Hirundo rustica) by subjecting nestlings to an immune challenge  that simulates a bacterial infection, and assessed the effects on growth, feather quality, expression of morphological (gape coloration) and behavioral (posture) begging displays involved in parent-offspring communication, as well as on food allocation by parents. Compared to sham-injected controls, LPS-treated chicks suffered a depression of body mass and a reduction of palate color saturation. In addition, LPS treatment resulted in lower feather quality, with an increase in the occurrence of fault bars on wing feathers. The color of beak flanges, feather growth and the intensity of postural begging were affected by LPS treatment only in females, suggesting that chicks of either sex are differently susceptible to the immune challenge. However, irrespective of the effects of LPS, parents equally allocated food among control and challenged offspring both under normal food provisioning and after a short period of food deprivation of the chicks. These results indicate that bacterial infection and the associated immune response entail different costs to offspring of either sex, but a decrease in nestling conditions does not affect parental care allocation, possibly because the barn swallow adopts a brood-survival strategy. Finally, we showed that physiological stress induced by pathogens impairs plumage quality, a previously neglected major negative impact of bacterial infection which could severely affect fitness, particularly among long-distance migratory birds.Parent-offspring conflicts lead the offspring to evolve reliable signals of individual quality, including parasite burden, which may allow parents to adaptively modulate investment in the progeny. Sex-related variation in offspring reproductive value, however, may entail differential investment in sons and daughters. Here, we experimentally manipulated offspring condition in the barn swallow ( Theoretical models of conflicts among family members posit that offspring are selected to obtain a larger share of parental resources than their siblings and to attract more care than would be optimal for parents to provide In altricial species from diverse taxa, offspring are entirely dependent on food provided by parents and solicit care by using morphological and behavioral ‘begging’ displays Given the abundance of parasites in natural environments, one important source of variation in offspring quality is parasite infection Another crucial source of variation in offspring reproductive value is sex, as males and females may differ in susceptibility to environmental and rearing conditions Hirundo rustica) nestlings was to evaluate whether an immune challenge, simulating an infection by a bacterial pathogen, affected body mass and feather growth, as well as the expression of morphological (gape coloration) and behavioral  begging traits involved in parent-offspring communication. In addition, we evaluated variation in parental allocation strategies towards offspring differing in condition, as affected by the immune challenge, as well as by contingent need of food, as experimentally altered by a short-term food deprivation, because parental decisions and begging behavior are also expected to vary in relation to both general condition and current hunger state of the offspring  (LPS chicks), an endotoxin extracted from the outer membrane of Gram-negative bacteria, and the other half with a saline control solution (control chicks). LPS is commonly used to elicit an immune response in the absence of a living pathogen and causes several hormonal and behavioral alterations (the ‘sickness behavior syndrome’) in birds e.g. .We predicted that LPS depressed body mass and resulted in lower plumage quality Finally, two days after injection, we experimentally tested the effects of the immune challenge on success in sib-sib competition between LPS and control nestlings. We measured the intensity of postural begging behavior, reflecting the degree of offspring need, and the change in body mass during feeding trials. Moreover, we counted the number of feedings provided by parents to each chick, in tests where pairs (‘dyads’) of same-sex and opposite-treatment brood-mates were set to compete for parental feedings The barn swallow is a small (ca. 20 g), insectivorous migratory passerine with biparental care of the offspring. Females lay 1–3 clutches of 1–7 eggs  per breeding season st, nests were visited daily to record breeding events. At day 7 (day 0 = hatching of the first egg in a nest) we ringed all the chicks from broods with three or more nestlings and collected a blood sample (ca. 80 µl) for molecular sexing by PCR amplification of the sex-specific avian CHD-1 gene following the protocol originally devised by Griffiths et al. The present study was carried out between April and July 2010 at two colonies  located near Milan . Starting from April 1Escherichia coli (055:B5 - L2880 Sigma-Aldrich) , when chicks have attained final body size and before the onset of pre-fledging mass recession ch) e.g. . Injectines e.g. . The remBefore LPS injection, we measured body mass to the nearest 0.1 g by an electronic balance and tarsus length with a digital caliper (to the nearest 0.01 mm). The length of all primary wing feathers (hereafter ‘primary feathers’) was also measured using a ruler (to the nearest 0.5 mm). As a proxy of wing length, we used the length of the third outermost right primary feather (‘feather length’ hereafter) Finally, on day 17 we counted the fault bars on both wings in a subsample of 75 LPS and 73 control chicks from 35 broods. To determine which fault bars were developed after LPS treatment, we measured the length of all the feathers where fault bars were found as well the distance between each fault bar and the tip of these feathers. Since we already knew the length of all feathers before the injection of LPS or PBS, this procedure allowed us to determine the number of fault bars appearing after the immune challenge.2 and every reading was obtained from an average of 15 scans. Each nestling was measured twice in two regions of the gape, corresponding to the left flange and the palate. The standard white was recalibrated before starting measurements of any next brood and the white periodically checked to verify 100% reflectance. Reflectance measures were repeated 2 and 3 days after the injection of LPS.On day 12 (before LPS injection), we recorded mouth coloration of 149 chicks (75 LPS and 74 control) belonging to 35 broods with a spectrometer powered by a deuterium-tungsten halogen light source (Avantes AvaSpec 2048). The reflectance (%) of the mouth was measured relative to a standard white tablet (WS-2). To prevent interference by stray light, the reflection probe was positioned inside a matte black plastic tube, cut at 45° in order to avoid specular reflection when the probe was applied to the mouth. The illuminated field was about 7 mmad hoc implemented macros for Microsoft Office Excel 2003. Brightness corresponds to the total reflectance of a given surface, chroma represents the spectral purity (saturation), while hue is the spectral location, representing the position of a spectrum in the color wheel, progressing from red to UV-A. Repeatability of the two reflectance measures of each gape region was high, with intraclass correlation coefficient 1,147 = 4.152; P<0.0001) and 0.893 . Brightness, chroma and hue were thus averaged between spectra before analyses.Color analyses were restricted to the 320–700 nm spectral window, corresponding to the typical visual range of passerines see . SpectraTo test for a difference in competitive ability between LPS and control chicks, we compared the intensity of postural begging, body mass gain and number of feedings received from parents during feeding trials within pairs of same-sex and opposite-treatment siblings  two days after experimental treatment. Pairs of same-sex chicks (dyads hereafter) were randomly chosen within each brood. The test was performed both before and after a period of food deprivation in order to analyze the behavior of nestlings and parents under different hunger conditions  a.m. (±30 min). At the end of the 1.5 h feeding trial, the focal nestlings were weighed again in order to record body mass gain, indicating individual food intake First, the two focal nestlings were weighed, individually marked on their head with two white spots, and left in the nest for a feeding trial while temporarily removing the other chicks, that were kept in a safe and warm place. All feeding visits of the parents were videotaped with a Sony DCR-SR72E camera, placed 2–3 m from the nest in a frontal position. Recordings started in the morning at 7.30 The number of feedings obtained by each nestling of the dyad were counted on video recordings using VLC Media Player 1.1.4 . The use of the number of feedings provided by parents to each chick during trials was intended to assess the ability of the nestlings in sib-sib competition as number of interactions won against their competitors. Moreover, because feeding rates do not account for variation in size of individual feedings, we also used body mass change during each trial as a proxy for food intake and the balance between benefits and costs of scrambling.Furthermore, three feeding events were randomly chosen to estimate the intensity of postural begging, which was scored on a four-levels scale ranging between 0 (the nestling did not beg) and 3 (the nestling begged by moving the open wide mouth with fully stretched neck and tarsi) The whole protocol was performed for 45 dyads  belonging to 39 different broods.The effects of immune challenge on nestling traits  were analysed using linear mixed models, which included as predictors two dichotomous fixed factors for treatment (LPS or control) and sex, their interaction, and the value of the trait of interest before LPS injection as a covariate. Nest identity was included in the models as a random intercept effect. In all analyses, we also included age at LPS injection as a covariate to account for small variation in age at measurement. We first run separate analyses for trait values recorded 2 and 3 days after LPS injection, and then analysed traits recorded in both days in the same model, while including an additional fixed factor (day of measurement), identifying the data collected in either day.The presence and number of fault bars on the wings after LPS injection were analysed in mixed models assuming a binomial and a Poisson error distribution, respectively. The presence and number of fault bars before LPS injection were also included as predictors in the relevant models to account for individual variation in the number of fault bars at the beginning of the test. Nest identity was included as a random factor in the models.The analyses of postural begging intensity, number of feedings and body mass gain during feeding trials were carried out using repeated-measures linear mixed models where nestlings were modeled as subjects. Food deprivation (before or after) was included as a dichotomous factor identifying the repeated measurements of each subject. Sex, treatment (LPS or control) and the interaction between treatment and food deprivation were included as fixed factors. Nest and dyad were included as random intercepts, and the effect of food deprivation was allowed to vary randomly between chicks (random slope model) lmer procedure of the lme4 package All analyses were run in R 2.8.1 (R Development Core Team 2008) with the When removed from their nest, nestlings were kept in a safe and warm place. At each measurement session each chick was handled only for few minutes and nests were never left without at least one nestling inside to avoid parental desertion. Blood samples were collected by slightly puncturing the brachial vein and the puncturing site was carefully disinfected. Injections of LPS and PBS were performed just below the pectoral muscle threading the tiny needle (30 G) approximately 3–4 mm inside the abdominal cavity and taking care to avoid damaging inner organs (which are easily visible through the skin at this age). No obvious negative consequences of handling nestlings were detected. Nestling mortality until fledging was very low (2 out of 102 LPS chicks and 2 out of 100 control nestlings), and unaffected by experimental treatment. During videotaping, we could not note any obvious effect derived from the presence of recording equipment on both parental and offspring behavior. The study was approved by the Ethical Committee of the Department of Biology, University of Milan, Italy.21≤2.339; P≥0.126).Before LPS treatment, no significant differences in body mass, tarsus or feather length were found between LPS and control nestlings , while the effect of treatment on feather length was non-significant . All the two- and three-way interaction terms between day of measurement, sex and treatment were not statistically significant , implying that the effects of immune challenge did not differ statistically between day 2 and 3 post-injection.Analyses run by including data from both day 2 and 3 post-injection in the same model confirmed the statistically significant effect of immune challenge on body mass . Conversely, post-injection, both the proportion of individuals with fault bars on wing feathers and the number of fault bars differed between treatments : fault b21≤2.456; P≥0.117 for all color variables).Before LPS treatment, no significant differences in brightness, chroma and hue of the palate and the flanges were found between LPS and control nestlings .On day 2 after treatment, chroma was affected by LPS. In the palate, chroma was significantly smaller in LPS than in control nestlings  or sex  were found on any color variable  as well as of the interaction between sex and LPS treatment on flange chroma . These models also confirmed the sex-related variation in the flange chroma  (see above). In addition, the other two- and three-way interaction terms between day of measurement, sex and/or LPS were not statistically significant , implying that the effects of LPS treatment were not significantly different between day 2 and 3.Analyses including data from both day 2 and 3 after LPS injection  confirmed the significant main effect of the immune challenge on palate chroma .In this experiment on barn swallow nestlings, we subjected chicks to an immune challenge with LPS, and assessed its effects on growth, feather quality and parent-offspring communication. Infection by Gram-negative bacteria can elicit an immune response and cause an ‘acute phase response’, entailing physiological and behavioral alterations Loss of body mass and reduced feather development and quality  following LPS challenge may have been caused by a smaller food intake. In passerine birds the acute phase response is associated with an increase in resting and somnolence as well as a reduction of behaviors associated with motility, like scrambling for food and sib-sib competitive interactions (the ‘sickness behavior syndrome’) A decrease in body mass may also have been caused by a reduction in parental feeding soon after the immune challenge, either because LPS chicks may appear of reduced reproductive value to parents, or because their nest-mates prevailed in sib-sib interactions for access to food. Although we admittedly could not discriminate between these interpretations, we favor the idea that reduced access to food was mainly due to reduced motility. This is the case because previous studies on barn swallows suggested that both parents and older/larger chicks seem to enhance access to food by smaller nestlings, as expected in a species adopting a brood survival strategy Our findings also highlighted a possible trade-off between growth and immunity Passer domesticus) A novel finding is that the immune challenge increased the occurrence of fault bars, that are commonly considered as evidence of low feather quality We found that saturation of gape color, which is a main component of offspring begging signals, was depressed by LPS. The coloration of the soft tissues of the gape is partly dependent on the presence of dietary carotenoids Parus major) experimentally infested by ectoparasites, with infested broods increasing their begging rate Female, but not male, LPS nestlings showed significantly higher postural begging scores than control nestlings, irrespective of food deprivation. These intense begging solicitations of LPS females were probably caused by their impaired condition, as reflected by negative effects of LPS on other traits. Similar results were found in a previous study of begging in nestling great tits  on hue, chroma and brightness of palate and flanges of barn swallows nestlings at both day 2 and 3 after LPS injection.(PDF)Click here for additional data file.


