical significance was assessed by the t-test with (n−2) degrees of freedom.Standardized miRNA hit data for 1,046 miRNAs in 415 uterine corpus tumors (designated as ENDOCA), 770 breast tumors (BRSTCA), 286 prostate tumors (PROSCA), 62 pancreatic tumors (PANCCA), 478 colorectal tumors (COLNCA), and 482 lung tumors (LUNGCA) were extracted. However, TCGA reported expression data on only 680 miRNAs in 970 ovarian tumors (OVARCA). We therefore generated two separate files, one without OVARCA containing 1,046 miRNAs for each tumor and one with OVARCA that was trimmed to contain only 680 OVARCA miRNAs. In all of the miRNA hit data we calculated the average number of hits per miRNA per cancer and then re-ordered them. We compared miRNA expression in all seven cancer types pairwise  using the Spearman Rank Order Correlation (ρ) , PicTar (pictar.mdc-berlin.de) and miRanda (microRNA.org). Targets appearing on at least two of the three sources were compiled for specific miRNAs and then supplemented with experimentally confirmed targets listed in TarBase (diana.imis.athena-innovation.gr). The mRNAs were then submitted for pathway analysis using The Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 online tool (Pairwise rank order scatter plots of miRNA expression among the seven types of cancer are shown in The Spearman Rank Order Correlations (ρ) among the six types of cancer for which ranks were assigned on the basis of 1,046 miRNAs are also shown in In the current study, we have presented a simple rank correlation analysis of miRNA expression in seven types of cancer: uterine, ovarian, breast, prostate, pancreas, colorectal and lung. Our results show that all seven cancer types exhibit significantly correlated miRNA expression patterns. Although highly significant, the least similar of the expression patterns was observed in comparisons with ovarian tumors, likely due to the fact that, among the seven cancers, only ovarian tumors are uniformly of the serous histologic type, which are known to be more aggressive with a less favorable prognosis than the adenocarcinomas typical of the other six cancers used in this study. Thus, histologic similarity is potentially an aspect of the higher average miRNA expression rank correlations among the other six cancers.The initial impetus for analyzing TCGA miRNA expression patterns originated from a previous study of miRNA expression in endometrial adenocarcinoma . The aimExamination of miRNA expression ranks of the other six cancers showed that the same ancient miRNA families found to be significantly over-represented among the most highly expressed miRNAs in endometrial cancer were also significantly over-represented in the six types of cancer. There are 162 miRNAs belonging to families that first appeared in animal genomes >380,000,000 years ago, i.e., prior to the emergence of land animals . MoreoveAmong the ‘younger’ miRNAs is the ubiquitous miR-21. This miRNA is ranked first or second in six of the seven cancer types examined in this study. miR-21 is well-known to play an important role in a wide range of normal and pathologic biological processes including development, inflammation, cardiovascular and pulmonary function and cancer . Among tet al (The results have shown that miRNA expression among different human adenocarcinomas is significantly correlated. Moreover, the ranks of the most highly expressed miRNAs in these cancers are occupied by representatives of evolutionarily ancient miRNA families. This pattern is the result of ancient linkages between regulatory miRNAs and mRNA targets essential to carcinogenesis. Thus, it is among these most ancient miRNAs and their targets that attention should be focused. Recently, Hamilton et al  discover


Oxidative stress induced by reactive oxygen species plays an important role in the etiology of many diseases. Dietary phytochemical products, such as bioactive food components and marine carotenoids , have shown an antioxidant effect in reducing oxidative markers stress. Scientific evidence supports the beneficial role of phytochemicals in the prevention of some chronic diseases. Many carotenoids with high antioxidant properties have shown a reduction in disease risk both in epidemiological studies and supplementation human trials. However, controlled clinical trials and dietary intervention studies using well-defined subjects population have not provided clear evidence of these substances in the prevention of diseases. The most important aspects of this special issue will cover the synthesis, biological activities, and clinical applications of marine carotenoids, with particular attention to recent evidence regarding anti-oxidant and anti-inflammatory properties in the prevention of cardiovascular disease. Oxidative stress (OS) and chronic inflammation are the major pathophysiological factors contributing to the development of cardiovascular diseases (CVD), such as hypertension, diabetes and atherosclerosis. Accumulating evidence suggests that a compromised antioxidant system can lead to excessive OS in cardiovascular related organs, resulting in cell damage and death .Emerging evidence suggests that interventions, including nutrition, pharmacology, and physical exercise, may activate expression of cellular anti-oxidant systems and play a role in preventing inflammatory processes in CVD . For thecontinuum of pathophysiological processes that advance from local redox imbalance to endothelial dysfunction, endothelial inflammation, and excessive vascular remodeling. Consequent cell damage contributes to atherosclerosis, coronary artery disease (CAD), stroke and myocardial infarction [Advances in pathophysiological research suggest that CVD represent a farction ,6. In pafarction . OX) can be counteracted by the activity of dietary free radical electron (“antioxidant”) acceptor molecules (such as β-carotene and ascorbic acid), which sequester free radical electrons and prevent the oxidation of LDL particles [The nutritional prevention of atherosclerosis with the use of natural antioxidants represents an important new frontier in the prevention and treatment of CVD. There is evidence that the production of oxidized LDL (LDLarticles . Many foarticles .Although a large body of research has focused on individual or small numbers of antioxidants, increasing circulating antioxidant capacity through increased consumption of antioxidant-rich fruits and vegetables is protective against cardiovascular disease. The available scientific evidence indicates that the link between oxidative stress, a proinflammatory systemic environment and cardiovascular disease is strong . This co


Understanding the functional mechanisms underlying genetic signals associated with complex traits and common diseases, such as cancer, diabetes and Alzheimer's disease, is a formidable challenge. Many genetic signals discovered through genome-wide association studies map to non-protein coding sequences, where their molecular consequences are difficult to evaluate. This article summarizes concepts for the systematic interpretation of non-coding genetic signals using genome annotation data sets in different cellular systems. We outline strategies for the global analysis of multiple association intervals and the in-depth molecular investigation of individual intervals. We highlight experimental techniques to validate candidate  regulatory variants, with a focus on novel genome-editing techniques including CRISPR/Cas9. These approaches are also applicable to low-frequency and rare variants, which have become increasingly important in genomic studies of complex traits and diseases. There is a pressing need to translate genetic signals into biological mechanisms, leading to prognostic, diagnostic and therapeutic advances. The quest for identifying sequence variants associated with complex traits including common diseases has been greatly facilitated by technological progress in high-throughput DNA analysis, such as genotyping arrays and next-generation sequencing, complemented by advances in bioinformatics First, GWAS have focused on common SNPs MAF > 5%). SNPs either individually or in combination typically explain only a small fraction of the genetic variance of most complex traits %. SNPs eSecond, the associated sequence variant identified in GWAS may in fact only be linked to, rather than itself be, the causal variant. This phenomenon is known as linkage disequilibrium (LD) Third, the vast majority (over 90%) of associated variants have been found to localize outside of known protein-coding sequences, thus impeding the direct interpretation