We report the presence of infectivity in erythrocytes, leukocytes, and plasma of 1 person with variant Creutzfeldt-Jakob disease and in the plasma of 2 in 4 persons whose tests were positive for sporadic Creutzfeldt-Jakob disease. The measured infectivity levels were comparable to those reported in various animals with transmissible spongiform encephalopathies. Prnp), no clear epidemiologic risk factors have been identified for sporadic disease forms. sCJD is not a uniform disorder in terms of clinical and neuropathological phenotype. sCJD cases are classified as type 1 or 2 according to the polymorphism at codon 129 of the protease-resistant prion protein (PrP) sequence  and to the electromobility of the proteinase K–resistant core of the abnormal PrP (PrPres)  is a low incidence disease (≈1 case per million per year) that occurs as either a sporadic (sCJD) or a familial/genetic (fCJD) form. Whereas familial disease forms are linked to a mutation in the prion protein gene , was identified in humans. Variant CJD was demonstrated to be caused by the agent that causes bovine spongiform encephalopathy in cattle  for the detection of the bovine spongiform encephalopathy agent. To demonstrate that tgBov also displays a high sensitivity to vCJD infection, we titrated to endpoint a vCJD isolate (10% brain homogenate) by intracerebral inoculation in this model (Tg110)  from 1 vCJD-confirmed patient were injected intracerebrally in tgBov mice. Similarly, plasma samples from 4 sCJD MM1 patients were inoculated with tgHu . The bloitration , panel Ares accumulation were observed in the tgBov or tgHu mice inoculated with phosphate-buffered saline or brain and plasma from healthy human controls. The 3 blood fractions from the vCJD-affected patient caused a positive result but low attack rate among tgBov mice (No TSE clinical signs or PrPBov mice . On the Bov mice , the gloBov mice .In tgHu mice, positive transmission was observed among mice inoculated with 2 of 4 plasma samples . The infres banding patterns observed by Western blot in animals challenged with brain homogenate and blood components were identical (In tgBov inoculated with vCJD and tgHu inoculated with sCJD, the PrPThe data reported here confirm the presence of infectivity in erythrocytes, leukocytes, and plasma from vCJD-affected patients and demonstrate unambiguously the presence of infectivity in the plasma of some, but not all, sCJD-affected patients. The infectivity levels that we measured in the tested vCJD and sCJD blood components were comparable to those reported in various TSE animal models. The number of cases included in our study was limited; a new experiment that would include a larger number of cases and different blood fractions from sCJD cases will be necessary to refine the data. However, these results represent a substantial input for assessing the risk for interindividual bloodborne transmission of sCJD and vCJD.Biochemical typing and PrP ORF sequencing of sporadic and variant Creutzfeldt-Jakob disease genes.


Medicago truncatula homolog expressed in endosperm at seed filling stage. These tandem duplicates could contribute on the protein/oil QTL of Chr 20. Our study suggests that non-shared gene contents within the duplicated genomic regions might lead to absence/presence of QTL related to protein/oil content.Understanding several modes of duplication contributing on the present genome structure is getting an attention because it could be related to numerous agronomically important traits. Since soybean serves as a rich protein source for animal feeds and human consumption, breeding efforts in soybean have been directed toward enhancing seed protein content. The publicly available soybean sequences and its genomically featured elements facilitate comprehending of quantitative trait loci (QTL) for seed protein content in concordance with homeologous regions in soybean genome. Although parts of chromosome (Chr) 20 and Chr 10 showed synteny, QTLs for seed protein content present only on Chr 20. Using comparative analysis of gene contents in recently duplicated genomic regions harboring QTL for protein/oil content on Chrs 20 and 10, a total of 27 genes are present in duplicated regions of both Chrs. Notably, 4 tandem duplicates of the putative homeobox protein 22 (HB22) are present only on Chr 20 and this  Glycine max (L.) Merrill] seed is a good source of protein and oil, it is grown widely throughout the world for its numerous uses, such as various edible products, animal feed and potential industrial applications , an undomesticated form of the current soybean, is distributed in East Asia, including China, Taiwan, Russian Far East, the Korean Peninsula, and Japan, the origin or domestication site of soybean is still in controversy  is the main resource for quantitative trait loci (QTLs) for various traits and linkage map with 20 soybean chromosomes (Chrs). Also, classical, allozyme and other genetic markers such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSRs) and single nucleotide polymorphism (SNP), are publically available. Starting with the genome sequences of the G. max cultivar . The second round of soybean whole genome duplication occurred approximately 13 million years ago and this polyploidy event contributes to the soybean genome structure ranging from near-identical, rather divergent to latter more divergent, leading to dynamic and massive genome rearrangement , homeologous blocks of duplicated segments were found in all 20 Chrs  with ancient and recent duplications demonstrates that soybean genome is complex . Over 61 QTLs are associated with the protein content in 17 different soybean populations  I] because of the highest additive effect across many mapping populations and multiple environments , which is reported as The accumulated genomic data can be used to identify functional genes of specific traits. Even this can provide a basis for predicted gene duplicates following modes of recent duplications. Here, in this review, we compared duplicated genomic regions, which are involved in seed protein content. Increased divergence after recent duplications resulted in the appearance or disappearance of QTLs related to protein and/or oil, suggesting gene retention/loss. Comparing gene and sequence divergence between recently duplicated genomic regions harboring a major QTL for seed protein content on Chr 20, 27 out of 81 genes were present in the homeologous regions of both Chr 20 and Chr 10. Several genes with over- and/or under- retained may be functional and contribute to seed protein content regulation. Therefore, the information of recently duplicated and diverged genes will provide insights into the identification of candidate genes of agronomically important trait.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.


Adherence to colonoscopy recommendations can reduce colorectal cancer (CRC) morbidity and mortality. However, little is known about the use of colonoscopy among persons at risk for Lynch syndrome prior to clinical genetic testing (CGT) and how notification of mutation carrier status affects screening behaviors. The goal of this study was to evaluate colonoscopy use and attitudes towards CRC screening among unaffected relatives of Lynch syndrome mutation carriers before pre-test genetic counseling (baseline) and 6 and 12 months after disclosure of the test results. The primary outcomes of interest were colonoscopy utilization, commitment to screening for colonoscopy, self-efficacy regarding screening adherence, and the benefits, barriers, and efficacy of CRC screening.110 unaffected relatives completed a psychosocial questionnaire prior to pre-test counseling, 81 had GCT and received test results, and 78 subsequently completed follow-up questionnaires at 6 and 12 months post-results disclosure (52 were mutation negative and 26 were mutation positive). Linear mixed models were used to evaluate group comparisons in change over time. In addition to the main effects of mutation status and time, the time by mutation interactions were included as fixed effects in the model to test for differences in average rate of change over time between the two mutation groups.While both groups were similar at baseline, after disclosure of results, a significantly greater number of those with a positive test result had a colonoscopy compared with persons who tested negative where colonoscopy utilization decreased significantly Figure .Analysis showed a significant positive movement through the stages of readiness to have colonoscopy in the mutation-positive group from baseline to 6 and 12 month post-disclosure compared to a significant negative movement through the stages of readiness in the mutation negative group (Table Our results show that CGT may motivate mutation carriers to have a colonoscopy after receiving test results and may also have an impact on their longer-term screening behaviors. In addition, CGT may have a positive impact on mutation carriers’ attitudes towards CRC screening.