Up to now seven different subtypes have been reported (PCH1-7). The incidence of each subtype is unknown. All subtypes share common characteristics, including hypoplasia/atrophy of cerebellum and pons, progressive microcephaly, and variable cerebral involvement. Patients have severe cognitive and motor handicaps and seizures are often reported. Treatment is only symptomatic and prognosis is poor, as most patients die during infancy or childhood. The genetic basis of different subtypes has been elucidated, which makes prenatal testing possible in families with mutations. Mutations in three tRNA splicing endonuclease subunit genes were found to be responsible for PCH2, PCH4 and PCH5. Mutations in the nuclear encoded mitochondrial arginyl- tRNA synthetase gene underlie PCH6. The tRNA splicing endonuclease, the mitochondrial arginyl- tRNA synthetase and the vaccinia related kinase1 are mutated in the minority of PCH1 cases. These genes are involved in essential processes in protein synthesis in general and tRNA processing in particular. In this review we describe the neuroradiological, neuropathological, clinical and genetic features of the different PCH subtypes and we report on  Mi. Mi20]. Prenatal diagnosis by ultrasound imaging is not yet possible in the second trimester of pregnancy, therefore molecular genetic testing is required for prenatal diagnosis in high risk pregnancies .PCH type 3  also known as cerebellar atrophy with progressive microcephaly (CLAM), is a very rare subtype of PCH Table . Only thPCH type 4 , ORPHA166063, MIM 225753) shares similarities with PCH2; however PCH4 is more rare and the disease course is more severe Table  20,38].,38.20,38Only one family with PCH type 5  has been described  ..54].Certain subtypes of the congenital disorders of glycosylation (CDG) disorders manifest with cerebellar hypoplasia as well. CDG-1a patients have generalized hypotonia, developmental delay, swallowing problems, failure to thrive and cerebellar hypoplasia, next to variable external dysmorphia and hematologic problems . CerebelPhosphoserine aminotransferase deficiency is associated with low CSF concentrations of serine and glycine. Clinically patients exhibit seizures, progressive microcephaly, hypertonia and psychomotor retardation. MRI shows cerebral atrophy, poor white matter development and vermal hypoplasia. The authors did not mention the size and morphology of the cerebellar hemispheres Table  59]. Al. Al59]. CASK). It can manifests with microcephaly, optic atrophy, sensorineural hearing loss and pontocerebellar hypoplasia ia Table  67-69].-69.CASK)Extreme prematurity < 32 weeks) in infants can also lead to cerebellar hypoplasia, due to disruption of normal brain development. One should be aware of this when a prematurely born neonate presents with these features  and consists of four protein subunits. Together they form one heterotetramic enzyme, consisting of two catalytic subunits (TSEN2 and TSEN34) and two structural subunits (TSEN54 and TSEN15) . RA. RA83]. unstable . Morpholrebellum .Alternative functions of RARS2 compromising the same biological pathway as the TSEN complex could also be the underlying mechanism in PCH ,78,81. FSEPSECS are associated with PCCA . The SEPSECS enzyme  completely abolish its function, as deprivation of selenocysteins is associated with lethality in mice [Mutations in e Figure  is invol in mice .AARS). This mouse has increased levels of mischarged tRNAs which lead to intracellular accumulation of misfolded proteins in neurons, this induces the unfolded protein response (UPR) and these mice develop a neurodegenerative phenotype, with Purkinje cell death and subsequent ataxia [TSEN mutation, but one can argue that UPR activation is an early event in the pathogenesis of PCH and that postmortem studies do not capture this event [The identification of mutations in genes involved in transcription and translation in neurological disorders shows that these processes are important in (developing) neurons ,89-94. Tt ataxia . Howeveris event .On the other hand it is possible that there is a time frame during embryogenesis in which there is an extra high demand for protein synthesis in neuronal tissue in the early post-migratory stage. Human brain tissue expresses relatively high overall levels of nuclear and mitochondrial encoded tRNAs, which might be due to higher levels of translation in the CNS compared to other tissue . MoreoveKARS, YARS and GARS lead to different types of Charcot-Marie-Tooth neuropathy [DARS2) mutations cause leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL). Patients with LBSL have profound white matter abnormalities in their cerebrum, pons and spinal cord. There are some similarities between LBSL and PCH, like cerebellar involvement and spasticity, but the differences are more evident. Patients have slowly progressive cerebellar ataxia, dorsal column dysfunction, occasionally a mild cognitive decline and the age of onset is usually during childhood or even later. It is unclear how mutations in DARS2 lead to LBSL [Why mutations in other genes involved in protein synthesis lead to completely different phenotypes remains unclear. For example, mutations in other genes involved in tRNA charging like uropathy ,100,101. to LBSL .SARS2) give rise to a multi-organ disease with Hyperuricemia, Pulmonary Hypertension, Renal Failure and Alkalosis (HUPRA-syndrome). Although there is failure to thrive and a global developmental delay, patients exhibit no neurological symptoms and brain ultrasound was reported to be normal [HARS2) were found in one family with ovarian dysgenesis and progressive sensorineural hearing loss (Perrault syndrome) [Not all mutations in tRNA synthetase genes result in a neurological phenotype. Missense mutations in the mitochondrial Seryl-tRNA Synthetase gene  .TSEN, RARS2 and SEPSECS, in which the cerebellum appears to be preferentially affected and other diseases , with a different disease presentation.In summary, the most likely explanation for the neurological phenotype in the disorders described above is that maturing neurons are more vulnerable for defects in protein synthesis than other tissues, but this does not explain the difference between disease presentation shared by mutations of During the last decade many genes involved in protein synthesis have been associated with different neurological diseases. Several genes of PCH subtypes have been described, in view of the shared function in tRNA processing, a defect in protein synthesis seems the most likely pathomechanism in PCH. Hopefully identification of new genes in PCH subtypes will provide further insight and lead us to a common disease pathway. Also further research on TSEN and/or RARS2 function in PCH models is necessary to elucidate the question why solely the brain, and specifically the cerebellum and pons are affected in PCH.The authors declare that they have no competing interests.All authors made substantial contributions and have given final approval to the version being published.


