As an example, we are only beginning to understand the relevance of non-coding genome to organism aging or the existence of an epigenetic memory with transgenerational inheritance. Addressing these topics will be fundamental for exploiting epigenetics phenomena as markers of aging-related diseases or as therapeutic targets.Aging is a complex process that results in compromised biological functions of the organism and increased susceptibility to disease and death. Although the molecular basis of aging is currently being investigated in many experimental contexts, there is no consensus theory to fully explain the aging process. Epigenetic factors, including DNA methylation, histone modifications, and microRNA expression, may play central roles in controlling changes in gene expression and genomic instability during aging. In this Hot Topic review, we first examine the mechanisms by which these epigenetic factors contribute to aging in diverse eukaryotic species including experimental models of yeasts, worms, and mammals. In a second section, we will emphasize in the mammalian epigenetic alterations and how they may affect human longevity by altering stem cell function and/or somatic cell decline. The field of  Aging has been defined as a process of cellular senescence of adult tissues that results in compromised stress response, greater homeostatic imbalance, and elevated risk of disease , the epiSacharomyces cerevisiae, whereas activation of Sir2 significantly extents yeast lifespan   . The insS. cerevisae Sir2, Sirt1, have been observed in premature aging of mice. In contrast to yeast Sir2, which specifically deacetylates histones, Sirt1 act as a deacetylase of other proteins than histones, such as p53 acetylation  have been identified. As an example of common functions, decreased levels of the most highly related to see next chapters). In this way, and similarly to worms, loss of trimethylation at H3K27 by donwregulation of the EzH2 histone methyltransferase in humans could be associated with aging-related defects, such as Hutchinson-Gilford progeria syndrome  showed hypermethylation of specific CpG islands, most of which are related to homeobox genes   of proteins in regulation of stem cell aging has been extensively studied. The best-known PcG regulator of adult stem cells is BMI1, a member of the Polycomb repressive complex 1 (PRC1) and a critical protein for self-renewal of blood and brain stem cells  enhances growth of HSCs under certain experimental conditions  and age in non-tumorigenic gastric epithelia , but rather as the product of several epigenetic factors working in concert. Early studies showed that global levels of K20H4me3 were increased in several organs of rats older than 30 months old (chromatin instability)  . Some authors have recently described a bimodal effect observed during aging in several human tissues: loci in CpG islands gained methylation with age, whereas loci outside these islands lost it  (Perhaps the most important question is: fection) . The preCan the epigenetic levels of specific genes be employed as markers of so-called bio-age? In other words: Is it possible to predict a person’s age based on their epigenetic status? An exciting new application of epigenetic studies has recently been developed to answer these very questions. Unfortunately, despite its promise, on the causal association between human aging and epigenetic changes remains in its infancy. Probably the greatest barriers to progress in this area are the long period of time required for assessing longevity in humans and the enormous degree of variation among individuals owing to environmental factors. To date, changes in specific epigenetic marks have been correlated with aging, but the ‘great hit’ in epigenetic control of aging will occur when we will be able to understand how epigenetic factors relate to each other. The integration of DNA modifications, histone marks, and alterations of non-coding RNAs will surely pave the way to defining reference epigenomes of aging and to identifying epigenetic alterations associated with aging and disease states.


Ednrb gene, slEdnrb, was introgressed into the F344 strain, almost 60% of F344-sl/slEdnrb pups did not show any symptoms of aganglionosis, appearing healthy and normally fertile. These findings strongly suggested that the severity of HSCR was affected by strain-specific genetic factor (s). In this study, the genetic basis of such large strain differences in the severity of aganglionosis in the rat model was studied by whole-genome scanning for quantitative trait loci (QTLs) using an intercross of (AGH-slEdnrb×F344-slEdnrb) F1 with the varying severity of aganglionosis. Genome linkage analysis identified one significant QTL on chromosome 2 for the severity of aganglionosis. Our QTL analyses using rat models of HSCR revealed that multiple genetic factors regulated the severity of aganglionosis. Moreover, a known HSCR susceptibility gene, Gdnf, was found in QTL that suggested a novel non-coding sequence mutation in GDNF that modifies the penetrance and severity of the aganglionosis phenotype in EDNRB-deficient rats. A further identification and analysis of responsible genes located on the identified QTL could lead to the richer understanding of the genetic basis of HSCR development.Hirschsprung disease (HSCR) exhibits complex genetics with incomplete penetrance and variable severity thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. As reported previously, when the same null mutation of the  RET proto-oncogene EDNRB) EDN3) GDNF) SOX10NRTNECE1ZFHX1BPHOX2BKIAA1279TCF4Hirschsprung disease (HSCR) is a congenital malformation characterized by the absence of intramural ganglion cells along variable lengths of the distal gut. Due to the lack of ganglia, the stool cannot be passed through the colon, and the bowel wall is dilated sl) is a spontaneous null mutation that has a 301 bp deletion in the rat Ednrb gene that results in the absence of a functional receptor protein slEdnrbsl mutation, further, introgressed this mutation into LEH and F344 strains to produce two congenic strains: LEH-slEdnrb and F344-slEdnrbsl/slEdnrb rats, only 20% of pups survived until weaning; whereas in F344-sl/slEdnrb rats, 100% of pups survived to weaning. Interestingly, almost 60% of F344-sl/slEdnrb pups did not show any symptoms of aganglionosis, appearing healthy and normally fertile and showing normal body weight gain. Thus, we concluded that variation in the penetrance and survival was attributable to distinct differences in the severity of aganglionosis, and resistant genes in the genetic background of F344 significantly modulated the severity of the aganglionosis phenotype.Many researchers have used inbred models to search the unknown susceptibility or modifier genes of aganglionosis slEdnrb-mutated rats and uses this variation to identify modifiers that are influencing the aganglionosis aspect of the phenotype. These studies have been facilitated by the ability to control genetic background in inbred lines of slEdnrb rats that are not possible in patient studies.This study focuses on the variation in aganglionosis between individual sl/slEdnrb rats showed aganglionosis phenotypes. In our previous study, we found that when the sl mutation was introgressed into the F344 strain, the phenotype of aganglionosis was strongly modified sl/slEdnrb rats at postnatal 14 day exhibited abnormal dilation of the intestines resulting from the absence of ganglion cells in a long segment beyond caecum. In contrast, in F344-sl/slEdnrb pups at postnatal 14 day, an enlarged small intestinal phenotype  was not found. We have confirmed that the variation in the expressivity of this disease between these two strains was caused from the extent of aganglionosis by whole-mount acetylcholinesterase (AChE) staining 2 (AGH×F344) sl/slEdnrb progenies. The F2 animals (n = 410) were produced by heterozygotes mating between AGH and F344 strains and then 96 sl/slEdnrb pups were selected to phenotype based on the difference in skin pigmentation pattern between homologous mutants and other genotype rats or genotyping for the slEdnrb mutation . The number of sl/slEdnrb pups was consistent with the anticipated 25% transmission ratio. Microscopic examination of sl/slEdnrb intestines stained by AChE was used to appraise the length of aganglionosis gut, then the extent of aganglionosis was calculated as a ratio of length of the aganglionosis intestine to the length of the entire large intestine used as a quantitative trait in individual animals. We also recorded the gross intestine weight and body weight of pups at postnatal 14 day, and to fully capture the difference between sick and healthy ones, the ratio of gross intestine weight (gross intestine weight/body weight) was calculated, which demonstrates the expressivity of megacolon directly. We found that there was a high correlation between the aganglionosis extent and the ratio of gross intestine weight in F2 populations  composed of each homozygote of AGH and F344, and the heterozygotes were nearly the same as that of the F1 progenies (1.08 in ratio of aganglionosis extent).The range of the aganglionosis extent for each progeny is presented as black characters in 2 progenies are arranged by size of the ratio of aganglionosis extent. The trait-value graphs in both males and females showed similar gentle curves, which suggests that the mild aganglionosis extent in F344- sl/slEdnrb rats are under the control of polygenic inheritance.In D2Mit5 marker position, showing the highest linkage to the severity of aganglionosis (LRS = 23.9), was designated ‘Lrag1 (Locus of resistance to aganglionosis 1)’. The epistatic interaction between markers also was searched by MapManager QTXb, but no significant interaction was found.Final results of interval mapping were considered suggestive, significant, or highly significant linkages when the threshold likelihood ratio statistics (LRS) were 9.9, 20.2, and 30.4, respectively. As shown in sl/slEdnrb modifiers on the severity of aganglionosis, we evaluated complete genotype information in the total F2 distribution. F344 alleles at modifier locus on chromosome 2 decreased the extent of gut length affected by aganglionosis .By bioinformatics methods combining genome annotation with literature searches, some biologically relevant genes within modifier intervals have been identified successfully The enteric nervous system (ENS) mostly derives from migratory vagal neural crest cells. A minority of the foregut ENS also arise from migratory anterior trunk neural crest cells of the posterior vagal region RET and EDNRB was found in an association study conducted on Mennonite family with the W276C mutations in the EDNRBRET and EDNRB pathways has also been demonstrated in mice +/−RET and slEdnrb, or +/−RET and sEdnrb genes, had no intestinal aganglionosis, whereas +/−RET mice with the homozygous sEdnrb or heterozygous sEdnrb/slEdnrb mutations showed megacolon In human with HSCR, the genetic interaction between mutations in sl/slEdnrb on rat chromosomes 2. This locus contains a known aganglionsis susceptibility gene, GDNF. The GDNF ligand activates the RET receptor through the assembly of a multiprotein complex, including the GDNF family receptor alpha1  molecule, which have important functions in the development and maintenance of sensory, enteric, sympathetic and parasympathetic neurons and a variety of non-neural tissues RET and EDNRB has been well described in human patients and confirmed in mice Gdnf ligand and Ednrb. Though we failed to find the sequence