Advances in the understanding and management of pulmonary arterial hypertension have enabled earlier diagnosis and improved prognosis. However, despite best available therapy, symptoms of exertional dyspnoea and fatigue are commonly reported and result in a reduced capacity to perform daily activities and impaired quality of life. Exercise training has demonstrated efficacy in individuals with other respiratory and cardiovascular diseases. Historically, however, exercise training has not been utilised as a form of therapy in pulmonary arterial hypertension due to the perceived risk of sudden cardiac death and the theoretical possibility that exercise would lead to worsening pulmonary vascular haemodynamics and deterioration in right heart function. Now, with the advances in pharmaceutical management, determining the safety and benefits of exercise training in this population has become more relevant. Only three studies of supervised exercise training in pulmonary arterial hypertension have been published. These studies demonstrated improvements in exercise capacity and quality of life, in the absence of adverse events or clinical deterioration. However, these studies have not utilised an outpatient-based, whole body exercise training program, the most common format for exercise programs within Australia. It is uncertain whether this form of training is beneficial and capable of producing sustained benefits in exercise capacity and quality of life in this population.This randomised controlled trial will determine whether a 12 week, outpatient-based, supervised, whole body exercise training program, followed by a home-based exercise program, is safe and improves exercise capacity and quality of life in individuals with pulmonary arterial hypertension. This study aims to recruit 34 subjects who will be randomly allocated to the exercise group  or the control group . Subjects will be assessed at baseline, 12 weeks and 24 weeks.This study will determine whether outpatient-based, whole body exercise training is beneficial and safe in individuals with pulmonary arterial hypertension. Additionally, this study will contribute to clinical practice guidelines for this patient population.ACTRN12609000502235Australia and New Zealand Clinical Trials Register (ANZCTR):  Pulmonary arterial hypertension (PAH) is characterised by pathological changes in the pulmonary vasculature that cause an increase in pulmonary vascular resistance (PVR) thereby restricting blood flow through the pulmonary circulation ,2. To maDespite advances in pharmaceutical therapies, which have resulted in improvements in exercise capacity, haemodynamics and outcome in patients with PAH , exertio2] or maximum heart rate [HR]) ) ,17,19-21te [HR]) ,21.Historically, exercise training has been discouraged as a therapy for patients with PAH due to the perceived risk that exercise could lead to sudden cardiac death or worsening pulmonary vascular haemodynamics, acceleration of vascular remodelling and deterioration in right heart function . Some evThe studies to date have demonstrated improvements in exercise capacity in this patient population ,15,25, hTo date, the studies have only reported the immediate effects of exercise training and thus it remains unknown whether the benefits are maintained following cessation of supervised training. It is also unknown whether an outpatient-based, whole body exercise training program is beneficial in patients with PAH and whether this type of program is capable of producing sustained benefits in exercise capacity and HRQoL.This study aims to investigate the benefits of a 12 week hospital outpatient-based, individualised exercise training program involving supervised exercise sessions, followed by a 12 week home exercise program, for a cohort of subjects with PAH.We hypothesise that:(i) A 12 week individualised, whole body exercise training program will improve:2; anaerobic threshold; endurance time measured during the constant-workload cycle ergometry test)• Exercise capacity (as assessed by peak VO• HRQoL(ii) Exercise training is safe and improvements in exercise capacity will be achieved without evidence of clinical worsening(iii) An additional 12 week home exercise program will maintain or further improve the benefits gained during the 12 week, supervised, outpatient program.This study is a prospective, single blind, randomised controlled trial.Thirty-four subjects will be recruited from the Western Australian State Pulmonary Hypertension Service, at Royal Perth Hospital, Perth, Western Australia.Subjects will be included in the study if they: have a confirmed diagnosis of idiopathic or familial PAH or PAH associated with connective tissue disorders, based on elevated pulmonary artery pressures measured by right heart catheterisation at rest; are in WHO functional class II or III ; are medically stable and have been on PAH-specific pharmaceutical therapy for 3 months prior to enrolment into the study, and, are willing to complete the supervised and home exercise training programs. Medical stability will be defined as the absence of recent changes in: specific PAH therapy ; inotropes (including digoxin) and diuretics; symptoms (including dyspnoea and fatigue); body weight and co-morbid conditions.Subjects will be excluded if they have: oxygen therapy requirements; significant musculoskeletal disease, claudication pain, neurological or cognitive impairment, psychiatric/psychological or mood disorders that may affect their ability to undertake exercise testing or training; a history of moderate or severe chronic lung disease; cardiac disease associated with cardiac failure, poorly controlled angina, unstable cardiac rhythm; involvement in a supervised exercise training program within the last 12 months.The flow of subjects through the study is based on recommendations from the Consolidated Standards of Reporting Trials (CONSORT) statement  and outl2 > or ≤ 70% of predicted values  and end tidal carbon dioxide [PetCO2] to describe gas exchange);(i) Ventilatory variables at isotime during the constant workload cycle ergometry test  Change in WHO functional class;(iii) 6MWD (measured from the best of two 6MWTs at each assessment);(iv) Worsening clinical status .Improvements in endurance time following exercise training have recently been reported in the PAH population . These aConservative sample size calculations have been performed using a two-sided Mann-Whitney U test, assuming that the actual distribution is a double exponential. Group sizes of 14 will achieve 91% power to detect a difference of 200 seconds between the null hypothesis that both group means are 600 seconds and the alternative hypothesis that the mean of group 2 is 400 seconds with estimated group standard deviations of 180 and 120 and with a significance level  of 0.01.A total of 34 subjects (17 in the exercise group and 17 controls) will be recruited to allow for 20% attrition of subjects from the per protocol analysis.Data will be analysed using SPSS software . Data that are not normally distributed will be transformed and if transformation fails to satisfy normality, non-parametric tests will be used. The primary and secondary outcomes in the exercise and control groups will be compared using two-way analysis of variance to account for group and time interactions. Statistical relationships between outcome variables will be analysed using Pearson's or Spearman's correlations as appropriate. Data will be analysed using (i) per protocol analysis and (ii) intention-to-treat analysis. Per protocol analysis will be restricted to the data from subjects who complete all assessments and for the exercise group who attend at least 75% of their exercise classes. The required number of sessions must be achieved within a period of 15 weeks for both the supervised and home exercise training programs, allowing for intercurrent illness and other factors that may impact on an individual's ability to attend sessions. The last-observation-carried-forward method will be used to impute missing data at 12 and 24 weeks for the intention-to-treat analysis . The BerThis study will be the first randomised controlled trial to evaluate the physiological, clinical and psychological changes that occur following outpatient-based, whole body exercise training in PAH. We hypothesise that exercise training will improve exercise capacity and HRQoL. Adverse events and clinical worsening will be monitored in order to address theoretical safety concerns regarding the potential deleterious effects exercise may have on the pulmonary vasculature and right heart function. Assessment of the subjects following 12 weeks of home exercise program will determine whether the subjects were able to safely maintain the benefits achieved during the supervised exercise training period. Furthermore, this study will contribute to clinical practice guidelines, which will have important implications for the future management of patients with PAH.The authors declare that they have no competing interests.LG, SJ, RF, PW, DL, KG and EG designed this study protocol. LG drafted the manuscript and SJ, RF, PW, DL, KG and EG contributed to the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/11/25/prepub