of their functional effects The challenge ahead is to carve out suitable strategies to gain insights into cell type-specific molecular processes and pathways underlying the discovered GWAS signals. In this article, we (i) describe principles for interpreting non-coding genetic signals using public genome annotation resources; (ii) discuss considerations when using annotation data sets in primary cells, cell lines, and – looking ahead – differentiated induced pluripotent stem cells (iPSCs); and (iii) outline current and emerging strategies to prioritize and experimentally validate candidate regulatory variants and genes.The genomic positions of GWAS index SNPs and proxy SNPs can be compared to the positions of biochemical events referenced in publicly available annotation resources to help tease out the functional variant(s) from the vast number of trait-associated variants in LD. Such biochemical events include sites of transcription factor and micro RNA (miRNA) binding, chromatin accessibility and modifications, DNA methylation, and many other types. A trait-associated variant that overlaps with a regulatory element may be functionally relevant. The overlap also directly suggests a hypothesis with respect to the mechanism underlying the association, which can be tested in experimental assays. However, there are a number of caveats to this approach that need to be correctly evaluated.First, it must be noted that a large degree of non-functional overlap can be expected, because of the widespread distribution of the biochemical events. Thus, it is necessary to use unbiased approaches to evaluate which of the overlaps are functionally relevant and which occur by chance Second, regulatory elements that influence gene expression may operate in a spatial- and temporal-dependent manner More and more genome annotation data sets are becoming available, including those created by the Encyclopedia of DNA Elements (ENCODE) Project Consortium The growing number of genome annotation data sets will enable the functional interpretation of GWAS signals in an increasingly context-specific manner. Alongside these, it will be necessary to refine computational methods to distil the vast amount of data into discrete segments of interpretable biological function. Segmentation approaches determine patterns and similarities between individual chromatin data sets to summarize them into a small set of “chromatin states” Genome-wide annotation data sets can be obtained in several cellular systems, mainly from primary cell cultures and transformed cell lines, while in the future, differentiated iPSCs are likely to become more prominent for this application Primary cells are directly representative of the tissues and organs from which they were isolated. However, the isolation of homogeneous cell populations is challenging, involving preparative procedures such as fluorescent-activated sorting of cells. Therefore, many primary cell populations are available in limited quantities for experimental assays. In addition, chromatin maps generated in primary cells and tissues represent only a snapshot of the development stage during which they were isolated.Cultured cell lines and transformed  cell lines mostly retain the characteristics of the primary tissue from which they were derived. However, chromosomal rearrangements, changes to chromatin structure, DNA methylation, and gene expression profiles may arise through, for example, serial subculturing of the cell lines +/CD20+ B cells in multiple sclerosis. Indeed, this approach may recognize pathogenic cell types without prior knowledge of disease-relevant molecular processes The prioritization of candidate regulatory variants at GWAS loci by means of publicly available annotation resources suffers from a trade-off between sensitivity and specificity general hallmark of regulatory potential, such as open chromatin provided by deoxyribonuclease I (DNase I)-seq, formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq, or the recently introduced assay for transposase-accessible chromatin (ATAC)-seq individual mark, such as transcription factor binding provided by chromatin immunoprecipitation (ChIP)-seq.Besides choosing a cellular system that is biologically relevant for the screening of candidate functional variants at GWAS loci, as outlined above, attention has to be paid to choosing a suitable genome annotation mark. In this respect, we advocate the use of a Sites of open chromatin are associated with most classes of active gene regulatory elements, and as such, are specific to cell type, developmental stage, and other influencing factors Alternatively, ChIP-seq of components of the gene expression machinery may also be useful for the annotation of candidate regulatory variants. For example, ChIP-seq of the transcriptional co-activator protein p300 has been shown to associate with active, cell type-specific enhancer sequences To gain biological understanding of the genetic variants associated with complex traits including disease susceptibility and outcome, at least two strategies have been pursued. First, the combined analysis and interpretation of all genetic signals identified in a GWAS C and secp < 5 × 10−8) or a subset of SNPs reaching a certain significance threshold (e.g. p < 0.05) may be tested for association with shared gene pathways and networks. Of course, identified associations are not necessarily causal and require further validation. Findings may also be biased towards well-studied and frequently reported gene pathways. Nonetheless, these bioinformatic tools may provide a powerful means of generating novel hypotheses regarding molecular processes involved in disease etiology. For example, an integrated pathway analysis approach recently highlighted the striking role for host responses to mycobacteria in inflammatory bowel disease Much biological insight from GWAS can be gained when multiple association signals are collectively co-analyzed. Rather than aiming to identify distinct molecular mechanisms, these bioinformatic approaches focus on connecting a selection of target genes and their products with knowledge databases, e.g. concerning tissue-specific gene expression signature, intracellular localization, citation in the literature (PubMed abstracts) or gene ontology terms Annotating GWAS signals with information on gene expression can yield a better understanding of the regulatory networks underlying the association. In studies on expression quantitative trait loci (eQTLs), the alleles of the index or proxy SNP are correlated with variation in transcript levels that are quantitatively measured in unrelated individuals using gene expression arrays or – more sensitively and accurately – RNA-seq. SNPs that show a strong correlation with expression levels for a specific gene are likely to mark an eQTL for that gene. Both local- and distal-acting eQTLs can be identified, but the identification of distal-acting eQTLs has been largely unfruitful due to the inherent limited statistical power of the approach Instead of measuring gene expression levels across individuals, allele-specific expression (ASE) analysis measures transcript abundance within an individual http://www.uk10k.org/), which should give sufficient account of low-frequency and rare sequence variation.Ascertaining an exhaustive account of common as well as low-frequency variants is an important, initial step in the in-depth molecular analysis of selected, individual GWAS intervals. However, only a fraction of all genetic variants are examined in GWAS. Genotype imputation exploits known LD patterns and haplotype frequencies from reference data sets to estimate genotypes for additional SNPs not directly assayed in the initial genome-wide scan After the comprehensive assessment of genetic variation at GWAS intervals, the variants are then overlapped with genome annotation marks in order to identify candidate functional variants. Importantly, mere annotation of genetic variants using epigenomic data sets does not prove molecular function and causality . Candidate regulatory variants that overlap with one or more annotation marks require substantial experimental validation. This should involve an integrated approach of multiple experimental methods to gain confidence in the observed effect. Most frequently applied in vitro cellular assays include luciferase reporter assays Regulatory variants potentially lie great distances from the gene(s) they control, functioning through long-range regulatory interactions Danio rerio) embryos using morpholinos (antisense oligonucleotides) has proved