Homo sapiens, namely its Genomics and Evolution, Transcriptomics, Proteomics, Interactomics, Oncogenomics, and Pharmacogenomics.Apoptosis is a critical biological phenomenon, executed under the guidance of the Apoptotic Machinery (AM), which allows the physiologic elimination of terminally differentiated, senescent or diseased cells. Because of its relevance to BioMedicine, we have sought to obtain a detailed characterization of AM Omics in omics databases of the web) as well as the High Throughput biomolecular analytical techniques.This project exploited the methodology commonly used in Computational Biology  and 110 MIR-encoding genes targeting them: some have a critical role within the system (core AM nodes), others perform tissue-, pathway-, or disease-specific functions . By overlapping the cancer type-specific AM mutation map in the fourteen most frequent cancers in western societies  to their transcriptome, proteome and interactome in the same tumour type, we have identified the most prominent AM molecular alterations within each class. The comparison of the fourteen mutated AM networks (both protein- as MIR-based) has allowed us to pinpoint the hubs with a general and critical role in tumour development and, conversely, in cell physiology: in particular, we found that some of these had already been used as targets for pharmacological anticancer therapy. For a better understanding of the relationship between AM molecular alterations and pharmacological induction of apoptosis in cancer, we examined the expression of AM genes in K562 and SH-SY5Y after anticancer treatment.In We believe that our data on the Apoptotic Machinery will lead to the identification of new cancer genes and to the discovery of new biomarkers, which could then be used to profile cancers for diagnostic purposes and to pinpoint new targets for pharmacological therapy. This approach could pave the way for future studies and applications in molecular and clinical Medicine with important perspectives both for Oncology as for Regenerative Medicine. However, because of the technological and conceptual limits that this type of study still presents, it seems appropriate to focus on more discrete entities such as the molecular machineries specifically responsible for a phenotypic phenomenon or a biomolecular function   and Viruses (both dsDNA viruses and retro-transcribing viruses) from the databases Homologene [Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio, Takifugu rubripes, Drosophila melanogaster, Caenorhabditis elegans, Dictyostelium discoideum, Bacillus subtilis, Methanococcus jannaschii). MA was performed with ClustalW [Histone H4 (a highly conserved protein), SOD2 (a protein with an average degree of conservation) and Fibrinopeptide (a protein characterized by a low conservation level) were used as evolutionary markers [To characterize the phylogenetic distribution of AM proteins, we collected AM orthologs from a large number of mologene , iProClamologene  and Metamologene . The seamologene . The totClustalW , while aClustalW  to obtaiClustalW . Histone markers . The evoln ratio values between +1 and -1 were considered not significantly changed [Gene expression data on AM genes were obtained from three datasets: Human Transcriptome Map (HTM), NCI60 Cancer Microarray Project, and Oncogenomics for cancer and normal tissues -49. Beca changed ,51. To f changed , which c changed .AM protein expression data were obtained from The Human Protein Atlas and NCI60 protein datasets ,55. Due Data on protein-protein and protein-DNA interactions were obtained from BIND  and HPRDBcr-Abl [2/95% O2 in a 37°C humified incubator. Imatinib  was provided by Novartis Pharma . A 10 mM stock solution was freshly prepared in sterile phosphate-buffered saline and diluted in RPMI 1640 medium before use. K562 cells, exposed to 1 μM imatinib and relative controls, were collected and analyzed at different time points . Human SH-SY5Y neuroblastoma cells, derived from a patient with a bone marrow metastasis [2 in air. For apoptosis induction, 3 × 106 cells were seeded into 75 cm2 flasks in 15 ml of culture medium. Fenretinide (dissolved in absolute ethanol) was added to cultures to a final concentration of 3 μM; an equal volume of ethanol (~0.3% of culture volume) was used to treat control cells. SH-SY5Y cells exposed to 3 μM fenretinide and relative controls were collected and analyzed at different time points .The K562 cell line was derived from a patient with CML in blast crisis and expressing the chimaeric non-receptor tyrosine kinase p210Bcr-Abl . Cell wetastasis , were gr5 cells) was used for flow cytometric analysis. Cell fluorescence was assessed with a Facscalibur flow cytometer  by using the CellQuest software (Becton Dickinson). Standard protocols