Identifying rare human metabolic disorders that result from a single-gene defect has not only enabled improved diagnostic and clinical management of such patients, but also has resulted in key biological insights into the pathophysiology of the increasingly prevalent metabolic syndrome. Insulin resistance and type 2 diabetes are linked to obesity and driven by excess caloric intake and reduced physical activity. However, key events in the causation of the metabolic syndrome are difficult to disentangle from compensatory effects and epiphenomena. This review provides an overview of three types of human monogenic disorders that result in (1) severe, non-syndromic obesity, (2) pancreatic beta cell forms of early-onset diabetes, and (3) severe insulin resistance. In these patients with single-gene defects causing their exaggerated metabolic disorder, the primary defect is known. The lessons they provide for current understanding of the molecular pathogenesis of the common metabolic syndrome are highlighted. Insulin resistance is implicated in the pathophysiology of the twin epidemics of type 2 diabetes and obesity. Both conditions are associated with a high burden of premature morbidity and mortality globally; however, the dependence of one on the other is not complete. Despite intensive efforts, the molecular mechanisms underlying the relationship between obesity, insulin resistance, and type 2 diabetes in the metabolic syndrome are incompletely understood. The accelerating discovery of single-gene defects resulting in rare types of diabetes, obesity, or severe insulin resistance over the past 20 years provides the opportunity to unravel the role of several key mediators in these separate groups of disorders through firmer cause-effect relationships. We summarise the key monogenic disorders that result in nonsyndromic obesity, pancreatic beta-cell diabetes, or severe insulin resistance and discuss how the insights they provide may be applied to the understanding of more prevalent metabolic disease  and cocaine and amphetamine-regulated transcripts (CART) [The downstream effect of leptin receptor activation is regulation of neuropeptides such as those from the proopiomelanocortin (POMC) and agouti-related peptide (AGRP) neurons. Leptin downregulates orexigenic (appetite promoting) neuropeptides such as neuropeptide Y (NPY) and agouti-related peptide (AGRP) but also upregulates anorexigenic neuropeptides such as s (CART) . It mighs (CART) , raisingLeptin also has an important reproductive function. The development of adequate adipose stores to allow puberty to begin is signalled by leptin to the hypothalamus. Thus leptin has a permissive effect on puberty . Furtherleptin gene which resulted in a truncated protein which was not secreted. They were morbidly obese with excess fat mass, hyperphagia, but no changes in resting metabolic rate or total energy expenditure adjusted for body composition. These children were the human equivalent of the obese (ob/ob) mouse, resulting from a recessive homozygous mutation in the leptin gene and confirmed a critical role for leptin in human appetite regulation. This was reinforced by the observation that replacement of recombinant human leptin in these children resulted in a rapid reversal of the hyperphagia, promoted weight loss, and normalization of body composition [Evidence for the important role of leptin in energy regulation in humans comes from the observations of O'Rahilly et al., who identified children with severe early onset of obesity who had undetectable levels of leptin . They weposition . Furtherposition . These iPOMC gene, which codes for a number of important proteins including adrenocorticotrophin hormone (ACTH) and melanocortin peptides (α and β melanocyte stimulating hormone (MSH)). Individuals have been identified with homozygous mutations of the POMC gene resulting in complete loss of function of all POMC-derived peptides [Further evidence for the central role of appetite regulation comes from other monogenic defects of the peptides . They prα-MSH or β-MSH peptides have been linked with early onset of human obesity depending on the location of the mutation [Further insights are provided by mutations of specific POMC-derived peptides. Point mutations that disrupt the mutation , 25, 26.MC4R). The melanocortin receptor MC4R is expressed on neurons in the paraventricular nucleus of the hypothalamus. Activation of MC4R results in release of anorexigenic peptides such as brain-derived neurotrophic factor (BDNF) [MC4R are found in up to 6% of severe childhood obesity [MC4R mutations is variable with mutations resulting in complete loss of function having a more severe phenotype. This is demonstrated by the variation in the degree of hyperphagia with ad libitum energy intake. Those with partial MC4R deficiency have less excess intake compared with those with complete deficiency [MC4R in energy balance. The importance of the melanocortin peptides is confirmed by the demonstration of severe obesity associated with mutations of the melanocortin receptor MCR. The me obesity , 28. Whificiency . This reMC4R mutations. Those with leptin deficiency have the greatest energy intake, those with inactive MC4R mutations are less hyperphagic, and those with partially active mutations have similar intake to those with treated leptin deficiency [However, the complexity of the system and the interrelationship of the signals are also demonstrated by the variability in the ad libitum energy intake seen when comparing those with leptin deficiency, inactive and partially active ficiency .The discovery of peptides and receptors critical to the pathways of energy balance opens the opportunity for therapeutic targets for common obesity. This is demonstrated by the effectiveness of the replacement of leptin in those with leptin deficiency . HoweverTrkB) [BDNF mutations also have impaired cognitive function, delayed developmental milestones, and hyperactivity. Those with loss of function of the TrkB receptor also have memory deficits.Other players in the central control of appetite have also been identified through rare genetic mutations resulting in reduced production of peptides such as brain-derived neurotrophic factor (BDNF)  or loss TrkB) . Both muLesson 2: Genetically Mediated Differences in Satiety Are Likely to Underly the Difference in Body Weight Seen in the Current Obesogenic Environment. Defects in single genes causing severe non-syndromic human obesity result mainly through impacts on appetite. The recent rapid rise in obesity epidemic is clearly linked with the technological and social advances which have reduced the need for strenuous physical activity at work and at home, with abundant cheap, high-density food propagated with aggressive advertising. Nonetheless, the persistence of lean people in the current obesogenic environment suggests that either these people are able to withstand the toxic environment through conscious choices about diet and exercise and/or that they may have a genetic predisposition to stronger satiety signals, which enable them to achieve their leanness largely through unconscious mechanisms [chanisms . Alreadychanisms , 8.Lesson 3: Proof of Key Components of Pancreatic Beta Cell Function and Responsiveness of Certain Genetic Etiologies to Oral Glucose Lowering Drugs Acting Distal to the Monogenic Defect. The beta-cell monogenic diabetes are characterised by genetic mutations that result in early onset diabetes in absence of obesity or insulin resistance [sistance –33. Mostsistance . The beta cell is the major component of the pancreatic islets of Langerhans, which collectively account for 1-2% of the total pancreatic mass. The normal human beta cell has 3 functions that are critical to achieve its contribution to maintaining normoglycaemia. Firstly, the cell must be able to “sense” ambient glucose levels thus allowing any insulin output to be appropriate to requirements; secondly, the beta cell must be able to manufacture and store insulin; and thirdly, the cell must be able to rapidly secrete this insulin when required. Dysfunction of any of these roles will result in impairments of glucose homeostasis see . TypicalHYMA1, involved in mRNA encoding) and pleomorphic adenoma gene like 1  can be identified in 80% of these patients [NDM refers to diabetes diagnosed in the first 6 months of life, which can be transient (approximately 70% of cases) or permanent, and is most commonly the result of a monogenic mutation , 35–37. patients . ATP channel genes , or ATP-binding cassette transporter subfamily C member 8 (ABCC8)) or insulin gene (INS) are the most common causes of permanent neonatal diabetes mellitus (PNDM), whilst KCNJ11 and ABCC8 mutations have been shown to account for a minority of cases of TNDM [ATP channels link the ATP produced by cellular glucose metabolism to potassium flux and thus membrane excitability [KCNJ11 and ABCC8 mutations cause an increase in the opening of the KATP channel resulting in hyperpolarisation, preventing beta cell membrane depolarization and insulin release [ATP channel. The resultant closure of these channels stimulates insulin secretion, thus bypassing the effect of the KCNJ11 and ABCC8 mutations. Further validation of the function of the KATP channel is provided by the excellent response of many patients harbouring KCNJ11/ABCC8 mutations to SU therapy; indeed, it is now standard practice for all patients with diabetes diagnosed before 6 months of age to undergo diagnostic testing for mutations in these two genes, with attempts to transfer those with confirmed mutations from insulin therapy to oral SUs [ATP channel dysfunction evident in extrapancreatic structures ) the use of a nonbeta cell selective SU (glibenclamide) is also effective in treating the neuromuscular phenotype [INS gene mutations do not respond to SU medications and currently require lifelong insulin therapy.Activating mutations of the K of TNDM , 42, 43.tability . KCNJ11  release , 42, 45.oral SUs , 46. Furhenotype . Cases oATP channel give rise to TNDM while others result in PNDM is unknown, but changes in pancreatic beta cell turnover or compensation at the level of the beta cell or whole body that is able to overcome the effects of these genes may be possible explanations [The reason why certain mutations within the Kanations .PTF1A, pancreatic and cerebellar agenesis) [IPF1, pancreatic agenesis) [GLIS3, congenital hypothyroidism, glaucoma, kidney cysts and hepatic fibrosis) [RFX6, pancreatic hypoplasia, intestinal atresia and gall bladder hypoplasia) [GATA6, congenital heart defects) [NEUROG3, congenital malabsorptive diarrhea) [NEUROD1, sensorineural deafness and visual impairment) [FOXP3, enteropathy and dermatitis) [EIF2AK3, exocrine pancreatic dysfunction, spondyloepiphyseal dysplasia, developmental delay, acute liver failure, osteopenia and hypothyroidism) [SLC2A2, aminoaciduria, proteinuria, short stature and rickets) [SLC19A2, thiamine responsive megaloblastic anemia) [Several more rare genetic etiologies of NDM have been identified, most of which are autosomal recessively inherited and are associated with extrapancreatic manifestations. These include pancreas-specific transcription factor 1A (genesis) , insulingenesis) , GLIS faibrosis) , regulatoplasia) , GATA-bidefects) , neurogeiarrhea) , neurogeairment) , forkheamatitis) , eukaryoroidism) , solute rickets) , and sol anemia) . The majority of infants with neonatal diabetes of diverse genetic etiologies are born with reduced birth weight, resulting from lower fetal insulin production, as fetal insulin is a major growth factor in utero .GCK gene result in a severe diabetes phenotype, presenting at birth as permanent neonatal diabetes mellitus [GCK mutations result in a higher concentration of glucose being required to stimulate use of the substrate and therefore insulin secretion. However, once this threshold is reached, insulin secretion therafter is relatively normal. Humans who harbour such heterozygous inactivating GCK mutations therefore demonstrate mild fasting hyperglycaemia but normal post meal time glucose levels [GCK mutations have the opposite clinical syndrome of hyperinsulinemic hypoglycaemia [Glucokinase (GCK) is a hexokinase enzyme that is found in the beta cell, liver, brain, and multiple other tissues in most mammalian species. GCK has many roles within expressing tissues but functions primarily as an ambient glucose sensor for the pancreatic beta cell . The enzmellitus , 65. Hete levels . Those wlycaemia .GCK mutations is approximately 0.1%, accounting for 20–50% of all cases of monogenic diabetes [GCK mutations [GCK mutation resulting in mild hyperglycaemia does not require any glucose lowering therapy.The population prevalence of heterozygous inactivating diabetes . Mild fadiabetes . Furtherutations , 69. ForLesson 4: Glucose Toxicity Is Not Seen in Those with Lifelong Mild Hyperglycaemia Resulting from Heterozygous GCK Mutations. The glucose toxicity theory proposes that continual exposure to modest increases in blood glucose over a long period of time could has adverse effects on beta cell glucose sensitivity and function [GCK mutation carriers has