Arabidopsis thaliana, a direct oxygen sensing system has recently been discovered, where a conserved N-terminal motif on some ethylene responsive factors (ERFs), targets the fate of the protein under normoxia/hypoxia. In Oryza sativa, this same group of ERFs drives physiological and anatomical modifications that vary in relation to the genotype studied. The microalga Chlamydomonas reinhardtii responses to low oxygen seem to have evolved independently of higher plants, posing questions on how the fermentative metabolism is modulated. In this review, we summarize the most recent findings related to these topics, highlighting promising developments for the future.Low oxygen stress often occurs during the life of green organisms, mostly due to the environmental conditions affecting oxygen availability. Both plants and algae respond to low oxygen by resetting their metabolism. The shift from mitochondrial respiration to fermentation is the hallmark of anaerobic metabolism in most organisms. This involves a modified carbohydrate metabolism coupled with glycolysis and fermentation. For a coordinated response to low oxygen, plants exploit various molecular mechanisms to sense when oxygen is either absent or in limited amounts. In  Plants and algae rely on aerobic respiration to produce energy. Oxygen availability is thus required and changes in its accessibility lead to drastic metabolic rearrangements. Ultimately, aerobic organisms die if the absence of oxygen is prolonged. A lower level of oxygen can result from environmental conditions, but also anatomical and tissue constraints. During their life, plants and algae are exposed to a range of oxygen concentrations that can vary from a fully aerobic state (normoxia) to oxygen deficiency (hypoxia) or the total absence of oxygen (anoxia) . Excessii.e., fermentation -hydrogenase enzymes, He light) .2 production pathway under dark anoxia. Recently, the effects of lesions in PFL1 [2 evolution, down-regulation of in vitro hydrogenase activity and lower HYDAs and PFR1 transcripts and protein levels in pfl1 mutants suggest that the H2 producing pathway cannot compensate for a loss of PFL1 activity. Instead of competing for the same substrate, PFL1 and PFR1 may actually work together to satisfy the physiological needs of the cell. ADH1 is the only enzyme responsible for ethanol production under dark anoxia in Chlamydomonas[adh1 mutant showed no increase in H2 evolution, but high levels of extracellular and intracellular glycerol. Synthesis of glycerol at the level of dihydroxyacetonphosphate (DHAP) would allow for NAD+ recycling in the mutant strain, thus maintaining viability  pool [Chlamydomonas[The metabolic restructuring observed for insertional mutants suggests that perturbation of the anaerobic metabolism activates unknown pathways that were set in place to regenerate reduced pyridine nucleotides and sustain survival. Additional work aimed at understanding how PQ) pool  and the PQ) pool –186. ProPQ) pool , are alsmydomonas; their pADH1 and HYDA2 transcripts has been shown to be independent of oxygen levels but is instead regulated by the circadian clock and/or cell cycles. PFR1 and HYDA1 expressions, on the other hand, are strongly influenced by oxygen tensions [Chlamydomonas.Several fermentative genes follow precise day-night fluctuations in their expression levels, which are not necessarily linked to the photosynthetic oxygen dissolved in the medium . Interestensions ,188. TheChlamydomonas and higher plants [HYDA1 and Ferredoxin 5 (FDX5) under copper (Cu) deficiency and hypoxia [HYDG, HYDEF and PFR1, also increase in response to Cu limitation [A comparative transcriptomic reconfiguration study by low oxygen stress suggests the independent evolution of the regulatory networks between r plants . While i hypoxia –193. Tramitation , suggestHYDA1[HYDA1 transcription, by tuning protein levels to metabolic needs [Chlamydomonas research and will help us in understanding how the fermentative metabolism is modulated and restructured in response to different environmental conditions.CRR1 binds the GTAC motif which is the core of Cu response elements (CuREs) ,195, andHYDA1. In spitic needs . The ideChlamydomonas as hydrogen-producing organisms makes the studies of this organism important, because Chlamydomonas are able to produce hydrogen only under anoxia [Chlamydomonas strains that can better tolerate anoxia would contribute to the efficiency of hydrogen production by this organism. These examples highlight the importance of research on plant and algae responses to low-oxygen. Several aspects of the molecular responses to hypoxia are still incomplete. These include the role of reactive oxygen species, the possible cross-talk and partially overlapping signaling pathways among different abiotic stresses, and, most importantly, the development of a set of new plant varieties that can guarantee elevated yields even under critical environmental conditions such as those resulting from waterlogging and flooding.Global climate change has a deep impact on the frequency and magnitude of precipitations. This causes severe events such as drought and floods  that havr anoxia . The dev


A systematic data analysis pipeline was developed and validated to prioritize and predict the pathogenicity of all genetic variants identified in each patient, which enabled us to reduce the number of potential pathogenic variants from approximately 1,200 to zero to nine per patient. Subsequent segregation analysis and in silico predictions of pathogenicity resulted in a molecular diagnosis in 36 RP patients, comprising 27 recessive, six dominant, and three X-linked cases. Intriguingly, De novo mutations were present in at least three out of 28 isolated cases with causative mutations. This study demonstrates the enormous potential and clinical utility of NGS in molecular diagnosis of genetically heterogeneous diseases such as RP. De novo dominant mutations appear to play a significant role in patients with isolated RP, having major implications for genetic counselling.Molecular diagnostics for patients with retinitis pigmentosa (RP) has been hampered by extreme genetic and clinical heterogeneity, with 52 causative genes known to date. Here, we developed a comprehensive next-generation sequencing (NGS) approach for the clinical molecular diagnostics of RP. All known inherited retinal disease genes ( Retinitis pigmentosa  is the most frequent subtype of inherited retinal disease and is clinically and genetically a highly heterogeneous disorder . Fifty-two genes are known to be associated with nonsyndromic RP, involving all modes of inheritance ) and a known (dominant) missense mutation in PRPH2 ]) /234 = 47%).USH2A and PDE6B appear to be slightly overrepresented in the mutational spectrum, as has been described previously [Hartong et al., Overall, the results of this study underscore the extreme genetic heterogeneity in RP by identifying putatively causative mutations in no fewer than 20 different genes . Mutations in In this study, we developed a comprehensive diagnostic tool for RP, consisting of a massive parallel sequencing approach for all known retinal disease genes, with systematic analysis and interpretation of all detected genetic variants. Our interpretation workflow provides a general approach for the interpretation of the vast amount of data generated by NGS-based molecular diagnostics. The enormous potential and clinical utility of NGS is highlighted by a high diagnostic yield achieved by the identification of mutations of all modes of inheritance, including de novo mutations. The high degree of (inherited and de novo) autosomal dominant and X-linked mutations confers a significant risk for transmitting the disease to the patients' offspring, thereby illustrating not only the molecular diagnostic power of NGS, but also the huge impact of this method on the affected families.The strength of this study lies in the parallel analysis of all known retinal disease genes in 100 RP patients with an unknown molecular cause of disease by NGS. Systematic variant prioritization reduced the initially high number of identified variants  by 99.7% to a number that is manageable to validate by Sanger sequencing (approximately four variants per patient). Combined with a systematic assessment of pathogenicity of the validated variants, this NGS approach resulted in a molecular diagnosis in no fewer than 36 patients. The usage of a completely unscreened, prospective cohort would probably have led to a more accurate estimation of the success rate of this approach. Although such prospective studies can be performed in a research setting, it is, however, impossible within DNA diagnostics, where every effort needs to be made to solve a sample in a timely fashion.Results for details), thereby outperforming previously used approaches such as traditional Sanger sequencing and APEX analysis [Hartong et al., Importantly, the 100 patients investigated here originally belonged to a cohort of 234 patients that had previously undergone selected genotyping [Collin et al., RP2 . Of the 78 isolated RP cases, a molecular diagnosis was established in 28 cases, including 20 families that harbored mutations in autosomal recessive RP genes and two male cases with mutations in X-linked genes. In two presumed isolated cases, mutations were found in autosomal dominant RP genes, but re-evaluation of both families revealed that one of the parents also had RP. Intriguingly, de novo mutations were proven to be present in three further isolated cases. Two of these were located in dominant genes, whereas in one patient, one of the two autosomal recessive mutations in USH2A occurred as a de novo event. Additional de novo mutations are likely in two other cases because these detrimental variants occur in dominant genes, whereas no phenotypes are reported for the parents. However, the absence of DNA of at least one parent hampers a definite conclusion. Although de novo mutations have recently been shown to play a major role in human diseases with reduced reproductive fitness [Hamdan et al., RPE65  mutations [Bainbridge et al., Apart from the ability to analyze all known RP genes simultaneously, thus increasing effectiveness of the genetic analysis, this approach has added value and superior clinical relevance because it allowed the identification of a substantial number of cases with apparent de novo mutations, and it has the potential to identify additional mutations potentially contributing to the phenotype—that is, to determine the cumulative mutational load. The 100 investigated samples comprised 78 isolated cases and 22 cases from recessive multiplex families. Among the 22 multiplex families, a molecular diagnosis was achieved in eight families: seven families carried mutations in autosomal recessive RP genes and one carried a mutation in the X-linked gene Further improvements in sequencing technology together with the identification of novel RP genes will undoubtedly boost the success rate of NGS-based diagnostic approaches in RP in near future. Recently, others have also reported on the use of NGS for the identification of disease-causing mutations in genetically heterogeneous disorders, including that of dominant [Bowne et al., Together, these studies support our conclusion that NGS will become increasingly important for the identification of disease-causing genes, and underscore the impact of this technology for the future of DNA diagnostics. For this, improvements in sequencing performance and interpretation should be combined with further cost reductions and improvements in turnaround times.