difference of coding region in Gdnf gene between both rat strains, we cannot completely exclude the possibility that Gdnf is a responsible gene because the non-coding regulatory region of Gdnf remains unknown which could affect the Gdnf expression in a specific timing that is important for the ENS development. Such case has been found in human with HSCR that a non-coding RET variant within a conserved enhancer-like sequence in intron 1 is significantly associated with HSCR susceptibility Gdnf in embryonic day 15.5. It remains unknown whether there is difference in the expression level at other developmental stage of ENS. The spacial and temporal control of gene expression in the complex process of ENCCs colonization of the gut is very important. Ednrb is genetically required in the mouse for ENS development in vivo from embryo day 10 to embryo day 12.5 RET and EDNRB signaling pathways only influenced the ENS, no impact on melanocyte, retinal choroid, and kidney development, which showed a tissue-specific interaction Gdnf interacting with Ednrb mutation in AGH strain resulted in the serious aganglionosis.In this study, we used quantitative trait locus (QTL) mapping to detect the genetic loci that contribute to differences in phenotypic variation of aganglionosis extent between F344 and AGH strains with the same null mutations. Using this comprehensive approach, we have successfully identified a modifier locus of Lrag1. Some of these approaches include comparisons of gene expression levels of F344 and AGH rats in intestine tissues using microarray and next-generation RNA sequencing technologies. This analytical combination that includes QTL mapping and gene expression profiles has proven useful in the selection of candidate genes.Using congenic techniques, we are currently attempting to generate rat strains that harbor QTLs from one selection line on the opposite line to investigate whether each allele has a different effect on the phenotype. At the same time, several approaches are currently being employed to identify candidate genes located on A lack of existing comprehensive information on the susceptibility genes and interaction between susceptibility loci or modifier loci contributing to HSCR disease in the genetic background makes it difficult to understand the genetic base for many cases of HSCR. However, our study localized chromosomal sites where the allelic differences in genes presented in F344 and AGH rats and strongly affected the occurrence and severity of HSCR using QTL analysis. This study provided the new evidence that Hirschsprung disease is the consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut.slEdnrb (AGH) slEdnrb (F344) 2 animals (n = 410), in which 96 sl/slEdnrb pups were selected to phenotype based on the difference in skin pigmentation pattern. Namely, heterozygous AGH-slEdnrb rats had pigmented heads, backs, and tails. In contrast, homozygous mutant rats had almost no pigmentation on their heads previously described albino (tyrosinase mutant) strain, albino F2 rats were genotyped to distinguish homozygote from heterozygote and wildtype by PCR. Animals were genotyped for slEdnrb mutation using primers (F-CCTCCTGGACTAGAGGTTCC and R-ACGACTTAGAAAGCTACACT) that flank the site of the 301-base deletion. PCR products were electrophoresed in 2% agarose gels to distinguish the wild (511 bp) and mutant (210 bp) alleles. To determine the aganglionosis extent by strain, AGH (n = 33), F344 (n = 35), F1 (n = 32) were raised. Animals were maintained in specific pathogen-free conditions with feeding and drinking allowed ad libitum. All research and experimental protocols were conducted according to the Regulation for the Care and Use of Laboratory Animals of Hokkaido University and were approved by the Animal Care and Use Committee of Hokkaido University .Heterozygous AGH/Hkv-http://uswest.ensembl.org). Amplified samples were electrophoresed in 10% acrylamide gels, stained with ethidium bromide, and photographed under an ultraviolet lamp.The genome-wide scan was performed using 96 intercross progenies. Genomic DNA was extracted from tail clips of these intercross progenies using a standard protocol and was subjected to a genome-wide scan at 10–30 Mbp resolution using 94 polymorphic microsatellite markers . PCR priThe guts from pups at postnatal day 14 were dissected as a single piece from the proximal esophagus to the distal colon. Mesenteric attachments and the pancreas were removed, and the guts were then processed for acetylcholinesterase (AChE) whole-mount staining using routine protocols to visualize enteric ganglia To identify the aganglionosis modifier loci, genotyping data and the ratio of aganglionosis extent were analyzed by MapManager QTXb slEdnrb modifier loci, the t-test was performed to compare the mean values for data sets.For comparison of allele effect at


The study of ancient skeletal pathologies can be adopted as a key tool in assessing and tracing several diseases from past to present times. Skeletal fluorosis, a chronic metabolic bone and joint disease causing excessive ossification and joint ankylosis, has been only rarely considered in differential diagnoses of palaeopathological lesions. Even today its early stages are misdiagnosed in endemic areas.Herculaneum victims of the 79 AD eruption, resulting from long-term exposure to high levels of environmental fluoride which still occur today.Endemic fluorosis induced by high concentrations of fluoride in water and soils is a major health problem in several countries, particularly in volcanic areas. Here we describe for the first time the features of endemic fluorosis in the Our observations on morphological, radiological, histological and chemical skeletal and dental features of this ancient population now suggest that in this area fluorosis was already endemic in Roman times. This evidence merged with currently available epidemiologic data reveal for the Vesuvius area population a permanent fluoride health hazard, whose public health and socio-economic impact is currently underestimated. The present guidelines for fluoridated tap water might be reconsidered accordingly, particularly around Mt Vesuvius and in other fluoride hazard areas with high natural fluoride levels. Fluorine is a widespread element in the earth's crust, being present in its ionic form of fluoride in a number of minerals, as well as in soil, water, plants, foods and even air Long-term intake of high doses of fluoride can have adverse effects on human health, including dental, musculoskeletal, reproductive, developmental, renal, endocrine, neurological, and genotoxic effects via recrystallization of hydroxyapatite into the more stable fluoroapatite, by which hydroxyl groups are replaced by fluorine ions In bones and teeth, fluorine ion exchange takes place Skeletal fluorosis is a chronic metabolic bone and joint disease caused by prolonged, excess ingestion of fluoride, mostly through water of endemic areas Even if skeletal fluorosis has been widely studied for more than 40 years, because some of the early clinical symptoms resemble those of osteoarthritis, the first clinical phases of skeletal fluorosis could be easily misdiagnosed HerculaneumHere we present the pathological condition of a significant group of victims caught by the 79 AD Vesuvius eruption , recentlDue to the peculiar burial conditions of these skeletons, specific attention has been paid to discriminate pathological vs. diagenetic alteration. Bone histology, porosity, and enrichment of chemical elements are diagenetic parameters that quantify the post-mortem osteoalteration. Bones buried for long periods absorb and accumulate fluoride from soil Herculaneum skeletons are particularly suited to palaeoepidemiologic investigation, which also has important implications for present-day populations. Detailed morphological, radiological, histological and chemical evaluation of the skeletal and dental features of these ancient people for the first time suggests that fluorosis was already endemic in Roman times. Our findings merged with currently available epidemiologic data strongly support the hypothesis of an enduring fluoride health hazard for the Vesuvius area population, whose public health impact is underestimated today.Being a unique cross section of the entire living population, the Herculaneum.This study was approved by the Ethics Committee for Biomedical Activities of the University of Naples, Azienda Ospedaliera Universitaria “Federico II”, Naples . The Superintendency of Pompeii granted field investigation and study of the human skeletal materials unearthed in the 1997–99 excavations of the water-front chambers at Herculaneum suburban area We analysed 76 human skeletons aged 0 to 52-years-old, excavated within the water-front chambers 5, 10 and 12 of the Herculaneum victims aged 0 to 52-years-old of both sexes, by Instrumental Neutron Activation Analysis (INAA) The bone amount of fluorine (F), sodium (Na) and calcium (Ca) was measured in the iliac crest and/or rib bones of 27 The fluoride content of volcanic ash was determined at the University of Notre Dame Fluoride Dating Service, using an ion-selective electrode (ISE) according to Shurr (1989) The degree of lesion involving spine and peripheral joints were evaluated by an ordinal scaling system. The degenerative changes were scored following the four-value scoring classification adopted by Jurmain i] on an ordinal scale  was assigned to each joint of individuals aged ≥15-years-old. The total measured score (Σ Pij = sum of scores of the j-th articulation in the i-th subject) was divided by the maximum measurable score assigned to each individual . The obtained relative and normalized skeletal lesion index (SLI) ranged from 0 to 1 :A 0-to-3 score  = 200.4 • age represents the rate of physiological intake per individual per year. This model shows that 99% of fluorine concentration variability is age-related (R2 = 0.961).The average bone fluorine concentrations varying between 14000 and 23300 ppm are a function of age as shown by the equation at age 0 . These vat age 0 , conside2 = 0.923), we obtained a new equation combining2 = 0.81) of the observed data with those expected. This indicates that the SLI index suitably describes the degree of joint lesions shown by the Herculaneum people as a result of the fluorine accumulating in their bones.Taking into account this last correlation and that the skeletal lesion index is fluorine concentration-related  clearly indicate fluoride poisoning. The fluorine concentration as a function of age  shows a Herculaneum, the difference by gender in skeletal lesion occurrence in relation to age would merit further investigation, given the higher occurrence of skeletal fluorosis in females >30 contrasting with the epidemiological evidence The regression line describing the annual variation rate of fluoride (ppm/years) shows a significant increase in fluoride concentration with age, and a correlation with the degree of pathological involvement of the spine and appendicular joints, as assessed by SLI index evaluation. This correlation of bone fluoride concentration with both duration of exposure and extent of bone lesions has been demonstrated in present-day patients affected by skeletal fluorosis, the severity of which was found to be related to the amount of fluoride incorporated into bone. Usually, fluorine can range from ca 500 to ca 3000 ppm in unaffected people, exposed to optimal fluoride intakes ≤1 mg/L Herculaneum, the concentration of fluoride in groundwater was found to be even higher (3.6 mg/L). But