no significant progressive glucose toxic effect on the beta cell.function , 71. No function . This isfunction , 72. ConGCK mutation [GCK mutation is high in pregnancy; treatment for “hyperglycaemia” in a pregnant woman with a GCK mutation may result in reduced birthweight if the foetus has inherited the mutation [GCK mutation status, current advice is to perform serial ultrasonography to monitor foetal growth with consideration of treatment only in those with accelerated growth  [Up to 17.8% of pregnant women are diagnosed with gestational diabetes on the basis of recent guidelines, and up to 10% of these women will have a mutation , 74. Howmutation . Conversutation) .Lesson 5: Exposure to Mild Hyperglycaemia in Utero Does Not Program Offspring to Have Reduced Beta Cell Function. There is animal and human evidence for maternal hyperglycaemia exposure in utero programming offspring to have beta cell dysfunction [function , howeverfunction .The significance of GCK in sensing ambient glucose has led to the hypothesis that pharmaceutical manipulation of this physiological mechanism in the form of glucokinase activators could present an effective method of treating hyperglycaemia as a result of type 2 diabetes . No addiThe hepatocyte nuclear factor (HNF) family of proteins are ubiquitous and function primarily as transcription factors. Whilst predominantly expressed within the liver, their key role in regulating both pancreatic beta cell growth and function is demonstrated by mutations leading to progressive beta cell failure and childhood or early-adult onset of diabetes , 80. HNF1A are the most commonly encountered form of monogenic diabetes with a minimum prevalence of 50–60 cases per million population  or 1-2% of all patients with diabetes [HNF1A mutation carriers will have diabetes by the age of 25, and >95% by the age of 40 [GCK-related monogenic diabetes, the risk of micro- and macrovascular complications appears to be comparable to type 1 and type 2 diabetes and is directly related to glycaemic control [HNF1A mutations have an increased all-cause and cardiovascular-specific mortality rate when compared with nonaffected relatives (hazard ratios 1.9 and 2.3 resp.) [HNF1A mutations  [HNF1A mutations frequently demonstrate high glucose lowering sensitivity to SU therapy [ATP receptor, thereby bypassing the metabolic pathways is rendered dysfunctional by an HNF mutation [HNF1A diabetes is made.Mutations in diabetes . Patientdiabetes . Penetrage of 40 . Progres control . Patient3 resp.) . The ristective) . Patient therapy –89. As nmutation . ConsequHNF1A directly regulates renal tubular expression of the sodium-glucose cotransporter (SGLT); HNF1A mutations consequently result in a low renal threshold for glucose  as a result of impaired tubular glucose reabsorption [1A/4A monogenic diabetes from type 1 diabetes when diabetes has been present for many years given that C-peptide progressively declines in type 1 diabetes but is retained in HNF1A/4A monogenic diabetes [sorption . This clsorption . Additiosorption . An hsCRdiabetes .HNF4A mutation results in a similar phenotype to the patients with HNF1A mutations and accounts for approximately 5–10% of all cases of monogenic diabetes. As with HNF1A mutation carriers, SU therapy remains an effective therapeutic strategy for these patients, but the elevated high density lipoprotein (HDL) levels and glycosuria are not seen in those with HNF4A mutations [Heterozygous inheritance of an utations . SomewhaHNF1B, which account for approximately 5% of cases of monogenic diabetes, lead to clinical syndromes more reflective of the widespread expression of this transcription factor [In contrast, mutations in n factor . Most caMitochondria are energy-generating organelles identifiable in most human cells . Each mim.3243A>G) [Mitochondrial DNA mutations can result in a wide range of multiorgan dysfunction with a number of recognised syndromes. Maternally inherited diabetes and deafness (MIDD) is the most commonly encountered mitochondrial diabetes syndrome and accounts for up to 1% of diabetes . The syn3243A>G) . These d3243A>G) . Most of3243A>G) . There i3243A>G) , and the3243A>G) .3243A>G) diabetes [Deafness in MIDD is sensorineural in nature and results from defects in the cochlear and affects the majority of those with mitochondrial  those that affect insulin signaling and (b) those that affect adipocyte development and/or function. INSR), leading to a global reduction of insulin receptor function in target cells. Mutations in the INSR gene result in a clinical spectrum of disease severity ranging from mild to severe [INSR gene, in which patients present in the peri-pubertal stage or beyond with acanthosis nigricans, dysglycemia  in the presence of severe hyperinsulinemia, oligomenorrhea, and hyperandrogenism in women. In men, the presentation is less obvious with only acanthosis nigricans and sometimes fasting hypoglycaemia. Men often remain undiagnosed even after the development of diabetes requiring high doses of insulin. The most common group of patients with monogenic insulin resistance have a mutation in the insulin receptor  anINSR mutation differ biochemically from patients with more common insulin resistance associated with the metabolic syndrome by having high levels of adiponectin, sex-hormone-binding globulin (SHBG), and insulin-like growth factor-binding protein 1 (IGFBP1) [Patients with (IGFBP1) . AdiponeLesson 7: Insulin Receptor Signalling on Pancreatic Islets Is Not Required for Beta Cell Compensatory Response to Severe Insulin Resistance. The severe hyperinsulinemia seen even in those with almost complete loss of insulin receptor function suggests that the insulin receptor on pancreatic islets is not necessary for beta cell functioning, as has been suggested by some studies in mice [ in mice . However in mice .Lesson 8: Acanthosis Nigricans and Ovarian Hyperandrogenism Are Likely to Be Mediated by Hyperinsulinemia Acting through Non-Insulin Receptor Pathways. Acanthosis nigricans (a velvety hyperpigmentation of the skin) and ovarian hyperandrogenism  are seen both in patients with severe insulin receptor mutations who have reduced activity of the insulin receptor as well as in those with common obesity-related insulin resistance. This suggests that these clinical features are likely to be mediated by insulin signalling through a noninsulin receptor pathway such as by stimulating the insulin-like growth factor-1 (IGF-1) receptor [receptor . Insulinreceptor , 110  has been identified accounting for the clinical features of acanthosis nigricans, ovarian hyperandrogenism, diabetes mellitus, and in contrast to those with insulin receptor mutations, both metabolic dyslipidemia and fatty liver in the proband [AKT2 highlights the importance of AKT2 in adipocyte differentiation as well as its role in the direct effects of insulin signaling. Surprisingly few mutations have been identified in genes encoding the multiple downstream components of the insulin signaling pathway. A mutation in v-akt murine thymoma viral oncogene homolog 2 ( proband . In addiAKT2 mutations but not in those with an insulin receptor mutation suggests that the pathway required for hyperinsulinemia to drive liver fat accumulation and atherogenic very low-density lipoprotein (VLDL) secretion depends on the insulin receptor but not on AKT2. This supports the concept of selective postreceptor insulin resistance, also relevant to the highly prevalent form of insulin resistance [The presence of metabolic dyslipidemia and fatty liver in those with sistance   Hepatic Insulin Resistance Occurs in Common Metabolic Dyslipidemia rather than Total Postreceptor Insulin Resistance. Although hyperinsulinemia in the context of normal blood glucose is often thought to be the hallmark of insulin resistance, the possibility of partial insulin resistance was not clear until studies of monogenic cases of insulin resistance were examined for the effect of hyperinsulinemia on actions such as suppressing hepatic lipogenesis [ogenesis . It has ogenesis . Those wogenesis   patients are recognized very early in life due to their near-total lack of body fat and strikingly muscular appearance . DespiteAGPAT2) and Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) account for the majority of cases and were identified through linkage analysis with positional cloning in affected families [AGPAT2 is highly expressed in the adipose tissue and is one of the critical enzymes involved in the biosynthesis of triglyceride and phospholipids from glycerol-3-phosphate [BSCL2 is highly expressed in the brain and adipose tissue and its protein seipin is thought to be involved in lipid droplet formation and adipocyte differentiation [AGPAT2 and BSCL2 mutation have similar metabolic abnormalities; however, there are certain distinguishing phenotypic differences. Higher prevalence of mental retardation and cardiomyopathy is seen patients with BSCL2 mutations, and the presence of lytic lesions in the appendicular skeleton in patients with AGPAT2 likely reflects the altered tissue expression and function of the two gene products [Autosomal recessive (either homozygous or compound heterozygous) mutations in several genes have been implicated in the pathogenesis of CGL. Mutations in two genes: 1-acylglycerol-3-phosphate-O-acyltransferase 2  [CAV1) [PTRF is involved in the biogenesis of caveolae which are invaginations of the plasma membrane found in many cell types but particularly abundant in adipocytes, muscle, and endothelia. PTRF regulates expression of caveolins 1 and 3, which are proteins found within caveolae, that are required for caveolae formation within many tissues [CAV1 is an integral component of caveolae, which binds fatty acids and translocates them to lipid droplets [Autosomal recessive mutations in polymerase 1 and transcript release factor (PTRF) , 121 and) [CAV1)  have als tissues . CAV1 isdroplets .BSCL2 mutations have the most severe loss of body fat including loss of fat from mechanical sites such as retroorbital, palm, sole, and periarticular regions, in addition to the metabolically active adipose tissue in the subcutaneous, intra-abdominal, intrathoracic, and other regions. However, those with AGPAT2, CAV1, and PTRF mutations do not have loss of mechanical fat. Both PTRF and CAV1 mutation carriers have preserved bone marrow fat, which is absent in those with AGPAT2 and BSCL2 mutations [Patients with utations .Lesson 11: Not All Fat Is Bad. Given that obesity is associated with insulin resistance both longitudinally within individuals and cross-sectionally between individuals, this suggests that adipose tissue itself causes insulin resistance. However, the exaggerated appearance of insulin resistance features in rare patients with congenital lack of adipose tissue suggests that a normal amount of adipose tissue is critical for metabolic health. The different adipocyte-related cellular functions of the various congenital generalized lipodystrophy genes suggest that the common severe insulin resistance phenotype  seen in these disorders is a result of the loss of adipose tissue rather than their specific gene functions in other tissues [ tissues .LMNA) gene [LMNA encodes the protein lamin A/C which forms part of the nuclear lamina, and mutations have been linked to many other disorders such as Charcot-Marie-Tooth neuropathy, progeria syndromes, restrictive dermopathy, limb-girdle muscular dystrophy, and mandibulo-acral dysplasia [PPARG), which is a nuclear hormone receptor most highly expressed in adipose tissue [AKT2 [CIDEC) [PLIN1) [CIDEC and PLIN1 are important in lipid droplet dynamics and regulate triglyceride mobilization.The first gene identified as causative of familial partial lipodystrophy (FPL) of the Dunnigan variety was lamin A/C (NA) gene , which rysplasia . The sece tissue . It is iue [AKT2 , cell-de [CIDEC) , and per [PLIN1) . Both CIIn all these FPLs, the failure of fat development is often not manifested until puberty and women are much more severely affected than men. This may relate to the higher adiposity seen in females than males, particularly with the onset of puberty. The phenotypic heterogeneity between the various genetic types of FPL is not clear; however, the partial loss of subcutaneous fat from the extremities is seen in all types. In the LMNA variety, the trunk is also affected but face and neck are spared, while there is preserved abdominal fat in the remaining types . DespiteThe lipodystrophies suggest that there is a critical amount of adipose tissue required for buffering episodic excess caloric intake by serving as a sump for free fatty acids and glucose. Loss of this capacity produces increased uptake in other metabolically less suited tissues such as liver, muscle, and pancreatic beta cells where harmful effects include fatty liver, dyslipidemia, and loss of insulin sensitivity and insulin secretion. An exacerbating factor may include the loss of adipocyte-tissue-derived leptin which signals adequacy of adipose tissue to the brain. Loss of leptin in CGL (and to a less extent FPL) suggests that whole body energy stores are low and leads to compensatory hyperphagia. The