Lysosomal storage diseases are inherited metabolic disorders caused by genetic defects causing deficiency of various lysosomal proteins, and resultant accumulation of non-degraded compounds. They are multisystemic diseases, and in most of them (>70 %) severe brain dysfunctions are evident. However, expression of various phenotypes in particular diseases is extremely variable, from non-neuronopathic to severely neurodegenerative in the deficiency of the same enzyme. Although all lysosomal storage diseases are monogenic, clear genotype-phenotype correlations occur only in some cases. In this article, we present an overview on various factors and processes, both general and specific for certain disorders, that can significantly modulate expression of phenotypes in these diseases. On the basis of recent reports describing studies on both animal models and clinical data, we propose a hypothesis that efficiency of production of compounds that cannot be degraded due to enzyme deficiency might be especially important in modulation of phenotypes of patients suffering from lysosomal storage diseases. There are several thousand inherited diseases identified to date (Korf and Rehm GBA1 gene (coding for the glucocerebrosidase), and resultant alterations in sphingolipid metabolism, may contribute to biochemical changes found in Parkinson disease. More recent studies indicated that a lack of the glucocerebrosidase activity compromises lysosomal protein degradation and causes accumulation of α-synuclein, which results in neurotoxicity  of cell, tissue and organ dysfunctions in LSD, it is well documented that clinical symptoms arising from accumulation of incomplete degradation of the same substance which hydrolysis is inhibited at various stages can be significantly different. For example, brain disease in patients suffering from both mucopolysaccharidosis type I (MPS I) and mucopolysaccharidosis type III  (MPS III subtypes A to D) results from accumulation of metabolites of heparan sulfate (HS), one of glycosaminoglycans (GAGs) . The spectrum of neuronopathic symptoms of this disease is from severe mental and cognitive disability to completely normal intelligence and behavior, including all intermediate phenotypes . In fact, deficiency of α-L-iduronidase has been initially described as two different diseases, Hurler disease (MPS I) and Scheie disease (mucopolysaccharidosis type V or MPS V). Subsequent studies demonstrated that both disorders are caused by mutations in the same gene, IDUA, and led to the conclusion that they are just two extreme clinical phenotypes of the same metabolic disease  may result in attenuated, non-neuronopathic phenotypes  result in impaired degradation of certain glycoconjugates and accumulation of globotriaosylceramide (Gb3) in various tissues including kidneys, heart, and the nervous system. As discussed by Ries and Gal , there iThere are 3 clinical forms of Gaucher disease (glucocerebrosidase deficiency): type 1 is restricted to visceral and/or skeletal involvement with no brain dysfunction, type 2 is a neuronopathic form characterized by severe central nervous system involvement in infancy, and type 3 is an intermediate form, with milder neuronopathy. In Gaucher disease, some clear genotype-phenotype correlations could be detected. The N370S mutation (in either homozygotic or complex heterozygotic form) is restricted to patients with type 1 disease, while homozygosity for the L444P mutation is highly associated with type 3 disease , one of GAGs. Amongst over 130 pathogenic mutations described in the ARSB gene, most are missense mutations  is a disorder caused by deficiency in N-acetylgalactosamine 4-sulfatase (arylsulfatase B) activity due to mutations in the In summary, while some genotype-phenotype relationships are evident in LSDs, there are plenty of examples that the same mutation may result in very different clinical symptoms and different progress of the disease. Moreover, level of residual activity of the deficient enzyme often does not correspond to the disease severity.Since genotype-phenotype correlations cannot be determined in many cases of LSDs, it became clear that other factors or processes must influence severity of symptoms occurring in particular patients. In fact, publications from last several years indicated that there are various physiological processes and environmental factors that can significantly modify the course of LSDs. Since mechanisms of these processes and actions of these factors have been excellently reviewed in past few years, we will present them only briefly in this overview, describing their crucial features and refereeing readers to previously published articles for details.Contrary to early assumptions on the mechanisms of LSDs, it is now obvious that various symptoms appear not only due to primary storage of undegraded compounds in lysosomes  and live together under very similar conditions, which is definitely not the case. Therefore, one may assume that there are processes specific to each disease which can significantly modulate its course and severity.Vast majority of LSDs results from an imbalance between synthesis and degradation of particular compounds. While the synthesis process is unaffected, the degradation is impaired which leads to accumulation of certain substances in lysosomes. About a decade ago it was speculated that various efficiencies of syntheses of particular compounds, which may occur in human population, could significantly influence severity of LSDs and result in different phenotypes of patients bearing the same mutations in genes responsible for degradation of these compounds . Neuronopathy of this disease is primarily due to demyelination of neurons. To learn about molecular mechanisms of this disease, a mouse model was constructed in which a homologue of the human Arsa gene, coding for ASA, has been inactivated  mutation (ASA-/-) did not reveal demyelination, and thus, characteristic symptoms of human MLD, like reduction in nerve conduction velocity, progressive paralysis, and peripheral neuropathy were not observed in these mice  is caused by deficiency in arylsulfatase A (ASA) and accumulation of sulfated glycosphingolipids, predominantly 3-H-1-benzopyran-4-one) , accumulated in: MPS IIIA and MPS IIIB, caused improvement of biochemical parameters and phenotypes of affected animals. This was demonstrated by chemical impairment of HS synthesis by the use of either rhodamine B ([9-(2-carboxyphenyl)-6-diethylamino-3-xanthenylidene]-diethylammonium chloride)  in one or two genes coding for enzyme(s) involved in HS synthesis, Ext1 and Ext2, which resulted in at most 2-fold decrease in the rate of production of this GAG. Therefore, reduction of HS by 30–50 % led to significant decrease in severity of the disease, including amelioration of the amount of disease-specific biomarker in the brain , but also on efficiency of production of the substrate(s) that cannot be efficiently degraded in lysosomes. More specifically, less efficient production should result in less severe symptoms. Do we have any evidence that similar phenomena may occur in humans suffering from LSDs? A few years ago, a report was published in which efficiency of GAG synthesis was measured in fibroblasts of healthy persons and patients suffering from various types of MPS . However, phenotypes of patients bearing mutations resulting in residual activities of affected enzymes depend on other factors, particularly various stresses caused by different agents . The summary on factors and processes determining severity of LSDs is presented schematically in Fig. Genotype-phenotype correlations in LSDs are clear for some types of mutations, particularly when


Peritonitis is a major complication of various disease entities in internal medicine, surgery, and gynecology and, despite treatment advances, remains associated with unacceptably high morbidity and mortality. Causes of peritonitis are manifold including infectious, autoimmune, or chemical processes. A precise knowledge of inflammatory mechanisms in the initiation and establishment of peritoneal inflammation is of pivotal importance for the prevention and treatment of peritonitis. This special issue aims to bring the molecular mechanisms of inflammatory processes in the peritoneal cavity into focus, regardless of the organ system or the specific syndrome they result from.Mediators of Inflammation, we are pleased to present to the reader a series of special features written by designated experts in the field.In this special issue of γ) in regulating peritoneal inflammation and fibrosis. Furthermore, a novel PEGylated Toll-like receptor 7 (TLR7) ligand might be of great value for treatment of mast-cell-mediated neutrophilic inflammation.Local production of proinflammatory mediators at sites of tissue and cell injury within the peritoneal membrane plays a critical role in the propagation of peritoneal inflammation. In addition to infectious causes, various forms of peritoneal damage, induced by surgical trauma and peritoneal dialysis, compromise peritoneal integrity and can ultimately lead to tissue damage, fibrosis, adhesions, or even in the most severe cases the development of encapsulating peritoneal sclerosis. Peritoneal mesothelial cells appear to play a major role in these processes as they are capable of expressing a raft of proinflammatory and immune-modulatory mediators that either directly mediate fibrotic remodelling or attract immune cells to the peritoneal cavity or participate in immune activation. Therapeutic modulation of this sequence of events could provide novel avenues to attenuate peritoneal inflammation and its resulting tissue damage. The present special issue presents novel data on this topic, particularly underscoring the potential impact of the peroxisome proliferator-activated receptor-gamma  is mediated by the dismutation of superoxide into hydrogen peroxide. To analyze whether SOD3 can regulate inflammation in the absence of functional NOX2 complex, T. Kelkka et al. used an elegant design, comparing anti-inflammatory effects of overexpression of SOD3 in Ncf1We would like to thank all contributors and reviewers for their support to this special issue and feel sure that readers will learn much about this important topic.Christoph AufrichtChristoph AufrichtWolfgang NeuhoferWolfgang NeuhoferNicholas TopleyNicholas TopleyMarkus WörnleMarkus Wörnle