despite this fact, dental fluorosis appears less severe when compared with that from Mehrgarh, most likely as a result of the lower need of hydration in the more temperate Mediterranean climate. The prevalence of mottled enamel alterations at Herculaneum parallels the pathological condition exhibited by the victims' skeletons. Thus, the occurrence of endemic dental hypoplasia appears correlated to high fluoride intake during life, inferred from the high amount of fluoride that we determined in the bones.The widespread occurrence of osteoarthritis, osteophytosis, enthesopathy and fractures is particularly high in comparison with other Roman and pre-Roman communities Herculaneum enamel mottling is associated with high levels of linear enamel hypoplasia (LEH), which occurs commonly in most ancient populations Herculaneum residents.In ancient This palaeoepidemiologic scenario has likely remained unchanged for the Vesuvius area population till today. Currently, the maximum fluorine concentration of the water-bearing stratum is close to the current Maximum Contaminant Level (MCL) of 4 mg/L of drinking water, and within the range of concentrations able to induce crippling skeletal fluorosis Herculaneum residents At present, in volcanic and other areas where groundwater is contaminated with fluoride of natural origin, communities with normal nutritional intake exposed to fluoride water concentrations of ca 4 mg/L and daily total intake of ca 14.0 mg/day show prevalence of skeletal and dental fluorosis equivalent to those observed for the Herculaneum unequivocally points to endemic skeletal and dental fluorosis induced by environmental fluoride poisoning, still occurring today. A clinical-epidemiological investigation in schoolchildren from the Vesuvian towns The overall epidemiologic scenario that we detected for the ancient inhabitants of A recent report by the National Academy's National Research Council (NRC) Herculaneum now indicate that fluorosis was endemic already during Roman times. This evidence and currently available epidemiologic data show a permanent fluoride health hazard for the population living around Vesuvius. At present, the major public health and socio-economic impact of this hazard is underestimated. For several years, the local authorities have allowed a maximum fluoride content of 2.5 mg/L in tap water throughout the area, a value that exceeds WHO as well as national guidelines.Our findings on the pathologic skeletal and dental features of the ancient residents of Effects on the skeleton are the best indicators of the toxic responses to fluoride and are considered to have direct public health relevance. According to WHO recommendations, in areas with high fluoride levels and warm climate it would be appropriate to lower the maximum value of 1.5 mg/L established for naturally occurring fluoride in drinking water. Therefore in setting guidelines for fluoride in the densely populated Vesuvius area, the following predisposing factors for fluorosis should be better evaluated: ambient temperature, volume of water intake, other trace elements in the water, a diet based on beverages and food preparation in naturally fluoridated boiled water, and water storage methods. Bearing in mind that progressively higher fluoride intakes lead to increasing risks of dental and skeletal fluorosis, the adoption of low-cost defluoridation methods should be seriously considered and encouraged.In evaluating all the possible health consequences of exposure to fluoride concentrations higher than the established WHO parameters, it should also be taken into account that the maximum fluoride content of 5.0 mg/L accepted in natural mineral waters protects only the population over 15 years old and only if there is no exposure to fluoride from other sources, as experienced by communities living in volcanic and other fluoride hazard areas.Table S1Assessment of ligaments and tendons calcification and ankylosis in the postcranial skeleton of specimens aged 1 to 52-years-old. 91.8% of the individuals show ossification processes in at least one of the long or flat bones , with clavicle the most involved bone (88.2%). Ankylosis, mainly detectable in spine, foot toe distal interphalangeal joint and manubriosternal joint, affects at least one of these three anatomical sites in 39.2% of the individuals.(DOC)Click here for additional data file.Table S2Occurrence of osteophytosis and spondyloarthritis in spine joints of specimens aged ≥15-years-old. Osteophytic lesions (moderate+severe+ankylosis) increase towards lumbar joints , with 27.6% overall occurrence. Spondyloarthritic lesions (moderate+severe+ankylosis) occur in 18.5% of the joints, with lumbar vertebrae the most affected (22.8%).(DOC)Click here for additional data file.


Clinical studies suggest the comorbidity of functional pain syndromes such as irritable bowel syndrome, painful bladder syndrome, chronic pelvic pain, and somatoform disorders approaches 40% to 60%. The incidence of episodic or persistent visceral pain associated with these “functional” disorders is two to three times higher in women than in men. One of the possible explanations for this phenomenon is estrogen modulation of viscerovisceral cross-sensitization. While a central site of this modulation has been shown previously, our studies suggest a peripheral site, the dorsal root ganglion (DRG). Estrogens have remarkably wide range of functions including modulation of voltage-gated calcium channels (VGCCs) and purinoreceptors (P2Xs). Significantly, inflammation dramatically alters purinoception by causing a several fold increase in ATP-activated current, alters the voltage dependence of P2X receptors, and enhances the expression of P2X receptors increasing neuronal hypersensitivity. Gonadal hormones are thought as indispensable cornerstones of the normal development and function, but it appears that no body region, no neuronal circuit, and virtually no cell is unaffected by them. Thus, increasing awareness toward estrogens appears to be obligatory. However, E2 has a multiplicity of actions: membrane, cytoplasmic, and nuclear: E2 modulates cellular activity by altering ion channel opening, G-protein signaling, and activation of trophic factor-like signal transduction pathways i i  stronglyδ-fiber nociceptors gives rise to the initial sharp feeling of pain while on the contrary unmyelinated C-fiber nociceptors gives rise to the later, duller, burning pain sensation due to their different morphology. The cell bodies of these axons are located in the DRG.Nociceptors were defined as receptors by Sherrington more than a century ago (1906) that respond selectively to stimuli that cause tissue damage. The nociceptors can be divided into two main groups based on anatomical and functional properties: Activity in myelinated A2 and P2X3) have been very well studied and acknowledged as molecular detectors of noxious thermal, mechanical, or chemical stimuli i in cultured DRG neurons [2+]i may represent a level of DRG activation to noxious cutaneous stimulation, while ATP-induced changes in [Ca2+]i may reflect the level of DRG neuron sensitization to noxious visceral stimuli since ATP is released by noxious stimuli and tissue damage near the primary afferent nerve terminals [Some molecular targets have been identified as key players in the activation and sensitization of visceral nociceptors, notably ASICs, TTX-resistant Na channels, and the TRPV1 receptor . TRPV1 r neurons . Capsaicerminals .2+ permeability [2+ concentration changes before and after receptor activation [The expression of transient receptor potential vanilloid 1 (TRPV1) receptors is widespread in several areas of the nervous system, but it is particularly strong in DRG. TRPV1 receptors are at the cellular membrane as well as in the membrane of intracellular calcium stores . For pain transmission, the pain/temperature sensitive TRPV1 receptor is highly expressed in nociceptive neurons of the peripheral nerve system. TRPV1 receptors are activated by a wide variety of stimuli, both exogenous  and endogenous , and they mediate a nonselective cationic current with particularly high Caeability . The pottivation . Signifitivation  indicati2. Several authors have suggested that the sex differences in pain sensitivity and the prevalence of chronic pain disorders may result from a malfunctioning endogenous pain inhibitory response rather than an increase nociceptive activity [2/3. Painful stimuli initiate action potentials in the peripheral terminals of DRG neurons evoking release of excitatory neurotransmitters such as glutamate  release and activated ion channel receptor is the direct recording of the receptor response to its agonist and antagonist on isolated cells expressing the receptor, by means of calcium imaging or patch clamp recordings techniques. Histological and functional studies implied that the important receptors involved in the transmission and modulation of nociceptive inputs are expressed at the level of the peripheral and/or central terminals of DRG neurons .The fact that homeostatic changes are similar in all “functional” disorders suggests a model in which alteration in the neuronal circuits in predisposed individuals is triggered by the similar pathophysiology. Both physiologic and psychological variables appear to play important roles in the development of functional syndromes, and psychologically oriented treatments have a role in their management. Patients have an altered perception of visceral sensation that is typically manifested as hyperalgesia, an enhanced perception of pain. Pain is the symptom that patients with functional disorders list as the most depressing and is a major factor for consulting a physician. The functional characteristics of primary sensory  neurons and their receptors are usually investigated by means of a wide diversity of experimental models, both  rodents . The usein vivo activation. An important advantage is that these neurons can be studied apart from endogenous signals. A large body of literature supports the idea that E2 modulates nociceptive responses in functional pain syndromes. Within the context of our hypothesis, E2 modulation of nociceptive responses depends on the type of pain, its durations, and the involvement of other antinociceptive mechanisms. E2 modulates DRG neurons response to ATP, suggesting that visceral afferent nociceptors are modulated by E2, which may explain the observed clinical and animal gender differences in visceral hypersensitivity and suggests a potential target for mediating nociception. Future direction of research should include more experiments to study the role of peripheral ERs in visceral nociception using in vivo models. Thus, from a public health perspective, the outcome of this study will have a substantial impact, because it will increase our knowledge of nociceptive functional diseases, such as IBS, interstitial cystitis, and chronic pelvic pain, and help achieve a deeper understanding of gender differences presented in clinical aspects of these symptoms associated with various psychiatric disorders. Only a thorough understanding of the mechanism implicated in these phenomena can truly contribute to the design of new and more efficient therapies. Many illnesses affect women and men differently. Some disorders are more common in women, and some express themselves with different symptoms. It is very well documented that differences between the sexes exist in the prevalence and severity of a broad range of diseases, disorders, and conditions. In calling for greater focus on gender-based biomedical research, the clinical and scientific communities will be better equipped to identify barriers to the achievement of knowledge about gender differences-including ethical, financial, sociological, and scientific factors. In developing countries, pain accounts for nearly 20% of all primary health care visits. Studies have shown that at least one-third of patients with pain also suffer from depression and it affects more women than men. The new approach in pain research and treatment should address a crucial question in women's health and in visceral nociception in particular. Reaching further, this research is a liaison between the basic science work and the clinical aspects that are addressed through other disciplines such as anesthesiology (pain management), gastroenterology, obstetrics, and gynecology.Although it is generally accepted that each primary afferent neuron is a single sensory channel, several studies have challenged that view and demonstrate that a population of DRG neuron can innervate both the viscera and somatic tissues. The design of the proposed studies using retrograde labeling from the uterus and colon will address the possibility that the same primary afferent can innervate both reproductive and gastrointestinal organs (DRG neuron will have both retrograde tract tracing dyes). This new subset of dichotomizing fibers provides a novel pathway for sensitization of one viscus by another. Adult DRG neurons in short-term culture retain the expression of receptors (P2X and TRPV1) [