resulting increase in ingested calories worsens systemic metabolic stress. Proof of this has been illustrated by the dramatic efficacy of leptin replacement in patients with most lipodystrophies , 131.The rare lipodystrophy syndromes support the adipose expandability hypothesis , 133. ThThe observation that loss of function of certain genes in humans leads to either severe obesity, early diabetes, or severe insulin resistance (with or without lipodystrophy) is very powerful in illustrating how defects in specific encoded proteins located predominantly in the brain, pancreatic beta cell, muscle, and or fat give rise to these distinct components of the metabolic syndrome. They challenge the view that environmentally driven obesity leads to insulin resistance which leads to type 2 diabetes, as supported by many cross-sectional and longitudinal epidemiological studies. The monogenic disorders provide particular insights that (1) there are clear biological driven origins of obesity affecting appetite, which predisposes to insulin resistance and type 2 diabetes, (2) specific defects in pancreatic beta cell function predispose to diabetes in absence of either obesity or insulin resistance, and (3) specific defects in insulin signaling or fat storage capacity can lead to severe insulin resistance in absence of obesity, which leads to diabetes through pancreatic exhaustion. All single-gene defects discovered to be responsible for human obesity have been identified to impact on appetite . Common ATP channel within the pancreatic beta cell, that regulate the amplification response to glucose or insulin exocytosis itself, is involved in type 2 diabetes [ Various monogenic pancreatic beta cell diabetes arising from key defects within the insulin secretory pathway results in diabetes, manifesting from early infancy to early adulthood. Certain monogenic subtypes of diabetes have resulted in dramatic success of transferring from insulin therapy to oral sulfonylurea therapy, based on the location of the genetic defect upstream from the target of the sulfonylurea drug. The confounding effects of obesity are less important in these disorders, so it is possible to translate some of these findings to the etiology of type 2 diabetes. Common genetic variants predisposing to type 2 diabetes have been identified in many beta cell targets overlapping with known causes of monogenic diabetes . Currentdiabetes . The assIn contrast to insulin resistance commonly observed in relation to obesity, patients with monogenic defects resulting in severe insulin resistance occur in absence of obesity or in the presence of generalized or regional lack of adipose tissue. Such lipodystrophic patients from diverse genetic etiologies in fat metabolism have a defect of triglyceride storage in adipose tissue, which results in lipid accumulation elsewhere in the body. This leads to severe insulin resistance associated with the usual accompanying characteristics of fatty liver and atherogenic dyslipidemia. At a more direct level, those who have a defect in their insulin receptor have very high levels of insulin resistance and frequently develop diabetes from compensatory beta cell exhaustion, yet are protected from fatty liver and atherogenic dyslipidemia. Patients with insulin receptor defects also differ biochemically from other types of insulin resistance by displaying elevated adiponectin, IGFBP1, and SHBG, which are all commonly suppressed in common insulin resistance or lipodystrophy-associated severe insulin resistance. This highlights the mechanisms behind partial insulin resistance or postreceptor selective defect in signaling through one arm of the insulin signaling pathway evident in common obesity-related insulin resistance.Several monogenic disorders manifesting the key components of the metabolic syndrome have enabled us to unravel the pathogenic link between the defined molecular defect (or groups of defects) and obesity, early diabetes, or severe insulin resistance, each without the confounding effects of all the other components. Moving forward, we should continue to discover additional monogenic causes for the remaining portion of patients with severe disorders who currently do not have any defined etiology, which will not only help improve their management but may uncover future novel and therapeutic targets for the increasingly common metabolic syndrome.


Exogenous plant-based cannabinoids (phytocannabinoids) and chemically related compounds, like the terpenes, commonly found in many foods, have been found to exert significant analgesic effects in various chronic pain conditions. Currently, the use of Δ9-tetrahydrocannabinol is limited by its psychoactive effects and predominant delivery route (smoking), as well as regulatory or legal constraints. However, other phytocannabinoids in combination, especially cannabidiol and β-caryophyllene, delivered by the oral route appear to be promising candidates for the treatment of chronic pain due to their high safety and low adverse effects profiles. This review will provide the reader with the foundational basic and clinical science linking the endocannabinoid system and the phytocannabinoids with their potentially therapeutic role in the management of chronic pain.The endocannabinoid system is involved in a host of homeostatic and physiologic functions, including modulation of pain and inflammation. The specific roles of currently identified endocannabinoids that act as ligands at endogenous cannabinoid receptors within the central nervous system (primarily but not exclusively CB Pain is an unpleasant, commonly occurring, and universal human experience; it is also a very complex phenomenon. The experience of pain and the resultant emotional state depends as much or perhaps more on the contextual circumstances  of the pain-inciting event as the intensity of the noxious stimulus. And a seemingly similar pain-producing event may be experienced (and communicated) quite differently from person to person, situation to situation, and among various cultures. The neurophysiology of acute pain due to a brief single noxious event is best understood. The nociceptive components of the peripheral and central nervous systems are highly refined to signal warnings of potential or actual tissue damage; reflex and conscious responses are usually adaptive for self-protection. Fortunately, most occurrences of pain are self-limited, resolving quickly with discontinuation of the noxious stimulus or in tandem with tissue healing or resolution of the insult to somatic or visceral structures.But pain that continues relentlessly due to on-going nociceptive stimulation from unresolved disease (nociceptive pain) or pathophysiological changes within the nervous system (neuropathic pain) serves little purpose. In contrast to acute pain, unresolved pain leads to subliminal and conscious reflex responses that are often maladaptive .1 It imp2The prevalence of persistent, debilitating pain is increasing, commensurate with advances in trauma care that allow survival after serious injury and medical care that slow disease progression, or transform imminently life-threatening diseases into chronic progressive conditions, or provide a cure but with residual morbidity. Similarly, the growing proportion of older individuals in economically developed and developing nations, and the propensity to develop chronic pain-producing conditions with advancing age , is leading to a high prevalence of chronic pain worldwide.Unfortunately, currently available analgesic medications and pain-modulating procedures are severely limited by combinations of low efficacy, excessive toxicity/risk/safety concerns, insufficient access to care, or unbearable cost. In patients with chronic pain, and especially neuropathic pain, “success” is measured in small increments of improvement among limited numbers of patients. In randomized clinical trials of analgesics for neuropathic pain, no more than half of patients experience clinically meaningful pain relief from pharmacotherapy.5The first portion of this article presents a very basic review of the pharmacology of the cannabinoids and endocannabinoid receptor system, drawing both from animal and human models.Extensive research and prolonged exposure to cannabinoids both in animals and humans have addressed important questions about safety. Cannabinoids have a very high therapeutic index. In fact, it is virtually unlimited insofar as fatalities have not been reported directly related to the toxicity of any cannabinoid, even with extremely high dosing. However, there are potentially severe cognitive, psychotomimetic, and substance abuse-related adverse effects associated with Δ9-tetrahydrocannabinol (THC) exposure that must be taken seriously, especially in young or cannabis-naïve patients.17In medicinal use, these adverse effects may be prevented or mitigated by avoiding THC entirely in favor of other non-psychoactive cannabinoids.21Based on relatively new but limited scientifically based literature, it is now only possible to speculate about mechanisms of action and what the future may hold for phytocannabinoids as effective analgesics across the vast and varied cohorts of people living with chronic pain. With that in mind, this review will proceed with a summary of what is known about different cannabinoid congeners on various types of pain (efficacy and tolerability) and the putative role of commonly available “generally regarded as safe” (GRAS) ingredients that may enhance the effectiveness of certain phytocannabinoids.Cannabis (i.e. marijuana and hemp) and structurally related synthetic analogues  but higher quantities of cannabidiol (CBD) which may offer a range of medicinal benefits without the cognitive effects and abuse potential associated with THC.23Cannabinoid refers to a pharmacological class of about 60 naturally occurring compounds (phytocannabinoids) found in plants of the genus Cannabis has a long and storied social and medicinal history dating back thousands of years.Cannabis cultivation and distribution, especially as these pertain to marijuana, have preoccupied governments from China, through India to Europe and Great Britain, and across the Atlantic to the Americas for centuries due to its inherent psychedelic intoxication.Cannabis have mostly been sought for recreational or ritualistic purposes, and are commonly derived from smoking dried plant material or its concentrated oily derivative, hashish. Even though hemp has minimal potential psychoactivity, it is nonetheless subjected to the same restrictions as marijuana in many jurisdictions.Cannabis and cannabinoids. Notwithstanding the momentous breakthrough represented by these reports in support of the potential value of cannabinoids for medical use, the health hazards of smoking coupled with the cognitive-behavioral effects of Cannabis have created political and regulatory obstacles worldwide, with regard to evaluating cannabinoids as medicines and mainstream health care professionals’ acceptance of Cannabis as a legitimate therapeutic agent. Fortunately, as the sciences of drug delivery and cannabinoid pharmacology have progressed in recent years, there are rapidly evolving technologies that will facilitate or enhance the medically indicated use of this pharmacological class of agents while overcoming the barriers imposed by unwanted or harmful psychoactive effects of Cannabis and smoking it as the only effective way to obtain adequate blood levels of cannabinoids.27Only recently have we gleaned scientific insight into several of the pharmacologically distinct cannabinoids and their effects at specific receptors within various animals and humans. In 1997 both the United States National Institutes of Health and the British Medical Association released reports on the potential therapeutic uses of 1  and CB2 , are differentiated by their physiological actions and locations within the body. These are members of the seven transmembrane G-protein coupled receptor superfamily which comprise the binding sites for almost half of all contemporary drugs.31The potential value of the cannabinoids for medicinal purposes arose from the discoveryThe endogenous cannabinoid system has been described as “an ancient lipid signaling network which in mammals modulates neuronal functions, inflammatory processes, and is involved in the etiology of certain human lifestyle diseases, such as Crohn’s disease, atherosclerosis and osteoarthritis. The system is able to downregulate stress-related signals that lead to chronic inflammation and certain types of pain, but it is also involved in causing inflammation-associated symptoms, depending on the physiological context.”321 receptor has been cloned from humans.1 receptors leads to dose-dependent and stereo-selective inhibition of adenylate cyclase activity, thus affecting memory, perception, and movement. The CB1 receptor appears to be responsible for the mood-enhancing effects of Cannabis as well as negative, dysphoria-inducing, and frank psychotomimetic effects in susceptible individuals.The CB1 receptor distribution has been well characterized in the human brain.1 receptors in these areas helps