ARF, significantly contribute to this event highlighting p53 reactivation as an attractive perspective for neuroblastoma treatment. In addition to its role in tumorigenesis, MYCN sensitizes untransformed and cancer cells to apoptosis. This is associated to a fine modulation of the MDM2–p53 pathway. Indeed MYCN induces p53 and MDM2 transcription, and, by evoking a DNA damage response (DDR), it stabilizes p53 and its proapoptotic kinase Homeodomain Interacting Protein Kinase 2 (HIPK2). Through the regulation of the HIPK2-p53 inhibitor High Mobility Group protein A1 (HMGA1) and the homeobox proteins BMI-1 and TWIST-1, MYCN establishes a delicate balance between pro- and antiapoptotic molecules that might be easily perturbed by a variety of insults, leading to cell death. MDM2–p53 antagonists, such as Nutlin-3, are strikingly prone to inducing death in MYCN-amplified neuroblastoma, by further pushing on HIPK2 accumulation. Here we discuss implications and caveats of exploiting this pathway and its connections to MYCN-induced DDR for a tailored therapy of MYCN-amplified neuroblastoma.The p53 oncosuppressor is very seldom mutated in neuroblastoma, but several mechanisms cooperate to its functional inactivation in this tumor. Increased MDM2 levels, due to genetic amplification or constitutive inhibition of p14 Neuroblastoma, one of the most common tumor of childhood, is an extraordinary challenging nosocomial entity due to its tremendous heterogeneous behavior ranging from spontaneous regression to very aggressive and inexorable progression. In 2009, the International Neuroblastoma Risk Group defined seven parameters  required to stratify neuroblastoma patients into tentatively homogeneous risk groups and estimated that the high-risk fraction  accounted for about 40% of their 8800 patients . About hp53 is a master oncosuppressor protein involved in protecting cells from genetic instability by inducing cell cycle arrest or apoptosis in response to cellular stress and DNA damage. Its genetic inactivation characterizes more than 50% of human cancers and its functional impairment occurs in most of the remaining part, highlighting the “reactivation of p53” as a treatment option potentially effective for an extremely broad range of human cancers. Indeed, p53 restoration already proved to be effective in promoting tumor regression in animal models and several strategies aimed at restoring p53 function  are being currently tested in clinical trials .p53 is very seldom mutated in childhood cancer of the central and peripheral nervous system and in primary neuroblastoma its mutation rate does not exceed 1–2% . However ARF impairs its activity on p53. Interestingly, ARF inactivation (due to deletions or promoter methylation) or MDM2 amplification are frequently found in neuroblastoma cell lines. Analysis of a small series (41 cases) of neuroblastoma tumors documented ARF inactivation or MDM2 amplification at diagnosis in about 30% of the cases, while five of six mutations detected on p53 were not present in the primary lesion, but appeared at relapse or after chemotherapy . The emerging molecular picture providing a rationale for this is depicted in the next paragraph.Although p53 expression levels may be directly repressed by miRNAs , deregulotherapy . Althougotherapy . (iii) Uotherapy . Of rele context , with obMYCN belongs to a conserved family of bHLHZip transcription factors that coordinate cell proliferation, differentiation, and survival as well as the interactions between cells and their microenvironment, including angiogenesis, invasion, and inflammation. Disruption of either c-Myc or N-Myc in mice leads to embryonic lethality due to growth defects in multiple organs and tissues. Additionally, c-Myc is one of the four essential genetic ingredients that efficiently convert fibroblast into pluripotent stem cells, confirming that Myc proteins play an unparalleled role in the regulation of stemness and cell proliferation. Therefore it is not surprising that direct or indirect deregulation of MYC expression occurs in the vast majority of human cancers.Primary deregulation of Myc expression or experimentally induced Myc overexpression also leads to apoptosis, largely (though not exclusively) via the ARF/p53 pathway, which initially appeared paradoxical for a prototypic oncogene. However, more recently it has been shown that many oncogenes  can trigger a DNA damage response (DDR) most likely connected to the replication stress and certainly involving the signaling cascades prompted by the ATM/ATR kinases, which in turn promote p53 accumulation/activation and apoptosis, cell senescence or stable inhibition of cell cycle progression. The DDR, therefore, has been envisaged as an intrinsic anti-cancer barrier that needs to be disrupted  for cell transformation to proceed .in vitro , unless MDM2 increase due to p53 transcriptional activation restores the balance, progressively silencing the pathway. The MYCN-dependent apoptotic-sensitive phenotype observed in vitro is mirrored in vivo by a high level of mitosis-karyorrhexis and by the very frequent initial response to the induction therapy observed in MNA neuroblastoma patients. Nevertheless, MNA tumors most frequently relapse, suggesting that several MNA cells find the way to escape apoptosis and are able to quickly reconstitute local tumor bulk and distant metastatic dispersion. While the increase in p53 mutation rate in relapsed and post-chemotherapy neuroblastoma indicates that inactivation of p53 is relevant for tumor progression, the small amplitude of the phenomenon (up to 15% mutation rate) confirms that the power of the p53 pathway to induce death under treatment is made functionally inefficient, at least in some cells. Thus, although the transient disequilibrium described above justifies the increased rate of apoptosis in MNA cells, it does not translate in a benefit for the patients, rather it pushes the selection of most resistant cancer clones. It is reasonable to believe that a more potent and more permanent unbalancing of the pathway leading to an increased activation of the proapoptotic components  could improve the outcome possibly also in vivo. Consistently, uncoupling p53 from MDM2, via small molecules MDM2-antagonists, results in massive cell death in MNA neuroblastoma cells either alone or in combined treatment with chemotoxic drugs  Oveic drugs . Furtheric drugs , with poWhile all of the above supports the hypothesis that the pharmacological reactivation of p53 might be an interesting therapeutic opportunity for MNA neuroblastoma, that further improving the molecular understanding of this pathway is also required to avoid that an oversimplification of the picture might cause unforeseen effects in patients. The increasing levels of complexity in the regulation of the pathway also call into play MDMX as a relevant modulator of p53 mono- or polyubiquitination by MDM2, which would alternatively cause its transcriptional inhibition and nuclear export versus proteolytic degradation, with relevant consequences on the biological outcome in normal and cancer cells . Nutlin-de novo p53 mutations associated with a multi-drug-resistant phenotype with an extremely high rate in several cancer cells, including neuroblastoma (Last, but not least, the inborn leaning to develop multi-drug resistance brought about highly effective death-inducing approaches in most aggressive tumors, should be considered. Indeed, continuous exposure to Nutlin-3 promotes appearance of blastoma . Togetheblastoma , two maiThe 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.


Sulfur is both crucial to life and a potential threat to health. While colonic sulfur metabolism mediated by eukaryotic cells is relatively well studied, much less is known about sulfur metabolism within gastrointestinal microbes. Sulfated compounds in the colon are either of inorganic  or organic  origin. The most extensively studied of the microbes involved in colonic sulfur metabolism are the sulfate-reducing bacteria (SRB), which are common colonic inhabitants. Many other microbial pathways are likely to shape colonic sulfur metabolism as well as the composition and availability of sulfated compounds, and these interactions need to be examined in more detail. Hydrogen sulfide is the sulfur derivative that has attracted the most attention in the context of colonic health, and the extent to which it is detrimental or beneficial remains in debate. Several lines of evidence point to SRB or exogenous hydrogen sulfide as potential players in the etiology of intestinal disorders, inflammatory bowel diseases (IBDs) and colorectal cancer in particular. Generation of hydrogen sulfide via pathways other than dissimilatory sulfate reduction may be as, or more, important than those involving the SRB. We suggest here that a novel axis of research is to assess the effects of hydrogen sulfide in shaping colonic microbiome structure. Clearly, in-depth characterization of the microbial pathways involved in colonic sulfur metabolism is necessary for a better understanding of its contribution to colonic disorders and development of therapeutic strategies. The crucial role of the microbiome in digestive processes and intestinal health is now fully integrated in gastroenterology research  production within the colon . The SRB use sulfate as a terminal electron acceptor for respiration, with the concomitant production of H2S , acetate-utilizing Desulfobacter spp. (9–16%), propionate- and hydrogen-utilizing Desulfobulbus spp. (5–8%), lactate-utilizing Desulfomonas spp (reclassified within the genus Desulfovibrio) (3–10%), and acetate- and butyrate-utilizing Desulfotomaculum spp. (2%). However, these observations are based on cultivation methods, which underestimate true bacterial diversity. More recently molecular-based techniques have been applied successfully to describing SRB diversity in various environments. SRB have thus far been detected and quantified from stool and colonic mucosa samples , cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST), three enzymes involved in HBilophila wadsworthia, a close relative of Desulfovibrio spp. , which is quickly absorbed by the mucosa or passes as flatus , Crohn's disease (CD), and ulcerative colitis (UC), afflict 0.1–0.5% of individuals in Western countries . Furthermore, H2S production in UC pouches was 10 times greater than that in FAP pouches -mediated proliferation  is a functional bowel disorder characterized by chronic abdominal pain, bloating, and abnormal bowel habits Mayer, . Diarrhe2S (NaHS) inhibits in vitro motor patterns in the human, rat and mouse colon and jejunum mainly through an action on multiple potassium channels  inhibited nociception induced by colorectal distension in both healthy and post-colitic rats (Distrutti et al., Consistent with this possibility, it was demonstrated recently that exogenous H2S, the sulfated compound with the highest potential influence on digestive health, can be generated from cysteine degradation as well as via dissimilatory sulfate reduction. The extent to which organic sulfur metabolism is operative in the human colon, how diet influences relative activities of the enzymatic pathways involved, and which organisms carry out these processes are poorly understood. In addition, the importance of the microbiota and the metabolism of sulfated bile acids are now established, and further work is needed to understand how dietary fat intake influences these pathways. With clear evidence that the human colonic mucosa is persistently colonized by SRB, the beneficial versus toxic effects of H2S need to be delineated. One possibility is that the mucosal microbiome is shaped in part through the differential susceptibility of mutualistic microbes to sulfide toxicity. Clearly, extensive work is needed to understand the primary pathways for the production of H2S and how their activities may influence both host and microbial components of the colonic ecosystem. This new body of knowledge may identify relatively innocuous approaches for the prevention or treatment of chronic colonic disorders.Microbial sulfur metabolism in the human colon is likely more extensive than has been previously recognized. For example, HThe 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.