The labile cell iron (LCI) composition varies with metal concentration and substances with chelating groups but also with pH and the medium redox potential. Although physiologically in the lower μM range, LCI plays a key role in cell iron economy as cross-roads of metabolic pathways. LCI levels are continually regulated by an iron-responsive machinery that balances iron uptake versus deposition into ferritin. However, LCI rises aberrantly in some cell types due to faulty cell utilization pathways or infiltration by pathological iron forms that are found in hemosiderotic plasma. As LCI attains pathological levels, it can catalyze reactive O species (ROS) formation that, at particular threshold, can surpass cellular anti-oxidant capacities and seriously damage its constituents. While in normal plasma and interstitial fluids, virtually all iron is securely carried by circulating transferrin , in systemic iron overload (IO), the total plasma iron binding capacity is often surpassed by a massive iron influx from hyperabsorptive gut or from erythrocyte overburdened spleen and/or liver. As plasma Tf approaches iron saturation, labile plasma iron (LPI) emerges in forms that can infiltrate cells by unregulated routes and raise LCI to toxic levels. Despite the limited knowledge available on LPI speciation in different types and degrees of IO, LPI measurements can be and are in fact used for identifying systemic IO and for initiating/adjusting chelation regimens to attain full-day LPI protection. A recent application of labile iron assay is the detection of labile components in intravenous iron formulations  In extracellular fluids, essentially all the iron is safely carried by the protein transferrin (Tf) that shields the Fe(III) from the environment and renders it virtually redox-inactive as well as non-exchangeable with, or displaceable by, physiological substances or metals. Thus Tf-bound iron (TBI) is a tightly bound form of ferric iron that by definition is ous iron . That coIn cellular compartments almost all (>95%) of the iron is also protein-bound, either directly to protein residues or via iron-containing prosthetic groups such as heme or iron sulfur clusters ISC; . Althoug3+ complexes as potential LCI candidates [e.g., nucleotides and even glutathione (GSH); Many attempts made to chemically define LCI in extracts from lysed cells led biochemists to implicate various Fein situ (cell or organelle) catalytic contribution of labile iron to basal as well as peroxide-stimulated cell reactive O species (ROS) formation and to inhibit iron-dependent cell functions  the reductive properties and chemical composition of the compartment in question and those in turn depend on cell metabolism . As expected for a dynamic cell parameter, the various LCI pools are likely to vary over time in response to chemical or biological stimuli as well as to metabolic demands/responses. For example, in human K562 or murine erythroleukemia cells, exposure to increasing TBI concentration results in a commensurate rise in cytosolic and mitochondrial LI indicating increased iron uptake by RME and ensuing release into cytosol  cell iron in shells of ferritin molecules  in a cellular milieu that is comprised of mM concentrations of organo-phosphates and -carboxylates, could possibly confer upon DHB a putative role as cell iron chaperon, carrier, or the like  cells, changes in cytosolic LCI levels occurred in response to increasing concentrations of TBI  compounds), and almost in parallel to changes in mitochondrial LCI , 2010. TFigure 1; alas2 (for XLSA), or glrx5 (for SA) or slc25a38 (for SA) but also in neuro-siderosis caused by the mutated gene fxn (for FRDA) or abcb7  and those can override the cell antioxidant defenses (3+ with 2:1 stoichiometry and (b) the bidentate deferiprone (DFP) that binds it with 3:1 stoichiometry. As in systemic siderosis, the targets of chelation are both within cells and in extracellular fluids, the goal of chelation is twofold: (a) metal detoxification by means of reducing the iron burden in overloaded tissues and (b) prevention of tissue iron accumulation, by maintaining a low level of LPI, namely the labile components of plasma NTBI .To the extent that a persistently elevated LCI is a risk factor for cell survival , metal dFigure 2. Such mode of metal redistribution can be accomplished by chelators with siderophore properties, namely membrane permeant chelators that have a combined accessibility and iron affinity for scavenging LCI in cell and organelles but also the ability to transfer the chelated metal to cell acceptors or the plasma iron acceptor Tf  or calcein green  are comprised of one or two metal chelating arms (aminodiacetate or iminodiacetate) linked to fluorescent probes  that undergo signal quenching upon binding of transition metals like Fe or Cu. The probes can be easily loaded into cells via membrane permeant precursors that are non-fluorescent and non-chelating, e.g., CALG-AM or CALB-AM; AM-acetomethoxy. These probes are hydrolyzed intracellularly, releasing the fluorescent, impermeant chelating CALG (binds iron 1:1) or CALB (binds iron 2:1) that interact (reversibly) with resident labile iron and undergo quenching commensurate with LCI concentrations. That mode of iron sensing is depicted schematically at the bottom of Figures 3 and 4 for CALB and CALG, respectively, whereby iron added to the free CALB or CALG quenches the FMS and addition of a relatively high concentration of a strong iron chelator restores the quenched fluorescence. In CALB-laden human hepatic HepG2 cells , the FMS fills the cytosolic space and binds a fraction of LCI that can be revealed upon addition of excess permeant chelator. Thus addition of DFR elicits a rise in fluorescence ΔF that corresponds to Fe released form the quenched CALB-Fe the chelator  that detect labile iron chanisms : (a) revchanisms . Changesfor CALG .Figure 4 depicts h9c2 cardiac cells double labeled with CALG (cytosol and nucleus) and RPA, a red rhodamine-phenanthroline iron-sensitive probe that targets potentiometrically (binds iron 3:1) to mitochondria. Addition of a permeant iron source reduces the fluorescent intensity of both CALG and RPA (indicating ingress of labile iron to the respective compartment) and subsequent addition of the permeant chelator DFP restores the quenched fluorescence signal of CALG, but not that of RPA . Parallel time-dependent changes in cytosolic and mitochondrial LCI compartments can be monitored by flow cytometry or fluorescence microscopy as means to assess iron transport into the respective compartments in response to addition of TBI or NTBI  that are revealed as changes in CALG fluorescence after addition of the permeant tridentate chelator . When time-dependent profiles are done on probe labeled cells exposed to different substrate concentrations, it is also possible to obtain the kinetic parameters of iron ingress into the respective LCI pools , by converting ΔF to LCI concentrations, as described elsewhere  and the fluorescence quenched histone-CALG-Fe  and incubated with the bidentate chelator DFP. As schematized in the lower panel, an increase in nuclear fluorescence elicited by added DFP denotes iron scavengery from the CALG-Fe moiety, whereas a decrease in RPA fluorescence denoted DFP-mediated redeployment of Fe to mitochondria. As some DFP-Fe might also egress from cells into a medium containing apo-Tf, iron can redeployed to Tf, that in turn can also deliver the metal to other cells. This is depicted in the right panel, whereby the medium from the h9c2 cells treated with DFP offered to iron-deficient erythroid MEL cells supported hemoglobin Hb synthesis.Targeted FMSs have also been used to demonstrate intracellular redistribution of iron (scavengery and redeployment) by addition of the chelator siderophore DFP in a cell model of iron maldistribution , 2011. A2O2 or other peroxides) the labile metal converts in situ the non-fluorescent dihydro-probes into the respective fluorescent probes. As with the other FMS, the contribution of LCI to the time-dependent generation of fluorescence is assessed by the degree of inhibition attained by addition of a permeant chelator , can be confusing and in practice oxymoronic. For example, plasma that does not contain pathological species contains in fact NTBI in the form of ferritin or iron-chelates generated during iron chelation therapy or polymeric iron that is given supplementary via parenteral routes and detected in plasma by NTBI assays. On the other hand LPI is a parameter that denotes, operationally, the level of labile iron species in native plasma/serum irrespective of treatment or medium composition, an asset that can also become a liability if plasma/serum is not properly stored or some components of serum that affect its redox properties undergo extreme modifications . Recent studies indicated dimeric and oligomeric iron-citrate complexes as potential NTBI candidates  the actual repertoire of iron permeant species  and their relative concentrations at different degrees of IO, (b) the composition of the medium milieu  and (c) the different transport agencies present in different cell types. Those agencies comprise a variety of transporters or carriers of metals or metal-complexes, ionic channels or even the means for non-specific adsorptive endocytosis. As most NTBI transport studies have only been done with model simulating substrates in protein-free media and with model cells, their relevance to the etiopathology of tissue iron accumulation in systemic IO remains to be experimentally demonstrated : (1) by extraction of plasma NTBI with iron mobilizing/extraction/chelating agents like NTA or DTPA followed by size filtration (to separate NTBI from TBI) and detection of the iron by iron complex formation and direct measurement of colored complexes or following HPLC analysis  poses some methodological problems. Two different approaches have tried to overcome them  analysis  or co-caanalysis  or (b) bFO FDFO; . For eitThe LPI/NTBI assays have been used extensively to monitor chelation efficacy in transfusional siderotic patients treated with different chelators  or hemocWith presently available methodologies based on FMS, it has become possible to obtain real time measures of LCI and their compartments in both physiological and pathological conditions  and of LPI/NTBI in body fluids as early marker of systemic IO and treatment efficacy. Of particular importance is the ability to assess biological responses to pharmacological measures  and correlate them with clinical outcomes. That area still needs further strengthening, particularly in establishing the thresholds of changes in LPI and LCI that define a given pathological state, either overt or impending.Unfortunately, hitherto, it has been difficult to define the chemical components of LCI and LPI in biological systems, largely because of their heterogeneous character and variable nature of the media they dwell in. The same pertains to hypothesized iron chaperons that might be operative in mammalian cells and play some physiological role in intracellular iron traffic and metabolism. Although LCI is the parameter closely associated with tissue iron accumulation and ensuing iron-dependent damage, from the clinical perspective, only LPI has thus far proven diagnostically therapeutically relevant for assessing impending/emerging IO and therapeutically, for initiating chelation and monitoring its short term and long term efficacy.