particularly insightful. In systematically switching-off candidate genes at GWAS intervals, several novel genes implicated in platelet formation were identified and successfully validated Once established, target genes may be further characterized with respect to the trait of interest. Here, traditional assays to characterize gene function can be used including gene knockdown using small interfering RNA (siRNA) or gene overexpression using adeno-associated viral vectors Investigating the molecular mechanism of individual GWAS loci is arduous, and arguably, scalable in vitro approaches are needed to experimentally validate candidate functional variants in a high-throughput manner. Indeed, progress has been made in massively parallel reporter assays, which use large-scale DNA synthesis and next-generation sequencing to simultaneously measure the reporter activity of many thousands of enhancer variants Despite remarkable technological advances for the discovery of genetic variation, the experimental tools to study the molecular mechanisms of candidate functional variants have seen only little progress thus far. To this end, we envisage the application of site-specific genome-editing techniques to be game-changing. Transcription activator-like effector nucleases (TALENs) BCL11A, a gene implicated in hemoglobin disorders TALENs comprise a FokI nuclease domain, which cleaves DNA in a non-sequence-specific manner, fused to a modular DNA-binding domain. The DNA-binding domain is composed of highly conserved amino acid repeats, transcription activator-like effectors (TALEs), which can be engineered to recognize specific DNA sequences. The engineered nucleases bind as a dimer to a target site, where they induce a DNA double-strand break. In turn, DNA damage response pathways are triggered, such as non-homologous end-joining (NHEJ) or homology-directed repair (HDR), which enable the precise introduction, exclusion or alteration of gene alleles at the target site. Bauer et al. applied TALENs to modulate the activity of an enhancer sequence critical for erythroid expression of CRISPR/Cas systems have recently emerged as an alternative to TALENs, vastly improving its cleavage efficiency and ease of implementation at reduced cost For genome-editing techniques, the issue of specificity and delivery is paramount. The desired genome modification should ideally occur with high frequency in the cell population and with no detectable off-target effects. Although rapid progress has been made in this respect http://www.hipsci.org/). This open-access resource will create iPSC lines derived from over 1,000 healthy individuals and individuals with genetic diseases by 2016. Moreover, the proposed approach would make currently available tools for the correction of disease-causing mutations more efficient, such as zinc finger nucleases (ZFNs) combined with piggyBac transposons in iPSCs CRISPR/Cas9 constructs can be introduced into human somatic cells and iPSCs (via transfection) as well as various model organisms  and rare (MAF < 1%) genetic variants (not captured by GWAS) may explain a substantial fraction of the genetic component of complex traits including common diseases We believe there is still substantial scope for performing GWAS in the coming years. Well-powered meta-analyses of GWAS detect novel small-effect association regions, and fine-mapping approaches as well as studies in ethnic subgroups refine existing ones. In parallel, we call for a boost in the number of investigations into the molecular mechanisms of confirmed associations. There is a pressing need to translate genetic signals of complex traits and diseases into molecular mechanisms, through both global meta-analysis of multiple GWAS intervals and in-depth mechanistic studies of transcription, chromatin structure and DNA methylation at individual GWAS intervals. This functional translation is crucial for the identification of novel “druggable” or reversible components and pathogenic pathways. This in turn has the potential to empower clinical care through, for example, improved risk prediction, biomarker identification, disease subclassification, drug development and dosing http://www.genomicsengland.co.uk/) and personal  whole-genome sequencing, an overwhelming number of sequence variants will be identified at non-coding regions with potential regulatory effects. Thus, efficient strategies for functional translation are urgently needed. To this end, we expect that CRISPR/Cas9 will play a pivotal role in unravelling molecular mechanisms for a significant number of trait-associated genetic variants. Although not discussed in detail in this article, we suggest that proteomic tools could also be integrated in the functional translation of GWAS findings. For example, the interaction dynamics between a trait-associated variant and a transcription factor complex may be characterized by mass spectrometry Despite the identification of over 2,000 robust associations with more than 300 complex traits and diseases http://www.godmc.org.uk/) will bring together scientists studying the genetic basis of DNA methylation and provide a centralized hub for coordinating data analyses.Non-genetic factors, e.g. epigenetic variation, have been suggested to have a substantial impact on complex trait etiology. Similar to GWAS, such epigenetic variation  can be assayed across many individuals and tested for association with a complex trait of interest in epigenome-wide association studies (EWAS)


Root and bulb vegetables (RBV) include carrots, celeriac (root celery), parsnips (Apiaceae), onions, garlic, and leek (Alliaceae)—food crops grown globally and consumed worldwide. Few data analysis platforms are currently available where data collection, annotation and integration initiatives are focused on RBV plant groups. Scientists working on RBV include breeders, geneticists, taxonomists, plant pathologists, and plant physiologists who use genomic data for a wide range of activities including the development of molecular genetic maps, delineation of taxonomic relationships, and investigation of molecular aspects of gene expression in biochemical pathways and disease responses. With genomic data coming from such diverse areas of plant science, availability of a community resource focused on these RBV data types would be of great interest to this scientific community.The RoBuST database has been developed to initiate a platform for collecting and organizing genomic information useful for RBV researchers. The current release of RoBuST contains genomics data for 294 Alliaceae and 816 Apiaceae plant species and has the following features: (1) comprehensive sequence annotations of 3663 genes 5959 RNAs, 22,723 ESTs and 11,438 regulatory sequence elements from Apiaceae and Alliaceae plant families; (2) graphical tools for visualization and analysis of sequence data; (3) access to traits, biosynthetic pathways, genetic linkage maps and molecular taxonomy data associated with Alliaceae and Apiaceae plants; and (4) comprehensive plant splice signal repository of 659,369 splice signals collected from 6015 plant species for comparative analysis of plant splicing patterns.http://robust.genome.com, provides an integrated platform for researchers to effortlessly explore and analyze genomic data associated with root and bulb vegetables.RoBuST, available at  In the present decade, rapid advancements in DNA sequencing and molecular genetics techniques have generated a wealth of plant genomics, genetics, and breeding data. Whole genome sequences of several plants  are now available and the sequencing of a number of additional plants is ongoing . The rapid proliferation of plant genome annotations generated by these efforts has led to the development of several data analysis platforms focused on specific plant groups - Gramene for grass genomics , SOL GenInterestingly, most of the plant data analysis platforms available today are focused on plant groups with high economic value and are associated with data collection from genome sequencing projects. Very few data analysis tools are available for less-studied "minor" and "orphan" crops with no ongoing genome sequencing efforts. Integrated data collection and annotation platforms focused on specific orphan crop families would be invaluable for researchers working on these crops.Root and bulb vegetables (RBV) represent one such group of orphan crops ,7. TheseSmallanthus sonchifolius), a member of the Asteraceae, and wild yam (Dioscorea villosa), a member of the Dioscoraceae, are included in the Tropical Plant Database [Plants that form storage