Cancer stem cells (CSCs) play a major role in local recurrence and metastatic spread in head and neck squamous cell carcinomas (HNSCC). Evidence suggests that cancer stem cells are resistant to conventional therapy. So the emerging concepts of the role of cancer stem cells in the pathobiology of HNSCC should be understood carefully to be able to create new paradigms in treatment plans. Despite the progress in diagnosis and advancement in management of head and neck squamous cell carcinoma (HNSCC), long-term survival rates have not improved very much over the past decade. Even after combining chemotherapy and radiation for advanced stage disease, the treatment outcomes have marginally improved . The proThe stochastic model suggests that every cancer cell is able to initiate new tumor growth equally.The alternate hypothesis is that every tumor contains a rare population of cells termed CSCs or cancer-initiating cells (CICs) –9.A tumor can be seen as an “organ” composed of transformed cells that interact with stromal cells within the tumor microenvironment . The proonly a small fraction of the cancer cells within a tumor have tumorigenic potential when transplanted into immunodeficient mice;the cancer stem cell subpopulation can be separated from the other cancer cells by distinctive surface markers;tumors resulting from the cancer stem cells contain the mixed tumorigenic and nontumorigenic cells of the original tumor;the cancer stem cell subpopulation can be serially transplanted through multiple generations, indicating that it is a self-renewing population.The most recent key features of the cancer stem cell hypothesis were described by Prince and Ailles in 2008 :only a sThe concept of CSCs first began 150 years ago when Robert Virchow, a German pathologist, found similarities between embryonic and tumor tissues . The CSCThe most common characteristics of CSCs were described as those cells having anchorage independent growth and metastasizing capacity, active membrane transporter activity, ability to survive for a long time, resistance to damaging agents, self-renewal capacity, active telomerase expression , and differentiation capacity to mature progeny .Gao in 2008 identified precancerous stem cells (pCSCs) in cancer and suggested that both pCSC and CSC might also serve as precursors of tumor stromal components such as tumor vasculogenic stem/progenitor cells (TVPCs). Thus, he suggested, the developing process of tumor-initiating cells (TIC) is initiated from pCSC leading to CSC and later cancer, a cellular process that parallels the histological process of hyperplasia/metaplasia (TIC) from precancerous lesions (pCSC) into malignant lesions (CSC → cancer) .β (TGF-β) superfamily and function as oncogenes and tumour suppressors sometimes. Sonic Hedgehog signalling pathway is a major regulator of some of the fundamental processes including stem cell maintenance, cell differentiation, tissue polarity, and cell proliferation [Some of the most important signals enumerated in maintaining stem cell proliferation in tumorigenesis are Oct-4, Notch, Wnt/Catenin, bone morphogenic protein (BMP), Sonic Hedgehog signalling pathway, Mushashi-1 (Msi-1), and so forth . Oct-4 iferation .CSC expresses specific markers that vary considerably depending on tumor type or tissue of origin—the discovery of a universal marker has not yet been made. General methods for the identification of CSCs in solid malignancies are similar to those strategies employed to differentiate normal stem cells from their differentiated progeny. The list of potential markers of CSCs includes CD133, CD44, CD24 (in combination with CD44) efflux of Hoechst or Rhodamine dyes, CD90, CD117, CD34, and CD20. This list also includes the efflux of vital dyes by multidrug transporters, the enzymatic activity of aldehyde dehydrogenase (ALDH), colony and sphere-forming assays utilizing specific culture conditions, and the expression of specific cell surface antigens known to enrich for stem cells [Xenotransplantation assays, the gold-standard for identification of CSCs, are used to assess the tumorigenicity and self-renewing potential of the putative CSC population.CD44, the most well-recognized CSC marker, is a large cell surface glycoprotein that is involved in cell adhesion and migration.ALDH  activity is known to enrich hematopoetic stem/progenitor cells and recently has been revealed to enrich cells with increased stem like properties in solid malignancies .The most commonly applied among them are as follows.Hypoxia instigates cell demise by apoptosis/necrosis, it also prevents cell death by provoking adaptive responses that, in turn, facilitate cell proliferation or angiogenesis, causative to tumor progression . In addiκB, PI3K/Akt/mTOR, Notch, Wnt/β-catenin, and Hedgehog signaling pathways have been recognized as important regulators of CSCs and EMT [Evidence till date is suggestive of the fact that these hypoxic areas within a tumor are considered as a niche where CSCs reside. It is stated that effects of hypoxia on stem cell function are directly mediated by the hypoxia inducible factors (HIF). HIF function, as a master transcription factor, regulates hypoxia responsive genes. The molecular relationship between HIF signaling pathway with the biology of CSCs and epithelial mesenchymal transition (EMT) remains unclear although NF- and EMT . The lin and EMT . By stim and EMT .α and HIF-2α are associated with tumor aggressiveness; HIF-1α promotes the phenotype and function of CSCs, whereas HIF-2α is highly expressed in CSCs; also known as TIC of neuroblastoma, glioblastoma, renal cell carcinoma, and breast carcinoma are associated with unfavorable disease outcome [Emerging experimental evidence indicates that hypoxia also plays an important role in the regulation of the phenotype and function of CSCs consistent with recent findings in glioma CSC. It has been shown that hypoxia is able to maintain the stem-like phenotypes in neuroblastomas and activate signaling pathways that are associated with undifferentiated phenotypes of normal stem cells, including sex determining region Y box 2 (Sox2), Oct-4, and Notch-1 signaling . Both HI outcome .The CSC niche main role is to dedifferentiate nontumorigenic cells into tumorigenic CSCs and to induce the EMT, leading to dissemination of tumor cells from the primary tumor and further seeding at the metastatic place. Supporting cells of these niche release a number of molecular factors that have been identified to control stem cell identity. These factors include components of the BMP, Notch, Wnt, JAK-STAT, and Shh signaling pathways, which provide intercellular cues that regulate stem cell identity and differentiation , 27.It has been unveiled that a premetastatic niche is established by the attraction of bone marrow derived cells to the future site of metastases by the secretion of factors from cancer cells . The nicEMT is a process by which epithelial cells lose their polarity and are converted to a migrant mesenchymal phenotype. In recent times, it has been considered as one of the fundamental steps that induces invasion and metastasis of tumors during cancer progression . SimilarTill date, very few studies have been conducted using HNSCC tissue for CSC marker identification. Markers such as aldehyde dehydrogenase (ALDH), CD133, and CD44 have been successfully used to identify highly tumorigenic cancer stem cells in HNSCC. Prince et al., in 2007, were the first ones to unveil the presence of highly tumorigenic, stem-like cells in HNSCC using primary tumor specimens as one-third of their samples . In a foLim et al. later isolated sphere-forming cells (squamospheres) from primary HNSCCs and characterized their stem cell properties like self-renewal, stem cell marker expression, aberrant differentiation, and tumor-initiating potential. Furthermore, these HNSCC-driven squamospheres appeared to be chemoresistant to cisplatin, 5-fluorouracil (FU), paclitaxel, and doxetaxel and showed increased levels of ABCG2, one of the ATP-binding cassette (ABC) transporters. So accordingly the functional analysis of these squamospheres may provide a new insight into the tumorigenic process of HNSCC . But irrOral mucosa consists of a number of distinct layers and has self-renewing capacity, such as bone marrow and skin. Mostly the basal layer cells are in the process of preparing for cell division, which later remain or move to a suprabasal position and become committed to terminal differentiation and stratification. The first hypothesis recommended that the CSCs in oral squamous cell carcinoma (OSCC) could be from local basal layer-derived adult SC or progenitor, which accumulate additional genetic alterations with time, within the tumor. The other proposed mechanism suggested the origin of CSCs to be either putative nonepithelial stem cell sources in the oral mucosa  or due to cell fusion between a hematopoietic stem cell and a mutated oral keratinocyte or can also originate through dedifferentiation of mature cells .After understanding the possible role of CSCs in HNSCC, Oliveira et al. tried to find the possible influences of these CSCs in oral squamous cell carcinoma using CD44 and CD24. They suggested in their result that the absence of immunoexpression of these two investigated markers can be used in combination with other clinicopathologic information to improve the assessment of prognosis in OSCC .Costea et al. in their hypothesis suggested a potential involvement of the stromal microenvironment OSCC progression. As already known, the activated fibroblasts (myofibroblasts) inside the tumor stroma stimulate the transformed keratinocytes, thus influencing stem cell division patterns and with further genetic alterations of these keratinocytes leads to evolution of more invasive clones . OSCC isCSCs seem to harbor mechanisms that are found to be responsible for therapy resistance in glioblastoma and pancreatic cancer, and recently in colon cancer as well . It has Hypoxia has been understood to play a key role in tumor progression and hypoxic tumor microenvironment in turn has a control over the CSCs. So, when the antiangiogenic agents are administered in combination with CSC-targeted drugs, more effective results are attained in cancer therapy, along with inhibiting hypoxia inducible factors (HIF) . CSCs arκB-inhibitors, nutraceuticals, and antioxidant therapy. NFκB degradation by inhibitor of κB (IκB) proteins may be a useful therapeutic solution under such circumstances. It has also been suggested that treatment with echinomycin blocks the hypoxia-induced stroma [So, the therapeutic aspect should lead to target hypoxia curbing these hypoxic niches by HIF-inhibitors, NFd stroma .The confrontation in the years to come will be how to determine the contribution of each model to tumor growth in a given patient and how we can use that information to design more effective therapeutic strategy. Identification of reliable markers is required to characterize CSCs in HNSCC and OSCC in particular as this could ensure the clinical effectiveness of future targeted treatments, possibly resulting in a more effective outcome.