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ljunghia pulleinei Womersley, a mesostigmatic laelapid mite originally found in association with the mygalomorph spider Selenocosmia stirlingi Hogg (Theraphosidae) in Australia, is redescribed and illustrated on the basis of specimens from the African theraphosid spider Pterinochilus chordatus (Gerstäcker) kept in captivity in the British Isles . The mite is known from older original descriptions of Womersley in 1956; the subsequent redescription of Domrow in 1975 seems to be questionable in conspecificity of treated specimens with the type material. Some inconsistencies in both descriptions are recognised here as intraspecific variability of the studied specimens. The genus Arachnyssus Ma, with species A. guangxiensis (type) and A. huwenae, is not considered to be a valid genus, and is included in synonymy with Ljunghia Oudemans. A new key to world species of the genus Ljunghia is provided.Parasitic mites associated with spiders are spreading world-wide through the trade in tarantulas and other pet species.  Androlaelaps pilosus Baker (Laelapidae) can be found on the hexathelid spider Macrothele calpeiana , the only European tarantula Pterinochilus chordatus Gerstäcker, the Kilimanjaro mustard baboon spider.Close inspection of spiders often reveals mites associated with various body parts. Although these associations are most frequently reported from tropical spider families, mites are not uncommon on temperate spider species. Deutonymphs of Astigmata mites and Heterostigmata mites can be found phoretic on spiders; larvae of the Prostigmata families Erythraedae, Trombiculidae and Trombidiidae (chigger mites) can be parasitic on spiders, while Mesostigmata mites in the family Laelapidae often occur both as immature stages and adults on spiders Ljunghia includes species that have established close associations with various mygalomorph spiders in Indonesia Ljunghia, which includes a description of a new species from a Central American mygalomorph spider kept in captivity in Spain, a key for their identification and an enumeration of their host species The laelapid genus Ljunghia species from the British Isles, reports of mites parasitizing captive spiders is a common occurrence, often owing to contamination Ljunghia.Although there is no published record of an Ljunghia pulleinei. Detailed observations of the most important morphological features of this mite allowed to discern more details than those reported in the original descriptions The main aim of this study was a morphological redescription of Ljunghia Oudemans, 1932: 204 Ljunghia selenocosmiae Oudemans, 1932; by monotypy Ljunghia  Fain, 1989: 158 Ljunghia rainbowi Domrow, 1975; by original designation Arachnyssus Ma, 2002: 8 Ljunghia guangxiensis Ma, 2002; by original designation Chelicerae chelate-dentate in female, with fixed digit usually reduced in size; cheliceral digits of male subequal in length, with curved spermatodactyl slightly exceeding the tip of the movable digit. Dorsal shield entire, not covering the whole dorsal surface, and with hypotrichous setation . Sternal shield with three pairs of setae, metasternal shields and setae often absent. A pair of genital setae present or absent, usually placed on epigynal shield. Anal shield relatively small, elongate, bearing three circum-anal setae. Leg setation holotrichous to markedly hypotrichous.Ljunghia was proposed by Oudemans Selenocosmia Ausserer in Sumatra. Oudeman's genus Ljunghia has gained broad acceptance Ljunghia and Metaljunghia. We agree with Moraza et al. that this subgeneric structure is not useful, and it is not used here The genus Arachnyssus was erected Selenocosmia huwena Wang, Peng & Xie  entire dorsal shield; (2) idiosomal setae very long, with tips reaching far beyond the insertions of following setae; (3) anus with anterior position to adanal setae; (4) coxae I–IV not armed with spines; (5) sternal shield saddle-shaped, with posterior margin deeply concave; (6) epigynial shield short, tongue-shaped; (7) anal shield drop-shaped; (8) epigynial and anal shields well separated A new separate genus Arachnyssus neglected the existence of the genus Ljunghia because there is no reference to this genus in his paper Arachnyssus can be found in LjunghiaArachnyssus is here regarded as synonymous with Ljunghia, and the two species, namely A. guangxiensis and A. huwenae are therefore, newly transferred to the genus Ljunghia.It is obvious that the author who erected Ljunghia pulleini Womersley, 1956: 591–593 Ljunghia pulleini – Domrow, 1975: 35–37 (in part: only specimens of the type series) Ljunghia pulleinei (emend. nov.) s. str. – Fain, 1991: 78–79 Ljunghia  pulleini – Fain, 1989: 159 et al., 2009: 125 (in part) Pterinochilus chordatus  kept in captivity in the Laboratory of Molecular Parasitology, School of Biological Sciences, Bangor University, Gwynedd, NW Wales. The mites were collected by one of the authors, MAP, following the technique described aboved; October 2006.4 females, 2 males – on Female. Dorsal idiosoma . IdiosomVentral idiosoma . PresterGnathosomal structures . AnterioLegs. All legs with a well developed pretarsus and ambulacral apparatus (including pulvillus and two claws), shorter than idiosoma. Leg segments without specific projections or macrosetae, with the chaetotactic pattern as previously described. Coxae IV associated with relatively thin and long tubular structures of insemination apparatus .Male . IdiosomLjunghia pulleinei was inadequately illustrated, the description itself was insufficient Selenocosmia stirlingi Hogg in South Australia; (2) specimens from a nemesiid spider of the genus Aname L. Koch found in South Australia; (3) specimens from an unidentified diplurid spider in Queensland. All three series were keyed out together by Domrow and, despite the presence of some morphological differences, indicative of a mixture of three species, they were declared to be conspecific Aname, which differ from the typical series mainly by the lack of the subterminal pair of setae on the dorsal shield, belong to the species Ljunghia aname, which was originally described as a new subspecies of L. pulleineiThe original description of Ljunghia pulleinei by Fain Unfortunately, the description and figures of adults given by Domrow Ljunghia pulleinei bearing the longer setae cannot be reliably identified in the newest key of the genus Ljunghia pulleinei s. str. has here been collected from the Kilimanjaro mustard baboon spider from East Africa and previously from the common whistling spider from Australia. This is the first time that one and the same Ljunghia species has been associated with two different host species suggesting that Ljunghia species are not strictly species-specific. Ljunghia is well known from mygalomorph spiders but has also been reported from a more primitive liphistiid Malaysian trap door spider belonging to the Mesothelae Ljunghia species have been retrieved from more liphistiid spiders from Vietnam and Thailand Ljunghia.Opisthonotal region of dorsal shield with strongly suppressed setation, only 2–5 pairs of setae present 2Setation of opisthonotal region moderately suppressed, 7–14 pairs of setae present 7Ljunghia aname Fain, 1991Opisthonotal region with two pairs of setae, podonotal region with 14 pairs of setae Opisthonotal region with at least three pairs of setae 3Ljunghia novaecaledoniae Fain, 1991Genital setae (st5) absent; podonotal region with 20 pairs of setae, male sternogenital shield with three pairs of setae (st1–st3) Genital setae present; podonotal region with at most 18 pairs of setae, male sternogenital or sternogenito-ventral shield with at least four pairs of setae 4Podonotal region of dorsal shield with strongly suppressed setation, only 11 pairs of setae present, opisthonotal region with 4–5 pairs of setae 5Setation of podonotal region moderately suppressed, 17–18 pairs of setae present, opisthonotal region with 3 pairs of setae 6Ljunghia bristowi Opisthonotal region with 5 pairs of setae, setae J4 absent Ljunghia rainbowi Domrow, 1975Opisthonotal region with 4 pairs of setae, setae J4 present Ljunghia africana Fain, 1991Podonotal region with 14 pairs of setae, setae j5 absent; setae j6 and z5 relatively short and subequal in length Ljunghia pulleinei Womersley, 1956Podonotal region with 15 pairs of setae, setae j5 present; setae j6 relatively long, obviously longer than setae z5 Metasternal setae absent; opisthonotal region with 8 pairs of setae 8Metasternal setae present; opisthonotal region with at least 11 pairs of setae 9Ljunghia hoggi Domrow, 1975Podonotal region with 17 pairs of setae; setae J4 present, minute; sternal shield subrectangular; male with sternogenital shield Ljunghia guangxiensis  comb. nov.Podonotal region with 20 pairs of setae; setae J4 absent; sternal shield saddle-like, deeply concave posteriorly; male with holoventral shield Ljunghia minor Fain, 1989Opisthonotal region with 14 pairs of setae; male with sternogenital shield bearing five pairs of setae (st1–st5) Opisthonotal region with 11 pairs of setae; male with sternogenital-ventral or holoventral shield bearing five pairs of setae together with a number of additional ventral setae 10Ljunghia selenocosmiae Oudemans, 1932Setae J4 not modified, subequal with most of other dorsal setae; male with sternogenital-ventral shield, ventral part of the shield with 5–7 setae Setae J4 reduced in length, conspicuously shorter than other dorsal setae; male with holoventral shield bearing at least 13 setae in ventral part 11Ljunghia luciae Moraza, Iraola & Alemany, 2009Podonotal region with 21 pairs of setae; setae J4 shorter, with tips not reaching the posterior margin of dorsal shield; holoventral shield of male with 20–21 setae in ventral part Ljunghia huwenae  comb. nov.Podonotal region with 20 pairs of setae; setae J4 longer, with tips reaching beyond the posterior margin of dorsal shield; holoventral shield of male with about 13 setae in ventral part Ljunghia pulleinei were obtained from the mygalomorph spider Pterinochilus chordatus kept in captivity in Gwynedd, North Wales. P. chordatus is an East African theraphosid species distributed in Ethiopia, Kenya, Somalia, Sudan, Tanzania and Uganda All examined specimens of The collection of mites was carried out on the living spider. Due to the aggressivity of the spider it was necessary to develop a technique to collect the mites attached to the dorsal parts of the cephalothorax and the abdomen. The spider was reared in a plastic container covered by a plastic lid. Small holes of 2 mm where made in the lid to allow breading but also to allow insertion of a wire to touch the mites. The tip of the wire was soaked in 100% glycerol (glycerin) and then directed inside the cage towards every single mite. Due to the sticky nature of glycerol, the mites were instantly glued to the tiny tip of the wire and extracted from the cage by slowly moving the tip out to avoid distressing the spider. Once outside the cage, the tip of the wire with a glued mite was submerged in 96% ethanol where the mites detached and became fixed and preserved for further analysis.The mites were mounted on permanent microscope slides using Swan medium. Illustrations were made by using a high magnification microscope equipped with a drawing tube. Measurements were made from slide-mounted specimens with stage-calibrated ocular micrometers. Lengths of shields and leg segments were measured along their midlines, and widths were measured at the widest point. Dorsal setae were measured from the bases of their insertions to their tips. Measurements are mostly presented as ranges (minimum to maximum). The terminology of dorsal and ventral chaetotaxy follows Lindquist & Evans