explain the absence of life-threatening effects on vital physiological functions associated with extremely high doses of cannabinoids.CB1 receptor occurs in the testis, and presynaptically on sympathetic nerve terminals.1 receptor mRNA has been identified in the adrenal gland, heart, lung, prostrate, bone marrow, thymus, and tonsils.37Besides the brain, the CB1 and CB2 receptors share considerable structural similarities, their distribution and activity diverge. Among other actions, including pain modulation, CB2 receptors are thought to serve an important role in immune function and inflammation.2 receptor activation reduces nociception in a variety of preclinical models, including those involving tactile and thermal allodynia, mechanical and thermal hyperalgesia, and writhing.2 receptors on microglia within the nervous system may explain the putative benefits of cannabinoids in reducing cytokine-mediated neuroinflammation.Although CB1 and CB2 receptors inhibit adenylate cyclase via interactions at the G-protein complex. However, their activation and consequent inhibition of various ion channels differs.1 or CB2 receptors, either separately or in combination by their respective endogenous or exogenous ligands, leads to varied physiological effects  receptors and may play a modulating role at other transient receptor potential (TRP) receptor types.1 receptors, via G-protein coupled inhibition of adenylate cyclase. These effects include antinociception, hypomotility, and reduced memory.45It appears that the endocannabinoid system is intimately involved in tissue healing in the face of inflammatory conditions, correlating clinically with prevention and treatment of inflammation-mediated pain.However, there appear to be distinct differences between anandamide and other cannabinoids with respect to their antinociceptive properties and other physiological effects which vary as a function of route of administration. It is not known whether anandamide acts at the same sites as phytocannabinoids to produce antinociception. The behavioral effects of THC and anandamide after administration suggest that they act, at least in part, in the brain and/or spinal cord. These studies suggest that anandamide is less potent and has a shorter duration of action than THC.461 and CB2 along with other non-cannabinoid receptors) have exploded in recent years, and insights reveal this area of pharmacology to be highly complex and dynamic. For instance, there is mounting evidence that endogenous and exogenous cannabinoids exert some influence on opioid, 5HT3, and N-methyl-d-aspartate receptors. These interactions suggest a role for endocannabinoids in homeostatic pain modulation (antinociception), thus their use as exogenous agents in pain management.48Studies have demonstrated that antinociceptive effects of cannabinoids are mediated through mechanisms distinct from those responsible for other behavioral effects. For instance, THC has additive analgesic efficacy with kappa opioid receptor agonists. This effect is blocked by kappa antagonism, but opioid receptor antagonism does not alter the psychoactive effects of THC.1 and CB2 receptors, respectively, stimulating the endogenous noradrenergic pathway which in turn activates peripheral adrenoreceptors, inducing antinociception. Other studies have demonstrated the expression of functional CB2 receptors in areas of human dorsal root ganglion (DRG) sensory neurons. CB2 receptor expression also has been demonstrated in the spinal cord as well as in other brain regions particularly relevant for nociceptive integration.52Most recently, Thiago et al.2 receptors in the analgesic effects produced by CB2 agonists.2 receptor agonists can evoke analgesia by triggering the release of beta-endorphin in response to the stimulation of CB2 receptors expressed in human keratinocytes.56These findings implicate CB2 receptors in antinociception has been demonstrated in inflammatory and neuropathic pain models. Investigations involving carrageenan-induced inflammatory pain in rodents demonstrate that activation of CB2 receptors by CB2-selective agonists suppresses neuronal activity in the dorsal horn via reduction in C-fiber activity and wind-up involving wide dynamic range (WDR) neurons.2 receptor protein or mRNA in inflamed tissues and in the dorsal root ganglion in neuropathic states.1 receptors mediate the analgesic effects of cannabinoids on visceral pain from the gastrointestinal tract.62The role of CBIt may now be concluded that cannabinoids play a role in endogenous (homeostatic) modulation of nociception, and that exogenous cannabinoids potentially offer some degree of analgesia in various pain states.Evidence continues to accumulate suggesting that cannabinoids can impact normal inhibitory pathways and pathophysiological processes influencing nociception in humans.78When cannabinoids lead to a reported reduction in pain, it remains unclear where the effects are triggered, or which aspect of the pain experience is most affected and under what circumstances. As well, different cannabinoids may lead to mechanistically different pain-relieving effects. For instance, a recent study of functional brain imaging in human volunteers investigated the means by which THC may influence pain resulting from capsaicin-induced hyperalgesia. The study results suggest that “peripheral mechanisms alone cannot account for the dissociative effects of THC on the pain that was observed. Instead, the data reveal that amygdala activity contributes to inter-individual response to cannabinoid analgesia, and suggest that dissociative effects of THC in the brain are relevant to pain relief in humans.”Cannabis by Raphael Mechoulam and colleagues in 1964 at the Hebrew University of Jerusalem, and they identified it as the major psychoactive component of Cannabis, with preferential binding at CB1 receptors.86The two best-studied cannabinoids implicated as having potential analgesic properties are THC and CBD . THC wasCannabis. It has virtually no psychoactivity compared against THC.1 and CB2 receptors. Limited pharmacodynamic effects due to relatively weak receptor binding (low affinity) may be overcome with higher doses of agonist. Whereas the dose-limiting factor with THC resides in the highly variable propensity among individuals to experience and tolerate negative affective, cognitive, and psychotomimetic effects, the ability of cannabidiol to behave as a CB1 receptor inverse agonist may contribute to its documented mitigating action on THC psychotomimetic effects. More recently it has been postulated that cannabidiol may exert its effects via inhibition of anandamide deactivation or otherwise enhancing anandamide signaling.88Cannabidiol is a major constituent of 2 receptors seems to account for its anti-inflammatory properties and both primary and secondary influences on pain.1 receptors by CBD. Anxiolytic effects of CBD may also be attributed to its agonist effect at the 5-HT1A receptor.91Cannabidiol agonist activity at CBCannabis sativa L. This formulation is approved in Canada, New Zealand, Israel, and several European countries  for the management of spasticity in multiple sclerosis (MS). There are several on-going trials on its efficacy in treating MS-related pain.Cannabis, hyperalgesic and cognitive effects become more pronounced and problematic, especially in cannabis-naïve individuals.A pharmaceutical combination product of THC and CBD now exists as an oral spray consisting of 27 mg Δ9-tetrahydrocannabinol and 25 mg cannabidiol per mL , extracted from The therapeutic role of cannabinoids in cancer treatment, in terms of effects on tumor cells and on cancer pain, is of great interest. Correlations have been found between cannabinoid receptor levels and endocannabinoid activity and cancer severity, pain intensity, and survival.991 or CB2 receptors or modulation of endocannabinoids will have to be targeted to achieve analgesia without dose-limiting side effects.103For treating refractory cancer-related pain, there is mounting evidence that cannabinoids may be a useful addition to current analgesic treatments. However, to realize the full potential of cannabinoids suggested by preclinical data, it is likely that peripheral CBCannabis chemotypes expressing high titers of each component for future study.The entourage effect is the term used to describe enhancement of efficacy, with related improvement in overall therapeutic effectiveness, derived from combining phytocannabinoids and other plant-derived molecules.Cannabis-derived terpenes include limonene, myrcene, α-pinene, linalool, β-caryophyllene, caryophyllene oxide, nerolidol, and phytol.A chemical class known as the terpenes shares a precursor molecule with phytocannabinoids; they are all flavor and fragrance components common to human diets. Terpenes have been designated “generally recognized as safe” (GRAS) by the US Food and Drug Administration and other regulatory agencies. Cannabis-based medicinal extracts. Of particular interest are the phytocannabinoid–terpene interactions that could produce synergy with respect to treatment of pain and inflammation. Phytocannabinoid–terpene synergy increases the likelihood that an extensive pipeline of new therapeutic products is possible from this age-old plant.Terpenes are quite potent and affect animal and even human behavior when inhaled in very low concentrations. They display unique therapeutic effects that may contribute meaningfully to the entourage effects of Cannabis pharmacology—and specifically analgesia—have been scientifically demonstrated. Preclinical and clinical data indicate that cannabinoids administered together are more effective at ameliorating neuropathic pain than the use of a single agent.106The synergistic contributions of cannabidiol to 2 receptor at nanomolar concentrations and acts as a full agonist. β-Caryophyllene and cannabidiol occur abundantly in Cannabis sativa. So this plant species produces at least two entirely different chemical substances able to target CB2 receptors differentially. While studies on the pharmacokinetics of β-caryophyllene are still on-going, it is already clear that this terpene is readily bioavailable. Unlike many polyphenolic natural products, it is not metabolized immediately but shows a Tmax >1 h after one single oral administration. Orally administered β-caryophyllene (<5 mg·kg−1) produces strong anti-inflammatory and analgesic effects in wild-type mice but not in CB2 receptor knock-out mice, which is a clear indication that it may be a functional CB2 ligand.107The terpene β-caryophyllene is found in a number of commonly available plants, including black pepper, cinnamon, clove, and other spices. It selectively binds to the CB2 receptor-dependent manner.2 ligands β-caryophylleneinhibits the pathways triggered by activation of the toll-like receptor complex CD14/TLR4/MD2, which typically leads to the expression of pro-inflammatory cytokines  and promotes a Th1 immune response that plays a critical role in neuroinflammation, sensitization, and pain.2 receptor. Indeed, in cases of intractable or difficult-to-control pain, combination therapy with small doses of opioid and non-psychoactive cannabinoid receptor agonists may be an alternative way to circumvent the undesirable side effects of opioids yet obtain far greater analgesic efficacy than achieved with cannabinoids alone.110On-going studies show that β-caryophyllene is effective at reducing neuropathic pain in a CBCannabinoids may have another therapeutic benefit in managing chronic pain, with regard to sleep. Not only does normalized sleep improve pain relief and mood disorders associated with both poor pain control and poor sleep patterns, but there is significant risk of sleep-disordered breathing associated with central nervous system (CNS) drugs used to treat chronic pain.112Cannabis among most patients and acceptability among health care professionals and regulatory agencies.The phytocannabinoids have efficacy in the treatment of various chronic pain conditions with greatest promise as a therapeutic adjunct in treating peripheral and central neuropathic pain and inflammation-mediated chronic pain. However, the smoked route of administration and the psychoactivity of THC—with associated concerns about abuse and long-term cognitive adverse effects—continue to pose serious and significant barriers to obtaining benefit from A formidable barrier to oral bioavailability resides in the pharmacokinetics of naturally occurring and synthetic cannabinoids, but relatively slow elimination may provide clinical utility through prolonged duration of therapeutic effects once these agents gain entry into the systemic circulation. The phytocannabinoids are metabolized rapidly in the liver, undergoing extensive hepatic first-pass metabolism.116Putting these pharmacologic, clinical, and societal issues together, the direction for the future resides in the development of orally administered, highly bioavailable, non-psychoactive phytocannabinoid products that also take advantage of the entourage effect, to provide the millions of people living with debilitating pain a comparatively safe and effective form of relief.