In Spain a significant number of individuals die from atherosclerotic disease of the coronary and carotid arterieswithout having classic risk factors and prodromal symptoms. The diagonal ear lobe crease (DELC) has beencharacterized in the medical literature as a surrogate marker which can identify high risk patients having occultatherosclerosis. This topic however has not been examined in either the medical or dental literature emanatingfrom Spain.The majority of clinical, angiography and postmortem reports support the premise that DELC is a valuable extravascularphysical sign able to distinguish some patients at risk of succumbing to atherosclerosis of the coronaryarteries. A minority of studies have however failed to support this hypothesis. More recently reports using B modeultrasound have also linked DELC to atherosclerosis of the carotid artery and another report has related DELC tothe presence of calcified carotid artery atheromas on panoramic radiographs.DELC is readily visible during head and neck cancer screening examinations. In conjunction with the patient’smedical history, vital signs, and panoramic radiograph, the DELC may assist in atherosclerotic risk assessment. Key words: Diagonal ear lobe crease, atherosclerosis disease, calcified carotid artery, atheromas, panoramic radiographs. Atherosclerosis is a major source of disability and death in Spain. In 2002, coronary artery atherosclerosis (CAA) resulted in 45,000 Spanish citizens suffering a fatal myocardial infarction and carotid artery atherosclerosis resulted in the stroke associated death of an additional 34,000 persons . Many ofPreliminary uncontrolled observations by a pulmonologist that a “positive ear-lobe sign”  was shown to be significantly more common (47%) among all age groups of patients (N=531) hospitalized in a USA coronary-care unit after an acute myocardial infarction than among age-matched control subjects (30%) with no clinical evidence of CAA .In 1978, Kaukola, an internist at University Hospital in Helsinki, Finland reported on the correlation between DELC and CAA  requiring coronary angiography at Helsinki University Central Hospital because of typical signs of CAA, that is angina pectoris and ischemic electrocardiographic changes  admitted to a medical-surgical unit during a two month period of time for the presence of DELC . In patiA Turkish study of 415 men and women (mean age 58.9 years) requiring coronary artery angiography for stable angina with a positive stress test, unstable angina, or history of prior myocardial infarct were also evaluated for bilateral DELC . There wDELC has also been shown to have predictive significance. In one prospective clinical study, of newly admitted hospitalized patients (N = 108), matched for sex, race, and age and having at least one DELC but free of CAA followed for an 8-10 year period. Cardiac event rates  for those with DELC were higher (10.4 events per 100 patient-years) than for those without DELC (1.4 events per 100 patient-years) . The difSimilarly, a British forensic necropsy study convened to determine the medical/legal cause of death of unselected consecutive adult cases (N = 303) demonstrated that the proportion of DELC in those aged ≥ 55 was significantly higher than in younger subjects (74% vs. 39%) . The proA Swedish forensic necropsy study of the death of 420 males and 100 females (mean age 56) residing in Linkoping, Sweden , demonstThere are numerous other studies which have demonstrated the very strong relationship between DELC and CAA, however there are confounding issues related to DELC such as its prevalence in different ethnic groups as well as the effect of known atherogenic risk factors. For example, as noted earlier, among northern Europeans coming to autopsy (mean age 60) the prevalence of DELC is 75%,17 far greater than the prevalence among native Japanese where it is only 5% . In San Over the past 35 years there have been a number of published studies which have failed to substantiate the relationship between DELC and CAA ,24. A nuIn a prospective study conducted in Ireland, researchers assessed the relationship between DELC and the severity of CAA as defined by the volume of myocardium at jeopardy of necrosis because of the anatomical location and number of stenotic lesions exhibited by 125 consecutive patients undergoing coronary angiography  requiring coronary artery angiography. The angiograms were classified as normal (no lesions detected), minor lesions (< 50% occlusion), and single-vessel (> 50% occlusion), double-vessel, and triple-vessel disease. CAA of varying severity was noted in 67% of individuals with DELC and 67% of individuals without DELC. There being no difference between the groups, the authors concluded that the results of their study did not support DELC as a marker of CAA  requiring coronary angiography at an acute care hospital in Erlangen, Germany . They deIn 2009 in a letter to the editor in the American Journal of Forensic Medicine and Pathology, Koracevic and Atanaskovic reported on their previously unpublished study which they had conducted 13 years earlier . These iIn the 1970’s it was postulated that DELC and CAA arose simultaneously because the ear lobe and heart are supplied by “end arteries” without the possibility for collateral circulation. Others suggested that the generalized loss and degeneration of elastin and tears in the elastic fibers seen in biopsy specimens taken from the ear lobes of persons affected with CAA reflected the microvascular disease that was also present in the coronary bed but in a separate histologic study degenerative changes in the ear lobes of patients having sustained a fatal myocardial infarction could not be identified  but skinIn 2007, Celik S et al., published the first report on the relationship between DELC and carotid artery atherosclerosis imaged by B-mode ultrasound among a group of apparently healthy Turkish subjects attending a medical clinic for a standard medical check-up . This auIn 2009, Shrestha I et al., evaluated 212 patients  in Japan requiring a B-mode ultrasound evaluation of the extracranial carotid arteries because of a prior history of cardiovascular/cerebrovascular disease or for pre-operative screening purposes . ApproxiIn 2010, in a Letter to the Editor of the Journal of Oral and Maxillofacial Surgery, a dental researcher prompted by Celik’s report (Shrestha’s manuscript was still in-press), reported on the results of an observational study on the prevalence of DELC in 10 consecutive neurologically asymptomatic patients  referred for confirmation of the presence of a calcified carotid artery atheroma (CCAA) on their dental radiographs Fig.  34). Th. Th34). The results of the above noted pilot study are important even though it was of small sample size and uncontrolled given that a team of dentists and neurologists, in an earlier controlled study (when the importance of DELC was unrecognized), followed a group (N = 46) of patients (mean age 68) with CCAA on their panoramic radiographs for an average follow-up period of 3.6 years . During this time, coronary artery and carotid artery atherosclerosis as evidenced by adverse vascular events such as angina requiring hospitalization, need for coronary artery revascularization surgery, non-fatal myocardial infarct, stroke, and transient ischemic attack were significantly more common among the study patients than among controls. These results demonstrate that radiographic CCAA are independent indicators of future adverse vascular events and that the preponde-rance of cardiovascular events rather than cerebrovascular events is consistent with recent research findings that, in the same individual, CAA is often far more advanced than CCAA ,38. FurtA likely association between diagonal ear lobe creases (DELC) and atherosclerosis of the coronary arteries (CAA) and more recently the carotid arteries has been demonstrated over the past 35 years by clinical, autopsy, and angiography studies though not completely substantiated. A series of alternative hypotheses have also been suggested, including that DELC is the result of aging and that the relationship with atherosclerotic disease is mere coincidence and even that DELC is an anatomic peculiarity of some ear lobes perhaps the result of a particular way of sleeping .We, however conclude that while more research in this area is indicated, it would be prudent for dentists when conducting a head and neck cancer screening examination to observe their patients’ ears for the presence of DELC and in conjunction with the medical history, vital signs and panoramic radiograph formulate a risk assessment and determine if medical consultation is indicated for evaluation of coronary and/or carotid artery disease.