While the morphology and function of cells of the exocrine and endocrine pancreas have been studied over several centuries, one important cell type in the gland, the pancreatic stellate cell (PSC), had remained undiscovered until as recently as 20 years ago. Even after its first description in 1982, it was to be another 16 years before its biology could begin to be studied, because it was only in 1998 that methods were developed to isolate and culture PSCs from rodent and human pancreas. PSCs are now known to play a critical role in pancreatic fibrosis, a consistent histological feature of two major diseases of the pancreas—chronic pancreatitis and pancreatic cancer. In health, PSCs maintain normal tissue architecture via regulation of the synthesis and degradation of extracellular matrix (ECM) proteins. Recent studies have also implied other functions for PSCs as progenitor cells, immune cells or intermediaries in exocrine pancreatic secretion in humans. During pancreatic injury, PSCs transform from their quiescent phase into an activated, myofibroblast-like phenotype that secretes excessive amounts of ECM proteins leading to the fibrosis of chronic pancreatitis and pancreatic cancer. An ever increasing number of factors that stimulate and/or inhibit PSC activation via paracrine and autocrine pathways are being identified and characterized. It is also now established that PSCs interact closely with pancreatic cancer cells to facilitate cancer progression. Based on these findings, several therapeutic strategies have been examined in experimental models of chronic pancreatitis as well as pancreatic cancer, in a bid to inhibit/retard PSC activation and thereby alleviate chronic pancreatitis or reduce tumor growth in pancreatic cancer. The challenge that remains is to translate these pre-clinical developments into clinically applicable treatments for patients with chronic pancreatitis and pancreatic cancer. The concept of fibrosis as an inert, reactive tissue has changed significantly in recent times. Indeed, it is now well recognized that fibrogenesis in the pancreas is an active, dynamic process that may be reversible, at least in the early stages. Importantly, there is unequivocal evidence to indicate that PSCs play a central role in pancreatic fibrogenesis  stained with gold chloride in perisinusoidal spaces in the liver. However, Kupffer was unsure at the time as to whether these cells were different from resident liver macrophages, and this resulted in considerable confusion in the field for several decades. Almost 75 years after Kupffer's first description of “sternzellen,” Ito Ito,  reportedin vitro tool for researchers to characterize the biology of PSCs in health and disease.As noted earlier, the pancreatic counterparts of HSCs were first described by Watari in 1982, . Watari examined the pancreas of vitamin A loaded mice by fluorescence microscopy and electron microscopy and described the presence of cells exhibiting a rapidly fading blue-green fluorescence characteristic of vitamin A in the periacinar areas of the pancreas . In addition to the production of ECM proteins, PSCs also produce matrix degrading enzymes  and their inhibitors  , more recent efforts have been directed toward other non-fibrogenic functions of quiescent cells. Accumulating evidence suggests that PSCs may function as (1) progenitor cells; (2) immune cells or (3) intermediary cells in cholecystokinin (CCK)-induced pancreatic digestive enzyme (exocrine) secretion. With regard to their possible progenitor function, Mato et al.  isolatedin vitro observations were supported by the authors' in vivo work using a mouse model of bile-duct ligation induced acute pancreatitis and a model of spontaneous chronic pancreatitis (WBN/Kob rats) in which they found that PSCs engulfed damaged parenchymal cells. Thus, the authors speculated that PSCs may exhibit a locally protective “innate” immune function to inhibit disease progression in early pancreatic injury. The role of PSCs in innate immunity is supported by the fact that the cells express Toll like receptors  which recognize foreign pathogen-associated molecular patterns (PAMPs) . Two recent studies have convincingly demonstrated the presence of CCK receptors 1 and 2 on human PSCs  have now convincingly demonstrated that when activated during pancreatic injury, PSCs play a critical role in the pathogenesis of pancreatic fibrosis.in vitro. The selection of putative activating factors for examination was based upon the knowledge that during the process of tissue injury, PSCs are likely to be exposed to factors such as: (1) alcohol and its metabolites acetaldehyde and fatty acid ethyl esters (FAEEs) ; (c) the proinflammatory cytokines TNFα, monocyte chemotactic protein (MCP-1) and IL1, IL6 and IL13 stimulate proliferation, αSMA expression and/or collagen synthesis in PSCs , all of which are capable of activating the cells via autocrine pathways  activates PSCs and this activation is prevented by the antioxidant α-tocopherol (vitamin E)  include hyperglycaemia, endothelin 1, COX-2, galectin 1 and fibrinogen.Having established the functional responses of PSCs to exogenous and endogenous factors, the logical next step for researchers in the field was to identify the intracellular signaling pathways mediating these responses, with the ultimate aim of developing approaches to target specific signaling molecules so as to interrupt PSC activation and inhibit abnormal fibrogenesis.in vitro (noted above), the LPS signaling pathway has recently been examined in PSCs. The cells express the LPS receptor TLR4 (toll like receptor 4) as well as the adapter molecules CD14 and MD2  pathway , p38 kinase and c-jun amino terminal kinase (JNK), as well as the nuclear transcription factor AP-1   signaling mediates the induction of quiescence of PSCs in response to retinol and its metabolites ATRA and 9-cisRA  and using immunohistochemistry for the presence of specific proteins. These studies have clearly established that in the fibrotic pancreas, areas that stain positive for collagen also stain for alpha smooth muscle actin indicating the presence of activated PSCs Figure . MoreoveThe expression of growth factors known to activate PSCs is also increased in chronic pancreatitis. Pancreatic acinar cells adjacent to areas of fibrosis exhibit strong staining for TGFβ, while such staining is absent in acinar cells remote from bands of fibrosis   of pancreatic fibrosis. Fibrosis has been produced in rodents via various approaches. Rat models described in the literature include: (1) trinitrobenzene sulfonic acid (TNBS) injection into the pancreatic duct  produced by injured acinar cells and/or inflammatory cells during the acute phase of the injury. Activated PSCs are the major source of collagen in fibrotic areas (confirming findings from in human studies). As with human chronic pancreatitis, expression of factors known to activate PSCs are all reported to be upregulated in experimental pancreatic fibrosis including PDGF and its receptor, TGFβ and two TGFβ regulated genes SM22α and Cygb/STAP, and oxidant stress.Although the above models have provided useful data, caution needs to be exercised in assessing their direct clinical relevance, since most have involved relatively non-physiological methods , to produce pancreatic damage. However, there is one rat model produced by chronic alcohol administration and repeated endotoxin exposure  also contribute to the PSC population. Two recent studies have used a gender mismatch and chimeric approach whereby green fluorescent protein (GFP) labeled bone marrow derived cells (BMDC) obtained from GFP transgenic male donor mice were transplanted into lethally irradiated wild type female rodents   has reported that PSCs in long-term culture (6 weeks) express the senescence marker senescence associated b-galactosidase  and are highly susceptible to immune cell-mediated cytotoxicity. Furthermore, the authors report that in a rat model of DBTC-induced pancreatitis, PSCs not only express activation markers, but also senescence markers leading them to speculate that inflammation, PSC activation and senescence are timely coupled processes in the injured pancreas. However, this study did not assess PSC apoptosis. Indeed, the relative contributions of the processes of apoptosis versus senescence versus reversion to quiescence in the removal of activated PSCs after pancreatic injury remain to be clarified.As the processes of PSC activation are becoming increasingly clear, the fate of activated PSCs is also attracting increasing attention. Three possibilities that have been considered include: (1) reversion to quiescence; (2) apoptosis; and (3) senescence. Partial reversion to quiescence has been described n et al.  have demin vivo . Several treatments/approaches have been reported to prevent/retard fibrosis in animal models, including: (i) Antioxidants - vitamin E (the subclass tocotrienol has been shown to induce PSC death via apoptosis and autophagy)  has established that the predominant cells responsible for producing the fibrosis in pancreatic cancer are PSCs  as well as in vivo   results in activation of PSCs  , while PSC proliferation is likely mediated by PDGF. Recent studies have also implicated cycloxygenase 2  .More recently, orthotopic models of pancreatic cancer have been described wherein human pancreatic cancer cells  with or without human pancreatic stellate cells (hPSCs) were injected directly into the mouse pancreas  secreted by PSCs  where they likely facilitate the seeding, survival, and growth of cancer cells. Indeed, similar observations have now been reported in a model of lung cancer  may play a role in the well documented resistance of pancreatic cancer to chemotherapy and radiotherapy . Improved understanding of PSC biology will underpin the development of novel therapies in the future for the treatment of chronic pancreatitis and pancreatic cancer.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.