Voltage- and Ca2+-dependent L-type Ca2+ channels (LCCs) allow for Ca2+ entry into the myocyte, which then binds to nearby ryanodine receptors (RyRs) and triggers Ca2+ release from the sarcoplasmic reticulum in a process known as Ca2+-induced Ca2+ release. The highly coordinated Ca2+-mediated interaction between LCCs and RyRs is further regulated by the cardiac isoform of the Ca2+/calmodulin-dependent protein kinase (CaMKII). Because CaMKII targets and modulates the function of many ECC proteins, elucidation of its role in ECC and integrative cellular function is challenging and much insight has been gained through the use of detailed computational models. Multiscale models that can both reconstruct the detailed nature of local signaling events within the cardiac dyad and predict their functional consequences at the level of the whole cell have played an important role in advancing our understanding of CaMKII function in ECC. Here, we review experimentally based models of CaMKII function with a focus on LCC and RyR regulation, and the mechanistic insights that have been gained through their application.Excitation-contraction coupling (ECC) in the cardiac myocyte is mediated by a number of highly integrated mechanisms of intracellular Ca One such signaling pathway is that involving the Ca2+/calmodulin (CaM)-dependent protein kinase (CaMKII), which targets and functionally regulates a number of proteins that are central to cardiac excitation-contraction coupling (ECC), and has been identified as a promising new target for antiarrhythmic therapy in patients with heart failure  and ryanodine receptor (RyR) regulation, and the mechanistic insights that have been gained through their application.Cardiac electrophysiology is a discipline with a rich and deep history dating back more than a half-century. Much of our understanding of fundamental biological mechanisms in this field comes from experimental research coupled with integrative mathematical modeling. The goal of this modeling has been to achieve a quantitative understanding of the functional mechanisms and relationships that span from the level of molecular structure and function to integrated cardiac myocyte behavior in health and disease. Advances in computational techniques and hardware have led to the recent development of many sophisticated large-scale models of heart tissue, in which these electrophysiological myocyte models are the fundamental building blocks. Some of the most fundamental advances in computational cell biology, including formulation of dynamic models of voltage-gated ion channels, mitochondrial energy production, membrane transporters, intracellular Ca2+. The junctional SR (JSR) is the portion of the SR most closely approximating  via voltage-gated LCCs and triggers the opening of RyRs, and hence JSR Ca2+ release, is known as Ca2+-induced Ca2+ release (CICR) and comprises the initial transduction event of ECC. Recent imaging studies have demonstrated that dyadic clefts are small, where junctions contain an average of 14 RyRs, with some clusters incompletely filled with RyRs, and with a large fraction of clusters closely spaced within 20–50 nm, suggesting that smaller clusters may act together to function as a single site of CICR  and Ca2+ transients, and detection of local Ca2+ release events known as Ca2+ sparks  to the CaMKII regulatory domain in response to beat-to-beat transient increases of intracellular Ca2+ concentration. In cardiac myocytes, an activated CaMKII molecule can undergo autophosphorylation by neighboring subunits at threonine amino acid residues in its regulatory domain, which allows the kinase to retain activity even upon dissociation of Ca2+/CaM  and the SR Ca2+-ATPase (SERCA). The model predicted that CaMKII plays a critical role in the rate-dependent increase of the cytosolic Ca2+ transient by increasing ECC gain, but that it does not play a significant role in rate-dependent changes of AP duration. Similar results were obtained in the human cardiac AP model of O'hara et al. (+ current (INa), and the transient outward K+ current (Ito1). The HRd model was later updated and used to investigate the role of altered CaMKII signaling in the canine infarct border zone , and that disruption of the regulatory system itself  leads to the most aberrant physiological phenotypes. Livshitz and Rudy . This model identifies Ca2+ alternans as the underlying mechanism for electrical alternans, both of which were eliminated with CaMKII inhibition, suggesting this as an antiarrhythmic strategy. Zang et al. , it clearly demonstrates that the multiple targets of CaMKII interact to produce net alterations of integrated cellular function which are difficult to predict from knowledge if its functional effects on isolated individual targets. Saucerman and Bers  but not in the cytosol is a key factor that would influence the way in which local signaling mechanisms would be incorporated into models that followed that included detailed reconstruction of dyadic Ca2+ dynamics. In one such model, Soltis and Saucerman . Recently, Morotti et al.  correlates well with a CaMKII-mediated shift in modal gating of LCCs . Following incorporation into the canine cardiac myocyte model , as shown in Figure 2O2 . The simulated increase in ICaL via oxidized CaMKII corroborates the experimental findings of Song et al. (in silico model of H2O2 exposure discussed here predicts that the fraction of LCCs gaiting in mode 2 will shift from 5% in control (absence of ROS) to 7% upon this increase in ROS. Using this model, the simulated diastolic RyR leak also increases in response to oxidative stress (~44%) primarily as an indirect consequence of increased Ca2+ influx via LCCs. Wagner et al. . They, however, provide additional evidence indicating that their observed increase in Ca2+ leak does not require the presence of CaMKII, suggesting an important role for CaMKII-independent mechanisms of ROS-mediated alteration of cardiac ECC as well.Along these lines, Foteinou et al.  have recg et al.  who demor et al.  recently2+ mismanagement leads to decreased scavenging resources and accounts for ROS overflow as is believed to occur in heart failure (Hill and Singal, As these ROS-mediated effects need to be further examined, future studies will focus on establishing quantitative links between cellular ROS and redox balance, CaMKII activity and function, ECC, and whole-cell electrophysiology. Recently, Gauthier et al. ,b develo2+ ions, CaM, CaMKII, LCCs, and RyRs interact in the cardiac dyadic junction both at rest and during triggered ECC events. On an intermediate scale, models have predicted the consequences of normal and abnormal CaMKII signaling on whole-cell Ca2+ cycling, effects of ROS imbalance, AP shape, and the generation of cellular arrhythmias such as EADs. Incorporation of these cellular models into higher scale tissue simulations has provided important insight into the relationship between CaMKII function, electrical wave conduction velocity, and the emergence of arrhythmogenic substrates in diseased tissue. The continuum of biological scales spanned by these models allows for the development of multiscale approaches whereby we can predict and understand the emergence of macroscale phenotypes as a consequence of CaMKII-mediated molecular signaling events. A great deal of evidence now implicates CaMKII as a nexus point linking heart failure and arrhythmias (Swaminathan et al., 2+ leak and/or increased likelihood of spontaneous Ca2+ release events and DADs, as the key mechanism underlying arrhythmogenesis. Others (Hashambhoy et al., 2+ levels and AP properties, including the appearance of EADs. These mechanisms may co-exist in heart failure, and it is likely that both play an important role. Moreover, CaMKII modulation of other targets such as PLB, INa, and K+ currents, many of which have been incorporated into the above cell models, adds additional layers of complexity to the task of interpreting and predicting the mechanisms by which CaMKII signaling alters cardiac function in health and disease. As a result of the breadth and diversity of CaMKII targets, strategies that involve broad inhibition of CaMKII activity will impact and alter the function of many cellular subsystems, with complex consequences that may not all be beneficial. As mathematical models of CaMKII signaling in the cardiac dyad and beyond are further developed, they will play a key role in identifying target-specific therapeutic strategies for improving myocardial function and reducing arrhythmias.Integrative modeling of cardiac ECC, cell signaling, and myocyte physiology has played a critical role in revealing mechanistic insights across a range of biological scales. With respect to CaMKII function at the smallest scale, models have shed light on how CaThe 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.