roots and bulbs are included in several web sites that have a taxonomical or regional focus. For example, turnip and rutabaga are included in the Eurpoean Brassica Database , yacon , and runs on an Apache (2.0.53) web server. Graphical display of genes, RNAs, intron-exon structures, splice junctions, and alternative splicing patterns were implemented in RoBuST using Gene Plot, Splice Signal Plot and AS Plot modules from the EuSplice  project.Sequence records of Alliaceae and Apiaceae plant families were downloaded from GenBank  by submiPlant-specific annotation data files ("gbpln*.seq.gz") were downloaded from GenBank and all GenBank gene records which belong to plant species from Kingdom Viridiplantae were extracted. Since we were interested in splice signal information, all single-exon gene records were removed from this data set. This left 348,081 GenBank records covering 6015 Viridiplantae plant species, each of which had multi-exon gene annotations. These were further parsed to extract position coordinates and sequences associated with gene, mRNA, exon and intron structure, and donor and acceptor splice signals.We submitted seventeen new carrot and garlic trait terms to Trait Ontology (TO), which is part of the Plant Ontology Consortium . All sevWe successfully mapped Gene Ontology (GO)  functionRoBuST provides a comprehensive platform for integrated visual analysis of various types of sequence annotations from 294 Alliaceae and 816 Apiaceae plant species. This includes genes, mRNAs, tRNAs, rRNAs, ESTs, splice signals, promoters, regulatory elements, signal peptides, transposons, molecular taxonomy sequences and several additional types of sequence elements , alternative splicing (AS), and alternative transcription termination (ATT) events in genes with multiple transcript isoforms. The graphical plots also display regulatory elements such as promoters, polyA, mature peptides, signal peptides, and all other sequence element types found within the gene or RNA sequence Figure .The results page also has several informational tabs which provide additional data on the gene or RNA of interest. The "Summary" tab provides general information on the sequence record such as gene/RNA name and description, organism name, GenBank Accession/ID and links to the original GenBank record. The "Sequences" tab, provides options to download sequences of gene, RNA, coding sequence (CDS), protein and other sequence annotations . If a gene has an exon-intron structure, then the splice signal sequences of each of its splice junctions can be viewed by clicking on the "Splice Sites" tab. Database cross-references to five independent external databases  Emboss open reading frame detection tool: Getorf; (5) VecScreen [RoBuST supports the diverse sequence analysis requirements of Alliaceae/Apiaceae research community by integrating the following sequence analysis tools to its user-friendly web interface. (1) Customized access to plant family-specific and species-specific BLAST  searchesecScreen  for deteecScreen  and SpidecScreen  for exonA comprehensive collection of annotated plant splice signals can be a valuable tool for analyzing splicing mechanisms in plants. For this purpose, we have created the Plant Splice Repository (PSR)—unique collection of plant-specific splicing data associated with 6015 plants species belonging to 443 plant families extracted from GenBank plant sequence records. PSR currently contains 659,369 donor and acceptor splice signal pairs Figure  and is aPSR can be mined to retrieve splicing annotations associated with an entire plant family, a specific plant species or even a specific gene of interest Figure . For anySpliceBlast-PL yl cysteine sulfoxide and allium flavor chemistry pathways of Allium sativum  functional annotations for 43 Alliaceae and 76 Apiaceae genes can be accessed and analyzed in the GO annotation browser in RoBuST Figure .For traits, we used the well-established Trait Ontology (TO) collection for defining carrot and garlic trait terms. Each carrot trait which matched existing TO trait terms was mapped to their specific TO ID and TO annotation. Since several carrot and garlic trait definitions were not available in TO, we submitted these new trait definitions to Trait Ontology. This resulted in the addition of 17 new trait terms to the TO collection Table .Trait and functional annotations can be queried through the ontology browser using general keyword searches or by specific searches using ontology terms or accession IDs. Users can view complete information for each ontology term as well as parent and child terms and their relationships Figure .We plan to expand the current data coverage in RoBuST in the future, and develop it into a one-stop resource for the root and bulb research community by providing comprehensive coverage of genomic data for all available root and bulb plant species. Since comparative analysis of genomic data from tuber forming plants and root and bulb plants could prove useful for understanding common mechanisms of bulb and tuber formation, we plan to link genomic information on tuber-bearing species from SGN to RoBuST. We will also incorporate interactive tools that can be used by root and bulb researchers to annotate sequences, phenotypes and traits, and to submit linkage maps, markers, molecular phylogeny data, pathways and other relevant data types which are of interest to the user community. Additionally, several comparative genomics tools will be made available in the next release of RoBuST and these tools will allow user-friendly cross-species comparative analysis of sequence annotations not only within the root and bulb plant group, but also against well annotated genomes such as rice, wheat, arabidopsis, barley, grape, potato and tomato. Since inter-database comparative analysis would be very useful in plant genome analysis and knowledge discovery, we will implement link integration of RoBuST data to Gramene and Ensembl. We will also provide gateways to ExDom  a databaSince the current sequence data in RoBuST is from a single data source—GenBank, we did not have data heterogeneity issues in this version release. However, we will be faced with data heterogeneity when we expand the RoBuST content to include data from different databases like SGN, Gramene and Ensembl in the next version release of RoBuST. To address this, we will adapt DAS protocols  in RoBuSThe plant research community in agriculture often has a commodity-specific focus. Consequently, the availability of detailed information from diverse scientific disciplines has long been of interest to crop scientists. As genomic data is the common language of all biological disciplines, a focused collection of genomic data for an individual plant species or family provides a valuable tool for researchers specializing in particular crops. This fact led to the development of GrainGenes, SolGenes, etc. To this end, RoBuST has been developed for plant scientists working on root and bulb vegetable crops. The array of genomic information on these relatively poorly studied plants is not extensive but even today over 47,000 GenBank sequence records have been generated for Apiaceae and Alliaceae alone. With the recent advancements in next generation sequencing technology, an increase in genomic data for root and bulb orphan crops in public databases is imminent. Furthermore, there is little doubt that the whole genome of one or more species in Apiaceae will be sequenced in the next few years, especially since many species in this family have genomes in the size range of rice and tomato. The extraordinarily large size of Alliaceae genomes will impede their whole genome sequencing projects, but that fact notwithstanding, much more sequence information of Alliaceae will certainly be generated. With the certainty that next-generation sequencing will become a common research tool, the management of these large sequence datasets is receiving much attention by genomicists. Discussion of next-generation plant genetics is already underway  and simihttp://robust.genome.com.RoBuST is freely available to all non-commercial users at AB designed the database and web interface, integrated the data and wrote the manuscript. DS and PSW provided annotated data, defined key functionalities of the resource and helped write the manuscript. BK, VM and PP worked on development and implementation of the database and web interface. PS and PWS conceived the project, AB coordinated it. PS participated in project design and critically revised the manuscript. All authors read and approved the final manuscript.