Herbivore induced plant volatiles (HIPVs) are specific volatile organic compounds (VOC) that a plant produces in response to herbivory. Some HIPVs are only produced after damage, while others are also produced by intact plants, but in lower quantities. Among the known functions of HIPVs are within plant volatile signaling to activate systemic plant defenses, the priming and activation of defenses in neighboring plants and the attraction of natural enemies of herbivores. When released into the atmosphere a plant's control over the produced compounds ends. However, many of the HIPVs are highly reactive with atmospheric oxidants and their atmospheric life times could be relatively short, often only a few minutes. We summarise the potential ecological and atmospheric processes that involve the reaction products of HIPVs in their gaseous, liquid and solid secondary organic aerosol (SOA) forms, both in the atmosphere and after deposition on plant surfaces. A potential negative feedback loop, based on the reactions forming SOA from HIPVs and the associated stimulation of sun screening cloud formation is presented. This hypothesis is based on recent field surveys in the geographical areas facing the greatest degree of global warming and insect outbreaks. Furthermore, we discuss how these processes could benefit the individual plant or conspecifics that originally released the HIPVs into the atmosphere. Further ecological studies should aim to elucidate the possible reasons for biosynthesis of short-lived volatile compounds to have evolved as a response to external biotic damage to plants. Most vascular plants constitutively emit volatile organic compounds (VOCs), but emissions may substantially increase and diversify under conditions of abiotic and biotic stress -4,8-dimethyl-1,3,7-nonatriene (DMNT) were susceptible and were largely defoliated -3-hexenal and (Z)-3-hexenyl acetate are compounds released after mechanical or other destructive damage to cell membranes , nitrate (NO3) or hydroxyl (OH−) radicals  being the first C6 GLV compound synthesized by the lipoxygenase (LOX)/lyase pathway -2-hexenal , 3-hexenol  and (Z)-3-hexenyl acetate (ester) -DMNT, for example, could originate predominantly from the MVA-pathway in herbivore-stressed plants, while the fungal elicitor alamethicin stimulates the biosynthesis of (E)-DMNT via the MEP-pathway -β-ocimene and (E)-DMNT peaked on day 3 -α-farnesene tended to be biphasic with peaks on days 2 and 4 after the start of larval feeding. Emission rates of the induced LOX products, (E)-β-ocimene and -α-farnesene were positively correlated with the number of larvae feeding  -β-ocimene showed an increase in infected plants on several days. The infection also increased the emission of sesquiterpenes and LOX products by factors of 175-fold and 10-fold, respectively -3-hexenyl acetate -3-hexenyl acetate have been implicated as providing a key signal -3-hexenyl acetate, which is the main GLV emitted by that species -β-caryophyllene has been shown to offer Arabidopsis thaliana flowers a degree of defense against a bacterial pathogen SOA particles could deposit on plant surfaces and have further ecological effects.The ecological functions of HIPVs described above have all been established experimentally. All rely mechanistically on the responses of a receiver organism to volatile emissions from a plant that has been subjected to a degree of stress or stimulation. While the volatile compounds remain intact they theoretically constitute a signal that can be detected and potentially perceived by organisms of the surrounding community. However, as soon as the chemicals leave the plant there are a range of potential fates or roles that could be played out. In the following section those potential adaptive roles have been classified as roles for intact HIPVs and for reaction products of HIPVs Figure . In pracIt has been shown that species specific semivolatile VOCs emitted constitutively by plants can be adsorbed to the surfaces of neighboring plants  levels in the atmosphere are high, as occurs in environments contaminated by smoke and exhaust gases from fossil fuel combustion, VOC oxidation increases ozone levels  and hydroperoxy (HO2·) radicals. This will lead to the conversion of NO to NO2, and thus, promotes O3 accumulation and the efficient regeneration of the OH radical. Both O3 and OH radicals can react with other VOCs in the atmosphere  are formed via reactions of isoprene, monoterpenes and sesquiterpenes with NO3 radicals in the presence of OH radicals and NO. Organic nitrates are removed from the air during precipitation events and thus plant VOCs help to remove NOx from the lower troposphere . Reduced performance of whiteflies was related to up-regulated pathogenesis-related protein (PR1) genes, increased phenylalanine ammonia-lyase enzyme activity and elevated concentrations of salicylic acid (SA), phenolics and condensed tannins (Cui et al., 3 levels may cause slight reduction in plant growth, but plant fitness could be improved by the reduced impact of aggressive plant pathogenic fungi such as Drechslera teres on barley (Plessl et al., 3 limits hyphal growth, sporulation and germination of conidia in many other plant pathogenic and saprophytic fungi (Tzortzakis et al., The ability of plants to control and even promote the formation of phytotoxic ozone in the lower troposphere may benefit plants e.g., by eliciting defense reactions which provide better plant resistance against fungal pathogens and herbivores (Sandermann et al., −1 (Kulmala, In the atmosphere, nano-scale aerosol particles are typically formed during the late morning and then grow throughout the day with growth rates of 1–20 nm hKulmala, . The smaKulmala, . NucleatKulmala, . The cheKulmala,  and a siKulmala, . FinallyKulmala,  through 3 and OH leading to formation and growth of atmospheric SOA particles. Insect damage to pine saplings can increase the emission rates of reactive monoterpenes and sesquiterpenes by up to 10-fold after bark feeding (Heijari et al., Chamber experiments (Joutsensaari et al., Earlier studies (Mercado et al., Some of the constitutively emitted plant VOCs may have much longer life times in ozone-rich atmospheres than some HIPVs that have life times of only a few minutes (Holopainen, The behavior of SOA particles inside a canopy and their deposition on plants and other surfaces is not yet sufficiently understood (Holopainen, Production of HIPVs, many of which are highly reactive in the atmosphere, is an evolved response in many plant species under biotic stress. When considering the chemical and physical properties of the HIPV compounds, a longer atmospheric life time of the compounds could possibly attract natural enemies over longer distances. However, the molecular concentration in the atmosphere will in any case be rapidly reduced due to increased distance from the point source of a damaged plant. The selection of highly reactive compounds as attractive signals in indirect plant defense does not necessarily maximize the attraction capacity of HIPVs (McFrederick et al., Increased emission of reactive HIPV compounds from plants under biotic stress will inevitably lead to SOA formation as shown experimentally (Joutsensaari et al., Recent evolutionary models incorporate phenotypes expressed in the external environment; however, there is still debate whether such traits generate dynamics that alter evolution Bailey, . Such moWe suggest that high HIPV emission rate capacity of Boreal conifer forest trees could be such a trait and that it may have an impact on SOA formation in insect outbreak areas (Berg et al., 3 at night. The secondary organic aerosol particles formed from the same HIPV may have different chemical composition and different biological functions depending on the time of day during SOA formation. The size of SOA particles in the atmosphere may also grow by adsorption of other organic vapors to their surface (Kroll and Seinfeld, 2 in the atmosphere (Kroll and Seinfeld, To return to our original question; in this review we have tried to demonstrate that the carbon fixed by a plant and then, particularly under biotic stress, released back to the atmosphere as volatile organic compounds, will have important roles in chemical, physical and biological processes during their life time. These may be facilitated by the HIPV compounds originally synthesized and emitted by a plant, or in the form of other chemical compounds after atmospheric reactions of the HIPVs. So far, we know only a fraction of the highly diverse potential routes and functions that the VOCs emitted by plants may have. For example, the carbon of highly volatile C10 monoterpenes and semivolatile C15 sesquiterpenes in the atmosphere could be bound to freshly nucleated SOA particles as a result of ozonolysis during the day time or oxidized by reactions with NOThe 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.