The use of biomarkers of brain injury in pediatric neurocritical care has been explored for at least 15 years. Two general lines of research on biomarkers in pediatric brain injury have been pursued: (1) studies of “bio-mediators” in cerebrospinal fluid (CSF) of children after traumatic brain injury (TBI) to explore the components of the secondary injury cascades in an attempt to identify potential therapeutic targets and (2) studies of the release of structural proteins into the CSF, serum, or urine in order to diagnose, monitor, and/or prognosticate in patients with TBI or other pediatric neurocritical care conditions. Unique age-related differences in brain biology, disease processes, and clinical applications mandate the development and testing of brain injury bio-mediators and biomarkers specifically in pediatric neurocritical care applications. Finally, although much of the early work on biomarkers of brain injury in pediatrics has focused on TBI, new applications are emerging across a wide range of conditions specifically for pediatric neurocritical care including abusive head trauma, cardiopulmonary arrest, septic shock, extracorporeal membrane oxygenation, hydrocephalus, and cardiac surgery. The potential scope of the utility of biomarkers in pediatric neurocritical care is thus also discussed. This includes AHT, CA, and other pediatric neurocritical care conditions where brain injury biomarkers are showing promise.c) after pediatric TBI  based on pre-clinical studies  represents a mechanism of secondary damage that has been receiving increased attention recently, particularly as new imaging modalities are revealing the scope of this process , the astrocyte markers S100β and glial fibrillary protein (GFAP), and the axonal injury marker MBP. After demonstrating robust increases in NSE and S100β in CSF in infants and children with severe TBI  vs. placebo (n = 11) before surgery in infants, plasma levels of NSE, S100β, cytokines, and C-reactive protein were assessed (Bhutta et al., Finally, several groups have tested serum biomarkers of brain injury in the setting elective cardiac surgery in children (Abdul-Khaliq et al., It is an exciting time for biomarker development and exploration of bio-mediators in pediatric neurocritical care and rewarding that after over 15 years of work in this area, use of these tools may become standardized and incorporated into routine clinical use for diagnosis, prognosis and other aspects of patient management. Assessment of bio-mediators and biomarkers in CSF and serum is also helping to define therapeutic targets and provide theragnostic value in monitoring treatment efficacy. Brain injury biomarkers may also guide patient stratification for clinical trials – to help define the best sample for future RCTs or help show treatment effects. This could be important given the many failures of trials in TBI and the heterogeneity of this and other conditions in pediatric neurocritical care. We look forward to the development of point of care technology for brain injury biomarker applications in pediatric neurocritical care.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.


Gorillagorilla lower second molars (M2) differing in wear stages. Our results show that in unworn and slightly worn M2s tensile stresses concentrate in the grooves of the occlusal surface. In such condition, the trigonid and the entoconid-hypoconulid crests act to reinforce the crown locally against stresses produced along the mesiodistal groove. Similarly, the protostylid is shaped like a buttress to suffer the high tensile stresses concentrated in the deep buccal groove. These dental traits are less functional in the worn M2, because tensile stresses decrease physiologically in the crown with progressing wear due to the enlargement of antagonistic contact areas and changes in loading direction from oblique to nearly parallel direction to the dental axis. This suggests that the wear process might have a crucial influence in the evolution and structural adaptation of molars enabling to endure bite stresses and reduce tooth failure throughout the lifetime of an individual.Most of the morphological features recognized in hominin teeth, particularly the topography of the occlusal surface, are generally interpreted as an evolutionary functional adaptation for mechanical food processing. In this respect, we can also expect that the general architecture of a tooth reflects a response to withstand the high stresses produced during masticatory loadings. Here we use an engineering approach, finite element analysis (FEA), with an advanced loading concept derived from individual occlusal wear information to evaluate whether some dental traits usually found in hominin and extant great ape molars, such as the trigonid crest, the entoconid-hypoconulid crest and the protostylid have important biomechanical implications. For this purpose, FEA was applied to 3D digital models of three  Since decades scholars have focused their attention on the morphology of the occlusal surface of human and non-human primate teeth to gain insight on the food items each species is more adapted to process and to improve our understanding of early hominin diets and dietary adaptations –5. Even However, during food processing teeth must solve another equally important function, namely they should be designed to resist failure while distributing forces produced during masticatory loading to their supporting structures –12. ThesResults from fracture mechanics suggest that, at least in great apes, dental material properties are less likely to be of concern than dental morphology  in the load-bearing capacity of the teeth ,14. WithLess attention, however, has been devoted to understand whether the external geometry of the teeth might optimize resilience on stress distribution. In an attempt to interpret the load-dissipation behavior of great ape molars, Macho and Spears  used twoDespite these works, little is known about the functional biomechanics of external dental features, mainly because all of the above studies have much simplified the complex three-dimensional (3D) geometry of the tooth either using sections (2D approach), or modeling the tooth as cone shape-like, or applying unrealistic occlusal loadings.Gorillagorilla lower left second molars (LM2) during maximum intercuspation tooth-to-tooth contact, which might be more damaging to the tooth crown than food-tooth contacts because of increased localized stresses. The above mentioned dental traits  are well expressed in gorilla molars, which are also characterized by tall cusps, long shearing crests and relatively thin enamel [2s differ in wear stages, we also aim to evaluate the effects of a reduced relief through wear on the stress distribution. Finally, a digital simulation was carried out to assess whether interrupting the continuity of the crests  might affect the pattern of stress distribution.In this pilot study we used 3D FEA  with a nn enamel , presuman enamel . As our Gorillagorilla female skulls from the Museum für Naturkunde, Humboldt Universität, Berlin, Germany were selected for 3D FEA. The three specimens  were selected both because of their complete dentition and because their LM2 differing in wear stage (after Smith [Three er Smith ): ZMB-311) and mesially to the socket of the lower left third molar (LM3). Consequently, we considered only the bone tissues surrounding the LM2. Segmentation of the LM2 dental tissues  and its supporting dental tissues  was carried out in Avizo 7  , only the external surface of the teeth was segmented. The final refinement of the digital models was carried out in Rapidform XOR2 .To reduce the size of the digital models, we cut the mandibles distally to the socket of the lower left first molar , where volumetric meshes , scaling of the volumetric meshes was not required.Boundary constraints were applied to the medial and distal cut surfaces of the mandible section following indications provided by Benazzi et al. . The loaThe stress state patterns were qualitatively and quantitatively compared according to the first maximum principal stresses criterion for brittle materials ,32,44,452 was digitally modified in HyperWorks software to study the relationships between the presence/absence of dental traits and the pattern of stress distribution. In the simulation (hereafter referred as ZMB-31435sim), the midtrigonid and entoconid-hypoconulid crests  and specimen ZMB-31626 LM2 (wear stage 3) tensile stresses are observed in the grooves of the occlusal surface. Sections of the enamel along the buccolingual and mesiodistal grooves , usually concentrate tensile stresses. Both specimens also experience tensile stresses in the groove between the two main buccal cusps (protoconid and hypoconid). However, in ZMB-31626 LM2 tensile stresses interest the entire buccal groove, ending at the level of the protostylid, which is well-developed and shaped like a shelf , the lingual groove, which bifurcates in correspondence of an accessory cusp (cusp 7), as well as the anterior and posterior fovea. The propagation of the tensile stresses along the mesiodistal groove is interrupted by the trigonid and entoconid-hypoconulid crests, which are particularly massive in ZMB-31435 LM2 .In ZMB-31626 LM1435 LM2 , 6A,B. I2 (wear stage 4) the force was applied on a larger portion of the occlusal surface when compared with ZMB-31626 LM2 and particularly with ZMB-31435 LM2, encompassing almost all the cusps. The tooth is basically subjected to compressive loads resulting in compressive contact stresses. Tensile stresses of low magnitude are only observed between the protoconid and hypoconid cusps, but they do not interest the buccal groove  bend the crown mainly about the buccolingual axis, inducing tensile stresses along the buccolingual groove. In such condition, the protostylid concentrates high tensile stresses and reduces tensile stresses at the thin-enamelled cervix, and thus it might protect the tooth from marginal cracking as previously suggested [2 protostylid fragments the tensile stresses in the double fold structure. The discontinuity of the slope of the buccal groove creates an irregular change in geometry, making the tensile stress locally concentrated around the irregularly geometrical area, which is relatively far away from the cervix. Specimen ZMB-31435 LM2 confirms that the protostylid is a dental feature subjected to tensile stresses, but it also emphasizes that poorly expressed protostylids are less suitable to reduce tensile stresses at the cervix. Most of the tensile stresses observed in ZMB-31435 LM2 during maximum intercuspation are concentrated in the central fossa  might be also important to reinforce the crown, limiting dangerous effect of high tensile stresses due to occlusal loadings.Homo specimens show intermediate occlusal reliefs and surface sloping between Gorilla and Pan [Finally, it is important to underline some limits of our FE analysis that should be addressed in future works. We have attributed isotropic property to the enamel, but it has been suggested that enamel should be considered anisotropic . We havend Pan , we expeTable S1(DOC)Click here for additional data file.Video S1The OFA calculates a relief-guided pathway of antagonistic tooth rows from collision detection, deflection and break-free algorithms for user-defined timesteps. The contact areas of maximum intercuspation have been chosen for applying loads in the FE models.(MP4)Click here for additional data file.Video S2The OFA calculates a relief-guided pathway of antagonistic tooth rows from collision detection, deflection and break-free algorithms for user-defined timesteps. The contact areas of maximum intercuspation have been chosen for applying loads in the FE models.(MP4)Click here for additional data file.Video S3The OFA calculates a relief-guided pathway of antagonistic tooth rows from collision detection, deflection and break-free algorithms for user-defined timesteps. The contact areas of maximum intercuspation have been chosen for applying loads in the FE models.(MP4)Click here for additional data file.