Secondary objectives were to determine the impact of continuous control of Pcuff, and cuff shape on microaspiration of gastric contents.The primary objective of this study was to determine the efficiency of a pneumatic device in controlling cuff pressure (Pcuff using a pneumatic device (Nosten®), and 24 h of routine care of Pcuff using a manometer. Target Pcuff was 25 cmH2O. Pcuff was continuously recorded, and pepsin was quantitatively measured in all tracheal aspirates during these periods.Prospective randomized controlled study. All patients requiring intubation and mechanical ventilation ≥48 h were eligible. The first 32 patients were intubated with tapered polyurethane-cuffed, and the 32 following patients were intubated with cylindrical polyurethane-cuffed tracheal tubes. Patients randomly received 24 h of continuous control of Pcuff . In addition, percentage of patients with underinflation (31% vs 68%) or overinflation (53% vs 100%) of tracheal cuff, and percentage of time spent with underinflation  or overinflation  were significantly (p < 0.001) reduced during continuous control of Pcuff compared with routine care.The pneumatic device was efficient in controlling Pcuff compared with routine care, or between patients intubated with tapered compared with cylindrical polyurethane-cuffed tracheal tubes.No significant difference was found in microaspiration of gastric content between continuous control of Pcuff in critically ill patients intubated with polyurethane-cuffed tracheal tubes.The pneumatic device was efficient in controlling PNCT01351259)The Australian New Zealand Clinical Trials Registry ( Microaspiration of contaminated oropharyngeal secretions and gastric content frequently occurs in intubated critically ill patients, and plays a major role in the pathogenesis of ventilator-associated pneumonia (VAP) . Aspiratin vitro studies, and microaspiration in intubated critically ill patients  versus 0.34% [0.02-11]) between patients with high airway pressures (n = 16) and those with lower airway pressures (n = 48), respectively.cuff were significantly lower during continuous control of Pcuff compared with routine care. Further, percentage of time spent with Pcuff 20–30 cmH2O, and mean Pcuff were significantly higher during continuous control of Pcuff compared with routine care. However, no significant difference was found in the incidence of microaspiration of gastric content between continuous control of Pcuff compared with routine care or between patients intubated with tapered-cuffed tracheal tubes and those intubated with cylindrical-cuffed tracheal tubes.The percentage of patients with underinflation or overinflation of tracheal cuff, percentage of time spent with underinflation or overinflation of tracheal cuff, and coefficient of variation of Pcuff in critically-ill patients intubated with polyurethane-cuffed tracheal tubes. Previous studies found similar results in animals and humans intubated with polyvinyl chloride-cuffed tracheal tubes [cuff was relatively high (25%). This could be explained by the physicochemical characteristics of polyurethane, namely its hydrophilic aspect, resulting in condensation formation especially during continuous control of Pcuff. In our study, small condensation formation was rarely observed in the pilot balloon during routine care of Pcuff. However, important condensation formation filling the whole pilot balloon was frequently observed during continuous control of Pcuff. The fact that evaporation is probably reduced in closed circuit during continuous control of Pcuff might explain this difference in condensation formation between routine care and continuous control of Pcuff. Further studies directly comparing efficiency of the pneumatic device in polyurethane and polyvinyl chloride-cuffed tracheal tubes are needed to confirm our results. Although previous studies have demonstrated the efficiency of the pneumatic device in controlling Pcuff in patients intubated with PVC-cuffed tracheal tubes, the current study allow the generalization of this observation to patients intubated with polyurethane-cuffed tracheal tubes. Given the growing evidence supporting the use of polyurethane-cuffed tracheal tubes to prevent microaspiration and VAP, our results are necessary before performing further studies aiming to evaluate the combined beneficial effects of using polyurethane-cuffed tracheal tubes and continuous control of Pcuff in preventing VAP.To our knowledge, our study is the first to demonstrate the efficiency of the pneumatic device in controlling Pal tubes ,21. The cuff, no significant difference was found in microaspiration of gastric content between continuous control of Pcuff and routine care. One potential explanation for this result is the optimal routine care provided during the study, and the use of polyurethane-cuffed tracheal tubes. These tubes have been demonstrated to significantly reduce microaspiration in critically ill patients [cuff on the incidence of microaspiration, and VAP. However, whilst our study was underpowered to detect a difference in microaspiration, it was correctly powered to draw a valuable conclusion on primary outcome, i.e. the efficiency of the pneumatic device in controlling Pcuff. In fact, the number of patients required to detect the estimated difference in percentage of patients with underinflation or overinflation of Pcuff, with an alpha risk of 5% and a power of 80%, was 32 per group. As 32 patients were included in each study group, our study is sufficiently powered to conclude that the pneumatic device is efficient in controlling Pcuff. This result would allow future studies to evaluate the impact of the pneumatic device on the prevention of complications related to underinflation and overinflation of Pcuff. Although our previous study has already suggested beneficial effects of continuous control of Pcuff in reducing microaspiration and VAP [cuff on prevention of cuff-related complications such as microaspiration, and VAP.In spite of efficient control of Ppatients ,6. Furth and VAP , it was cuff a trend towards lower pepsin level and percentage of tracheal aspirates positive for pepsin was found in patients intubated with tapered-cuffed tracheal tubes compared with those intubated with cylindrical-cuffed tracheal tubes. Further, in spite of significantly higher percentage of time spent with underinflation of tracheal cuff in patients intubated with tapered-cuffed tracheal tubes compared with those intubated with cylindrical-cuffed tracheal tubes during routine care, microaspiration of gastric content rate was similar in the two groups. This result also suggests better sealing with tapered compared with cylindrical tracheal cuffs. However, our study was probably underpowered to detect a significant difference in microaspiration rate between the two groups.Some significant differences, including percentage of time with underinflation and overinflation were found between patients intubated with tapered-cuffed tracheal tubes compared with those intubated with cylindrical-cuffed tracheal tubes. These differences could be explained by different cuff shape between the two groups. No significant effect was found of tracheal cuff shape on microaspiration of gastric content. However, during continuous control of Pin vitro studies found tapered-shaped tube cuff to considerably improve sealing characteristics of polyvinyl chloride tube cuffs [in vitro study reported that tapered polyurethane