Human asthmatic disease is described as a chronic inflammatory disease of the airways. However, the underlying immunological processes involved in asthmatic disease are not fully understood. Animal models have been used to uncover patho-physiologic mechanisms which relate to human disease, leading to discovery and evaluation of therapeutic targets.We have temporally characterized two models of asthma in mice, an acute and a chronic House Dust Mite (HDM) model to elucidate comparative pathways in each model and ultimately link these to human disease. Specific mechanisms of asthma were evaluated including: mucus overproduction, Th2 cytokine response, airway remodeling, airway constriction, and IgE production.The acute HDM model in female Balb/c mice is a 10 day consecutive intranasal challenge with house dust mite extract. The model has an early phase cytokine response shown through significant increases of G-CSF, TNFa, IP-10 and KC in the lung on day 1 which could be linked to mechanisms driving inflammation such as chemotaxis of neutrophils and macrophages/monocytes. The height of the inflammatory phase in the acute model is at day 7 with resolution seen by day 16. In contrast, the chronic HDM model is a 5 week model in female Balb/c mice, challenging intranasally 5 times per week with house dust mite extract. The chronic HDM model reveals significant mucus overproduction and airway remodeling characteristics such as significant upregulation of soluble collagen in the lung and tissue inhibitors of metalloproteinase -1 (TIMP-1) which has been shown to be increased in sputum and lung biopsies from asthmatic patients.By modeling the essential mechanisms involved in asthma through either the acute or chronic HDM model; we will more fully understand human disease and discover superior therapeutics to combat asthma.


The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway. It has been recently demonstrated that a small population of tumor cells, with stem cell-like features (GSCs), can initiate tumor development It has been shown that the tumor suppressor protein PTEN is involved in cell cycle regulation, through the modulation of different signaling pathways, either in cancer in general and in brain tumor in particular The aim of the present study was to shed light on the role of PTEN pathway on cell cycle regulation in GSCs through a cellular differentiation model. Phosphoproteomics profiling of different GSCs lines, using reverse phase protein microarrays (RPMA), indicates a heterogeneous basal activity of PTEN and related molecules. Transcriptomics profiling of GSCs induced to differentiate demonstrates specific molecular changes in PTEN positive cells, that are not observed in cells lacking its activation. The most modulated transcripts suggest a post-transcriptional regulation model, which involves cdc25a as the main target.This study has been approved by Institute of Neurosurgery, Catholic University School of Medicine, Rome, Italy. Before surgery patients provided written consent.Tumor tissue samples were collected from 8 patients with primitive brain tumor undergoing complete or partial surgical resection at the Institute of Neurosurgery, Catholic University School of Medicine, in Rome, Italy. A diagnosis of glioblastoma multiforme, established histologically according to the WHO classification 5 cells were plated in matrigel (1∶25 in stem cell medium) coated flasks and induced by growth factors deprivation and supply of 5% serum in the growth environment. After 14 days, cells were checked for differentiation markers by flow cytometry and for cellular morphology. Cytofluorimetric analysis was performed using a FACS Canto flow cytometer (Becton Dickinson) to assess the expression of CD133, Sox2 and nestin (NES), as stem cells markers, and glial fibrillary acidic protein (GFAP) and beta 3 tubulin (TUBB3), as differentiation markers (Miltenyi Biotec antibodies). Cellular immunophenotype was also assessed by means of a laser confocal microscope . Primary antibodies against Sox2, GFAP  and TUBB3 (Millipore) were diluted in 1% BSA to a final concentration of 5 μg/ml and incubated for 1 h.GSCs were obtained from freshly dissociated surgical specimens cultivated in a serum-free medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (FGF), as previously described RPPA was performed as previously described Samples transcriptomes were characterized using Affymetrix GeneChip Human Gene 1.0 ST arrays. Total RNA was extracted and processed according to manufacturer procedures. CEL files were quality checked, acquired and processed using Affymetrix Power Tools with RMA normalization option. Preprocessed data are available as PTEN positive and PTEN negative groups of samples were defined using unsupervised hierarchical clustering of three dimensional  protein expression profiles. Euclidean metric was used for distance calculations. Differences in gene expression profiles were evaluated using paired t test . Association between gene expression levels of candidate transcripts and PFS or OS were assessed using a Cox proportional hazards regression model. Clustering, plots and t statistics were produced using R statistical environment 14 days after induction, differentiated cells show glial fibrillary acidic protein (GFAP) and beta 3 tubulin (TUBB3), while they lose the expression of CD133, Sox2 and nestin (NES) when assessed by cytoflourimetry. During the induction, cells proliferate to reach a count of 2,5 millions on average.Neurospheres show the expression of Sox2 while differentiated cells, which lack its expression, show the expression of the differentiation markers GFAP and TUBB 3 .The analysis of phosphoproteomics profiles shows that GSCs may present differences, in basal conditions, in the activation status of PTEN pathway. Its global activity has been checked by measuring the protein expression levels of PTEN and of its downstream molecular partner AKT, which behaviour can be regulated by specific phosphorylation events. In particular, we analyzed the presence of activatory S473 phosphoepitope of AKT. Three cell lines  showed proteins profiles which are related to the lack of PTEN pathway activity  while all the other lines showed profiles which indicate PTEN pathway activation . AccordiSamples were grouped according to the above-described molecular criterion and cells were induced to differentiate. Gene expression profiles in both groups were analyzed comparing differentiated and undifferentiated samples, in order to identify transcripts that are specifically modulated during differentiation. Profiles were analyzed comparing each differentiated sample with its corresponding baseline; then, transcripts that showed a statistically significant modulation in a paired test were selected. Transcripts having less than 2 fold of modulation in logarithmic scale in more than 30% of samples were discarded because the overall intensity of variation wasn't considered relevant in sample population. Results are presented in These transcripts showed no significant associations between expression levels and patients or specimen characteristics .When PTEN active and inactive cell lines are compared, differences are observed in the extent of modulation observed in previously selected transcripts. While Dub3 and mir21 are significantly modulated (with upregulation and downmodulation respectively) in PTEN active cell lines, their transcripts variation is far less evident than in PTEN inactive ones. Wee1 is significantly downmodulated in both groups.Stem cells tightly regulate cell cycle progression in order to remain quiescent inside their cellular niche with progress occurring only when external stimuli indicates the need for cell growth or for the replacement of damaged or senescent cells. This process is so crucial that involved molecules are subjected to multiple levels of regulation. Cell-cycle transitions can be often the results of coordinate factors which involves rapid turnover of crucial regulatory proteins through the ubiquitin-proteasome system. These mechanisms are shown to be frequently altered when observing cancer cell proteomes, and dysregulation of these pathways is clearly linked to the acquisition of neoplastic phenotype.β-TRCP-dependent turnover . DUB3 can interfere with this process also inhibiting ubiquitin flagging of Cdc25a. During mitosis, Cdc25 protein is stabilized by the complex Cyclin-B/Cdk1 through phosphorylation. This post-translational stabilization is negatively controlled by the Wee1 kinase, which can inhibit Cyclin-B/Cdk1 activity.A paradigmatic example is represented by the cell division cycle 25A (Cdc25A) protein, which is often found expressed in high concentration in many cancers By comparing GSCs to the differentiated counterparts, we observed that transcripts levels of Cdc25a, APC protein complex, SCF protein complex and Cyclin-B/Cdk1 do not vary after differentiation. Conversely, DUB3 mRNA is strongly upmodulated in differentiation and this could promote cell cycle progression, with the stabilization of Cdc25a, even in presence of DNA damage. Coherently, Wee1 appears to be one of the most downregulated genes, suggesting the release of Cyclin-B/Cdk1 from its inhibitory control, thus triggering the activation of Cdc25a in mitosis. Furthermore, it is interesting to observe that the mature form of miR-21 appears to be one of the RNAs that significantly fade during differentiation. miR-21 has been observed to target directly cdc25a in human cancer cells According to that observed in PTEN positive GSCs, we propose a model in which Cdc25a is persistently transcribed during each of the cell cycle phases. In GSCs, its corresponding protein level is negatively regulated by its post-translational regulators, which contribute to maintain cells in interphase. The initial phase of the commitment to differentiation is the release from cell cycle progression control by the inhibition of cdc25a degradation by the activity of the augmented concentration of Dub3. This protein counteracts both the effects of APC/Cdh1 and of ubiquitination triggered by DNA damage checkpoints, providing cells the ability to progress through S and G2 phases. The stabilization of cdc25a through G2 and mitosis is also permitted by the diminished inhibitory effect of Wee1, which is strongly downregulated in differentiation, on Cyclin-B/Cdk1 complex. The downmodulation of miR-21 likely contributes also to the overall increase in cdc25a protein level by removing the post-transcriptional inhibition based on translation efficiency.In this model, Dub3 and Wee1 are involved in cell cycle regulation in a cellular phenotype specific way. Dub3 promotes cell cycle progression in committed cells, although its transcript level remains low in stem cells. Conversely, Wee1 resulted expressed in stem cells, suggesting that its activity provide the ability to avoid G2/M transition.While recent literature Figure S1Hierarchical clustering of samples according to their mean PTEN, AKT and pAKT protein expression levels.(TIFF)Click here for additional data file.File S1Table reporting whole genome transcriptomic modulation. Each column report samples's fold change along differentiation.(XLSX)Click here for additional data file.