Congenital heart disease (CHD) is the most common birth defect in humans. It is a leading infant mortality factor worldwide, caused by defective cardiac development. Mutations in transcription factors, signalling and structural molecules have been shown to contribute to the genetic component of CHD. Recently, mutations in genes encoding myofibrillar proteins expressed in the embryonic heart have also emerged as an important genetic causative factor of the disease, which implies that the contraction of the early heart primordium contributes to its morphogenesis. This notion is supported by increasing evidence suggesting that not only contraction but also formation, mechanosensing, and mechanotransduction of the cardiac myofibrillar proteins influence heart development. In this paper, we summarize the genetic clues supporting this idea. The heart is the first organ to form in vertebrates during embryogenesis, and cardiac contraction initiates shortly after the cardiac tube is formed. The ever increasing needs of the peripheral tissues of the growing embryo for nutrients and oxygen are met gradually by the blood circulation induced by early cardiac contraction. The heart primordium has to perform its function for most of its own development, and this unique feature has profound implications in the relationship between form and function during cardiogenesis: factors that compromise early cardiac function result in altered morphology, and heart malformation usually has an important impact over the function of the organ. Defective heart development results in congenital heart disease (CHD), the commonest congenital defect in humans and a leading cause of infant mortality worldwide. CHD is a complex disease, as multiple genetic and environmental factors have been implicated in its pathogenesis. As in most complex diseases, most cases are sporadic, although a relatively small fraction of cases show familial segregation, following Mendelian patterns of inheritance. The study and identification of the genes causing the familial forms of the disorder can also give important information about the genetic component of the more common, complex variety.β-cardiac myosin heavy chain, MYH7) was shown to cause another cardiac disease, hypertrophic cardiomyopathy (HCM) [Genetic linkage is a technique that identifies genes responsible for inherited traits solely based in their position on chromosomes without prior knowledge of their product or its function. More than two decades ago, by genetic linkage analysis, a mutation of a gene encoding a myofibrillar protein (hy (HCM) . Since thy (HCM) .NKX2-5 and GATA4) encode transcription factors. Subsequently, mutations in genes that encode other types of protein, like signalling and structural molecules, have been described in families with Mendelian CHD, and common variants have been identified as predisposing factors for non-Mendelian CHD in large cohorts of sporadic cases  [The first genes shown to be responsible for familial cases of CHD  .α-cardiac myosin heavy chain, MYH6). Subsequently, we and others have shown that mutations in other genes that encode other myofibrillar proteins can cause defective cardiac development [Some relatively recent developments in the search for the genetic origins of CHD were initiated by an unexpected observation: a causative point mutation in chromosome 14 was identified in a large family with an autosomal dominant atrial septal defect (ASD), one of the most common forms of CHD [elopment –10. TradIn this paper, we discuss recent findings that indicate the importance of the functionality of the myofibrils, specifically their formation, contractility and mechanotransduction, in several aspects of cardiogenesis.Cardiac development is a complex process, by which an initial primordium consists of a straight tube of cardiac muscle (myocardium), lined by a single layer of endothelial cells (endocardium) , bends tIn vivo studies in chicken and mouse have shown that tropomodulin is an essential component of the myofibrils required for its proper formation [ormation , 19. Thiormation . Once trormation .Mice with targeted mutations of Tmod1 exhibit disruption of myofibrillogenesis, absence of cardiac contraction, looping defects, trabeculation defects, thinning of the ventricular wall, and impaired chamber formation , 21, 22.tnnt2) and sarcomeric actin (cfk) in zebrafish induce endocardial cushion and valve formation defects [Tnnt2) causes looping and endocardial cushion defects in mice [amhc) results in deficient ventricular development [X. tropicalis (myh6) [Myl7) gene [Analysis of spontaneous and experimental mutations in animal models has highlighted the role of myofibrillar proteins in cardiogenesis. In vertebrates, contraction of the cardiac primordium through interaction between the thick and thin filaments of the sarcomere occurs very early in development, well before the circulation of blood is needed to sustain the tissues of the embryo. It has been proposed that this initial activity of the cardiac myofibril apparatus functions as a stimulus for adequate heart development . Deficie defects . Targete in mice . In the elopment . Defectielopment  and malfs (myh6) . Abnormal7) gene .α-myosin heavy chain gene (MYH6) causes a dominant form of ASD [MYH6 was identified as the predominant sarcomeric disease gene for ASD [α-cardiac actin gene (ACTC1) have been identified as causative in other familial and sporadic cases of ASD [ACTC1 gene have been found in cases of septal defects associated with ventricular noncompaction [β-myosin heavy chain gene (MYH7) have been identified [MYH6, ACTC1, and MYH7 also cause hypertrophic and dilated cardiomyopathy [The aforementioned finding by linkage that a mutation in the m of ASD  has been gene MYH causes am of ASD  an otherm of ASD . Recentl for ASD . Additios of ASD . Differempaction  . A chromosomal translocation with a breakpoint near the ANKRD1 gene was discovered in another patient with the same abnormality [Titin, the largest myofibrillar protein, spans half of the sarcomere as it links its outer limit (the Z-band) with its centre (the M-band). This protein is composed of a series of springs of variable resistance that provide elasticity to the myofibrils and also contains segments that act as mechanosensors , 34. Theormality .Mechanical stimuli are first transmitted to the membrane of the cardiomyocytes (sarcolemma) through the extracellular matrix. Myofibrils are bound to the membrane of cardiomyocytes via specialized focal adhesion complexes concentrated in the costameres. These structures link the sarcolemma to the Z-bands of the sarcomeres that form the peripheral myofibrils . The mecIt has been shown that the focal adhesion complexes at the costameres can react differentially according to the rate and direction of the strain applied to the cell. Prolonged static strain over cardiomyocytes causes FAK to translocate from the focal adhesion complex to the nucleus . TransveFAK gene induces ventricular septal defects (VSDs) and deficient cardiomyocyte proliferation in mice, which suggests that the role of FAK as a mechanotransducer is also important for cardiogenesis and specifically for ventricular septation [FAK transcripts by antisense morpholino induced looping defects in X. laevis [ERK2 gene has been described in patients with VSD and PTA [PXN gene in mice results in abnormal heart structure [Deficiency of FAK in produces a general defect of mesoderm development in mice  and, speeptation . Knockdo. laevis . Upon ac. laevis  and are . laevis . Moreove and PTA . Ablatiotructure . These dε) is necessary and sufficient to induce FAK autophosphorylation [α-skeletal actin [ε [In contrast, animal models of mutations of genes encoding other proteins known to act as mechanosensors in the cardiac Z-band and the costameres do not display cardiac malformation. In response to variations in mechanical stress, muscle LIM protein (MLP) translocates to the nucleus , where irylation , and it rylation  and to trylation , where ial actin . Humans al actin , 59, TCAal actin , 61, or actin [ε  do not sMutations in many genes that encode proteins involved in the formation, contraction as well as force sensing and transduction of the myofibrils produce phenotypes that include impaired cardiac development in animal models and humans. It is being increasingly recognized that the early activity of the embryonic heart has a significant impact in the morphogenesis of the organ. The sensing and transduction of the forces it generates modify the gene expression of the early myocardium, which elicits remodelling, proliferation, and possibly apoptosis. Thus, we suggest that gene candidate approaches designed to discover new genetic determinants of Mendelian CHD or common variants predisposing to the nonfamilial CHD should consider genes involved in these signalling pathways.