cuff was more efficient than cylindrical polyurethane cuff in larger tracheal diameter in preventing fluid leakage [Two recent be cuffs ,13. Howe leakage . Differe leakage . Furthercuff was tightly correlated to airway pressure [cuff during continuous control of Pcuff was not significantly different between patients with high airway pressures and those with lower airway pressures, suggesting that the pneumatic device is efficient in patients with high airway pressures, such as those with severe asthma or ARDS. However, no definite conclusion could be drawn in this subgroup because of the small number of patients (n = 16).No significant difference was found in airway pressures between patients intubated with tapered compared with those intubated with cylindrical tracheal cuffs. Previous studies demonstrated that Ppressure ,23. The pressure ,24,25. Tcuff on outcomes such as VAP or tracheal injury. However, in order to adjust for patient-related confounders, such as tracheal size and aspect, airway pressures, and tracheal tube size, continuous control and routine care of Pcuff were performed in the same patient. Therefore, it was not possible to evaluate these outcomes. Further, it was mandatory to evaluate the efficiency of the device in controlling Pcuff before performing studies on its impact on prevention of complications. Finally, randomization for tracheal cuff shape was performed per period and not per patient. However, intubation is often an urgent procedure. Therefore, it is very difficult to perform randomization per patient in such a study. In addition, the impact of cuff shape on microaspiration was a secondary outcome.Our study has some limitations. First, this study was performed in a single ICU. Therefore, our results could not be generalized to other ICU patients. Second, our study did not evaluate the impact of continuous control of Pcuff in critically ill patients intubated with polyurethane-cuffed tracheal tube. Further studies are needed to determine the impact of continuous control of Pcuff and tracheal cuff shape on microaspiration, VAP, and tracheal injury.We conclude that the pneumatic device is efficient in controlling Pcuff in critically ill patients intubated with polyurethane-cuffed tracheal tube.• The pneumatic device is efficient in controlling P• Conical cuff shape could be beneficial in preventing microaspiration in critically ill patients.cuff and tracheal cuff shape on microaspiration, and VAP in patients intubated with polyurethane-cuffed tracheal tubes.• Further studies are needed to determine the impact of continuous control of PICU: Intensive care unit; Pcuff: Cuff pressure; VAP: Ventilator-associated pneumonia.SN: Covidien (advisory board), other authors: none.EJ, ADur, and SN designed this study. EJ, FZ, JD, MB, and SN collected the data. ADuh performed statistical analyses. EJ, and SN wrote the manuscript, and all authors participated in its critical revision. EJ, and SN had full access to all data in the study and had final responsibility for the decision to submit for publication. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/13/50/prepub


A continuous peripheral nerve block (cPNB) is provided in the hospital and ambulatory setting. The most common use of CPNBs is in the peri- and postoperative period but different indications have been described like the treatment of chronic pain such as cancer-induced pain, complex regional pain syndrome or phantom limb pain. The documented benefits strongly depend on the analgesia quality and include decreasing baseline/dynamic pain, reducing additional analgesic requirements, decrease of postoperative joint inflammation and inflammatory markers, sleep disturbances and opioid-related side effects, increase of patient satisfaction and ambulation/functioning improvement, an accelerated resumption of passive joint range-of-motion, reducing time until discharge readiness, decrease in blood loss/blood transfusions, potential reduction of the incidence of postsurgical chronic pain and reduction of costs. Evidence deriving from randomized controlled trials suggests that in some situations there are also prolonged benefits of regional anesthesia after catheter removal in addition to the immediate postoperative effects. Unfortunately, there are only few data demonstrating benefits after catheter removal and the evidence of medium- or long-term improvements in health-related quality of life measures is still lacking. This review will give an overview of the advantages and adverse effects of cPNBs. Since its first description in 1946, cPNB has evolved from a case report of a needle inserted through a cork taped to a patient's chest to a wide-spread and validated analgesic technique in the postoperative setting . The indDespite this evidence of value, the hypothesis that regional anesthesia has an overall beneficial and long-lasting effect on patient outcome following surgery still remains difficult to prove and has been challenged, especially in times with reduced resources. For more than 30 years, regional anesthesia has challenged anesthesiologists to determine whether it offers real benefits over other types of anesthesia, such as preserving cognitive function after major surgery compared to general anesthesia, improving long-term joint function and rehabilitation leading to earlier return to work, reducing costs, reducing the need for blood transfusions and increasing patient-reported outcomes such as satisfaction, quality of life, and quality of recovery.The implications of regional anesthesia after major surgery are multifaceted and not fully elucidated, thus requiring future studies with a focus on evaluating issues like implicating roles of mismanaged pain, environmental factors, stress responses associated with the perioperative period, long-term followups focusing on joint mobilisation and quality of life. This paper will elucidate the benefits of cPNB over intravenous analgesia and single-shot perineural blocks (sPNB) according to the published literature and show some aspects for possible future directions.In western countries, 50–70% of in hospital patients suffer from moderate-to-severe pain after surgery , and 40%Sterility is of utmost importance for the performance of cPNB independently of the technique performed (neurostimulation or ultrasound). The American Society of Regional Anesthesia and Pain Medicine (ASRA) recommends antiseptic hand washing, sterile gloves, surgical mask and hats, and the use of alcohol-based chlorhexidine antiseptic solutions . FurtherShort bevel needles are widely accepted as standard practice in regional anesthesia. The literature is not conclusive to support the notion that nerve injury is minimized using short bevel needles –44. The  use a proper technique with logical  and reproducible landmarks –59; accept only responses with previously described high success rate  , 57, 63; inject only at following nerve stimulator setting (with still weak motor response visible): amplitude width: 0.1 ms, frequency: 2 Hz, and impulse: 0.3–0.4 mA; use the correct block for the planned surgery  and ultrasound guidance for catheter placement is controversially discussed in the literature, and often the difficulties are major compared to the single use of either technique , 140–142In certain situations ultrasound might be, at least theoretically, superior to nerve stimulation for example when sensory nerves are the target , after lThe most commonly used local anesthetics for cPNB are bupivacaine and ropivacaine, and both seem to provide adequate analgesia without major toxicity. However, ropivacaine is known to be more “motor sparing”  and lessTo prolong the effect of local anesthetics, the addition of different drugs to local anesthetics has been suggested for sPNB and cPNB like opioids , 166, epPreliminary evidence does not suggest a rigid local anesthetic delivery system for all cPNB. According to the literature, total local anesthetic dose and not its concentration or its delivery rate mainly influences clinical effects, although this issue is currently disputed in the literature , 171–176Reducing the basal rate might reduce the risk of toxicity and the reduction of motor and sensory block could prevent the risk for patient falls . Until prospectively collected data are available, possible dose recommendations based on randomized controlled studies and clinical experience are given in For the outpatient setting, many types of pumps are on the market: reusable and disposable, nonelectronic and electronic, basal-only and basal-bolus capable , 182. NoDifferent case reports suggest beneficial effects of cPNB for different indications , 187, buAlthough single-injection nerve blocks (sPNB) also provide excellent analgesia, cPNB increases the flexibility of both duration and density of local anesthetic effect depending on the chosen dose. SPNB offers a good pain therapy for up to 24 hours but for this duration a dense motor block and important sensory loss must be taken into account. In situations like trauma surgery, where compartment syndrome might be masked by these dense and long-lasting blocks, these properties are not wished by the surgeon. However, there is not enough evidence to support the thesis that patient-controlled analgesia with opioids or regional analgesia delay the diagnosis of compartment syndrome provided patients are adequately monitored , 189. CoMost of cPNB benefits are mainly dependent on successfully improving pain control, reducing opioid consumption and its related side effects and increasing patient satisfaction . Like foFor shoulder and upper arm surgery, the interscalene nerve block (ISNB) has become a standard procedure in specialized centers . This teThe benefits for elbow surgery using continuous infraclavicular perineural block is well validated , but gooFemoral or posterior lumbar plexus infusions are well validated for hip , 207 andAlso if lumbar epidural provides generally equivalent analgesia to femoral perineural infusion for both, hip and knee arthroplasty cPNB offers a more favorable side effect profile avoiding the risk of epidural hematoma during anticoagulant administration , 207. ThFor foot and ankle surgery, the use of cPNB has clearly demonstrated its superiority to other analgesia regimens. It has been shown to decrease hospital costs and length of stay . SeveralThe effects of the short-term use of cPNB on functional outcome after orthopedic surgery are still controversially discussed. Ilfeld et al. reported in a retrospective study that in the 3 days after a total shoulder arthroplasty, a continuous interscalene block using ropivacaine 0.2% was associated with increased range of shoulder motion due to the complete analgesia provided . These rDespite the overwhelming evidence highlighting cPNB benefits during local anesthetic infusion, there exist few data demonstrating benefits after catheter removal. These data include improved analgesia a few days after removal , 216 andThese results are in accordance with the review by Liu and Wu analyzing the effect of analgesic technique on postoperative patient-reported outcomes . AuthorsTherefore, more studies focusing on the medium- or long-term improvements in health-related quality-of-life measures are needed , 237. Postoperative cognitive dysfunction (POCD) is an important issue in an aging society. Postoperative cognitive dysfunction is a poorly defined condition as numerous studies use different definitions and neuropsychological tests to detect potentially altered short- or longer-term memory, motor control or information processing following anesthesia. There is evidence that altered intraoperative physiology may influence POCD and longer-term outcomes. A meta-analysis of 21 studies on POCD and postoperative delirium (POD) found no effect of anaesthesia type on the odds ratio of developing POD  . HoweverApart from the classical postoperative setting, cPNB has been successfully introduced in the ambulatory surgery setting for both adults , 191 andContinuous peripheral nerve blocks offer the opportunity to deliver effective pain therapy at home and are considered to be a valid alternative to opioid-based analgesia and its related side effects . HoweverAdditionally, patients with known hepatic or renal insufficiency are excluded from ambulatory cPNB to avoid possible local anesthetic toxicity . Obese pThe advantages of sPNB over general anesthesia related to cost-effectiveness in the operation theatre have been recently described by Gonano et al. for shoulder arthroscopy . AuthorsUnfortunately, there are no studies comparing sPNB and cPNB regarding cost-effectiveness. However, the duration of hospitalization  or even The impact of cPNBs on analgesia has been described in many RCTs and has been elucidated above . The relAmbulatory shoulder arthroplasty and 23-hour-stay knee and hip arthroplasty using ambulatory continuous interscalene, femoral, and psoas compartment nerve blocks, respectively have been reported , 250. However, although postknee arthroplasty inflammation is reduced after a continuous femoral nerve block . the cPNTwo prospective investigations involving more than 2.100 patients suggest that the incidence of cPNB-related complications is very low and comparable to sPNB techniques , 252. HoThese complications are frequently seen in clinical practice and include catheter obstruction, catheter dislodgement, fluid leakage, disconnection from the pump system, infusion pump malfunction, and skin irritation or allergic reactions to the sterile catheter dressing , 256.These seldom but possible complications include inaccurate catheter tip placement resulting too far from the target nerve and therefore lacking successful analgesia,  or in anMost studies reporting infection in relation with cPNB derive from hospitalized patients even if data for large outpatient series are increasing. Although the reported rates of inflammation (3%-4%) , 253 andRisk factors for infection in the setting of cPNB include lack of or inappropriate antibiotic prophylaxis, and axillary or femoral catheter location . HoweverAlthough life-threatening catheter-related infections and sepsis have been reported in the literature , 272, noSterility precautions while filling the infusion pumps are of utmost importance. In a recent case report of a severe, deep cellulitis, evolving to mediastinitis was attributed to contaminated infusate from a pump which was manipulated in nonsterile conditions . TherefoAlthough neurologic injury associated with cPNB is usually transient ranging from 0.3% to 2.0% , it remaThere are also cases of long-term or permanent nerve injury in patients after perineural infusion . In fiveOnly few reports of nerve injury in patients sent home with cPNB are described in the current literature. However, pressure injury due to insensate extremity has been implicated as a likely cause . TherefoA further important neurologic concern associated with in- and out-patient cPNB is the risk of falls. Williams et al. reported recently 1.7% of outpatients treated with continuous femoral nerve block fell due to insensate lower extremity despite adequate instructions how to behave at home . Ilfeld This complication elucidates the potential benefit in providing low-concentration blocks that preserve more motor function and proprioception. Moreover, the desire to offer complete analgesia should be balanced against the risk of falling. Therefore, the recently published study dealing with multimodal analgesic approaches including regional anesthesia can offer a solution to achieve excellent analgesia while preserving motor function .Most investigators report basal hourly infusion rates of 5 to 10 mL/h using dilute solutions of either ropivacaine or bupivacaine , 34. Desv1.7is the main channel for pain transmission in the peripheral nerves [v1.7 local anesthetic would increase the ambulant use of cPNB with probably a remarkable impact in health costs.Local anesthetics act on the voltage-gated sodium channels . The Navl nerves , 287. SeLiposomes as reservoirs for drugs are biocompatible due to their biodegradability and low toxicity . They caThe Literature provides a plethora of information involving cPNB, but some aspects of perineural infusion remain controversial: the optimal catheter insertion modality, the optimal technique for each indication, the infusates offering the best safety, standardized local anesthetic delivery regimens, and optimization of continuous ambulatory infusion to reduce possible risks . The optimal analgesic technique for many surgical procedures has to be further elucidated, and cPNB must be compared with possible new analgesic techniques/regimens . The new application areas for local anesthetic such as anti-inflammatory effects/anticancer effects , 295 havThe socioeconomical aspect of anesthesia remains an important issue, and the role of cPNB for in hospital and at home use and their effects in the healing process and the readmission of the patients at work after surgery must be clarified. Prospective research addressing the above-mentioned issues can maximize the potential benefits and minimize the potential risks of cPNBs.


Olfactory bulb (OB) periglomerular (PG) cells are heterogeneous with respect to several features, including morphology, connectivity, patterns of protein expression, and electrophysiological properties. However, these features rarely correlate with one another, suggesting that the differentiating properties of PG cells may arise from multiple independent adaptive variables rather than representing discrete cell classes. We use computational modeling to assess this hypothesis with respect to electrophysiological properties. Specifically, we show that the heterogeneous electrophysiological properties demonstrated in PG cell recordings can be explained solely by differences in the relative expression levels of ion channel species in the cell, without recourse to modifying channel kinetic properties themselves. This PG cell model can therefore be used as the basis for diverse cellular and network-level analyses of OB computations. Moreover, this simple basis for heterogeneity contributes to an emerging hypothesis that glomerular-layer interneurons may be better described as a single population expressing distributions of partially independent, potentially plastic properties, rather than as a set of discrete cell classes. Olfactory bulb (OB) periglomerular (PG) cells are heterogeneous with respect to several features, including morphology, synaptic connectivity, patterns of protein expression, transmitter identity, transcription factor expression, and electrophysiological properties  kinetics derived from PG cell recordings  and delayed rectifier potassium current (IK) ] ] , a calcium-dependent potassium current [IK(Ca)] , Vm is the membrane potential within that compartment, Ex is the net reversal potential of the permeant ion(s), and Φ is the temperature coefficient (Table ICAN and IK(Ca) currents also were functions of the internal perimembrane calcium concentration, which was governed by calcium influx and by the constitutive diffusion rate of calcium out of the 0.1 um deep perimembrane region.Membrane current kinetics were implemented in NMODL   electrophysiological properties depicted in Figures 1A–D of McQuiston and Katz . The fi was neve2 ohmic cation conductance expressed in the single dendritic spine—changed accommodating and single-spiking neurons to instead produce non-accommodating spike train responses. In a network context, nicotinic neuromodulation of PG cells enhances their feed-forward inhibition of mitral cells, contributing to the sharpening of odor representations within OB  current itself, with a short post-LTS hyperpolarization punctuated by a modest deinactivation of the A-type potassium current. A roughly similar dynamical pattern of currents underlaid the LTS responses that included a decrementing burst of spikes, although the absolute dynamical levels of these conductances were reduced by roughly a factor of ten . The large cation conductance generated and maintained the plateau, declining along with the gradual decline in perimembrane calcium concentration until it could no longer be sustained. The calcium-dependent potassium conductance was primarily responsible for the termination of the burst of spikes following depolarizing input, but otherwise also followed the declining perimembrane calcium concentration. Unlike the much more common LTS responses, the role of the calcium T current in these persistent plateau potentials was negligible. Due to the absence of constraining pharmacological data regarding this response profile, it was not investigated further in the model.In contrast to the simple and LTS-based responses described above, the persistent plateau potential responses replicated in Figures The ionic basis of LTS responses in PG cells was experimentally determined using pharmacological manipulations. Specifically, blockade of sodium influx by choline replacement of external sodium or by intracellular infusion of the fast Na channel antagonist QX314 did not eliminate the LTS from PG cells  models are capable of replicating the global response patterns of complex neurons, even when single-compartment models fall short , extending dendrites to either contact the axons of primary olfactory sensory neurons  or not , marker expression , and presumptive neurotransmitter  (Kosaka and Kosaka, If this single heterogeneous interneuron population hypothesis is correct, then the fundamental experimental question shifts from “what are each of these cell types for?” to “what factors cause or require the expression of particular markers or properties within neurons of this [single] population?” That is, the observed pattern of mixed feature correlations suggests that PG cell heterogeneity may arise from multiple distinct and interacting processes rather than representing a discrete hierarchy of defined cell classes, and that certain nominal class markers may signal relatively transient neuronal states rather than durable identities. For example, while the calcium binding protein markers expressed heterogeneously among PG neurons have not typically been associated with particular functions, recent work has implicated calbindin-D28k, particularly, in some forms of synaptic plasticity Schmidt, . ConsequThe 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.