were followed to determine annexin-positivity with an Annexin V-FITC Apoptosis Detection Kit .To detect apoptosis or necrosis, one aliquot . The array consisted of 96 primers for 84 genes of AM core protein-encoding genes, plus five housekeeping genes and three RNA and PCR quality controls. PCR cycles were performed according to the manufacturer instructions. The relative level of mRNA expression for each gene in each sample was first normalized to the expression of housekeeping genes  in treated samples and then normalized with respect to the level of cDNA expression in control samples according to the 2CT -ΔΔmethod [Total RNA was extracted with Trizol (Invitrogen) from treated cells and untreated controls after trypsinization (for the neuroblastoma cell line) and centrifugation (for both lines) at 1200 rpm for 10 min. Total RNA (3 μg) was reverse-transcribed using SuperScript II and random hexamers  . cDNA  with the exception of CASP5 and NALP8, not found in the mouse, and the CASP12 pseudogene that turns out to be an expressed gene in both the chimpanzee and the mouse  that are involved in apoptosis-like phenomena  of AM proteins has allowed us to establish the evolutionary trend of AM . However, there is a small group of proteins (< 10%) characterized by a medium to high degree of conservation with a v value ranging between that of histone H4 and SOD2. These conserved proteins are involved in cell cycle regulation , signal transduction , protein metabolism and post-translational modifications   of AM proteins ranges between 4,430 daltons isoform 6 of E2F6) and 527,622 daltons (BIRC6). About 20% of these proteins have a MW of approximately 20 kD  with a frequency higher than 20% in about 50% of cancers. This also occurs with AM genes at 1q23 and 1q32 (about 40% of cancers). On the contrary, AM genes at 8p21 and 8p22 are located in regions that are loss-prone in 35% of cancers Figure . The mosAccording to PubMeth, DAPK1, PYCARD, RB1, and TP73 are methylated in at least half of our fourteen cancer models Table . AccordiBy performing the normalization and discretization of the expression of AM protein-encoding genes in breast, colon, kidney, leukaemia, liver, lung, neuroblastoma, ovary, pancreas, prostate, skin, stomach, and thyroid, we identified those that are transcriptionally deregulated in these tumours Figure . ExpressBy searching the database VITA, we determined the expression profile of MIRs predicted to target AM proteins in colon, kidney, lung, pancreas, prostate cancers Figure . In partOverlapping the chromosomal mutation map to AM expression profiles in each cancer model showed that 28% of AM genes with transcriptional alterations were located in genomic regions potentially mutated in neoplasia. The highest rate of overlapping was found in prostate cancers  and neuroblastoma , whilst the lowest was in lung  and liver cancers  Figure .By performing the normalization and discretization of AM protein expression in cancers of the breast, colon, kidney, leukaemia, liver, lung, neuroblastoma, ovary, pancreas, prostate, skin, stomach, thyroid, we identified a set of proteins that are dysregulated in these tumours Figure , Panel AAnalysis of the relationship between the link number of a gene to genome mutations and transcriptome alterations allowed us to discover that the genes with more links to mutated genes are more likely to be dysregulated in tumours Figure . Our anaBy plotting the available data related to drugs targeted at the AM network, we found that most of AM proteins targeted by drugs were characterized by high connectivity; particularly, there was a highly significant association between the betweenness of these proteins and their being targets of drugs  Figure .CT -ΔΔmethod, the 4 h point showed no significant change of AM genes expression with respect to the control, while the same analysis at the 24 h point revealed remarkable modifications in the transcriptome: these consisted of an up regulation of the genes that trigger or positively control the apoptotic mitochondrial pathway , caspases , or their positive regulators. More specifically, the following pro-apoptotic AM genes reached a peak of expression at this time point: APAF1, BCLAF1, BIK, TNFSF10, and TNFRSF25 , at the point with the highest percentage of annexin positive cells (26%) (24 h), and at the conclusion of the process (72 h). Expression of pro-apoptotic genes GADD45A, TNFRSF1A, and TNFRSF10A peaked at 24 h. Similar to K562 cells, the real-time expression data in treated SH-SY5Y cells showed a massive activation at 72 h of the caspase family genes CASP1, CASP4, CASP5, CASP10, and CASP14, including CASP8, that is emi-methylated in the SH-SY5Y cell line and has been reported to be induced after fenretinide treatment [A time course FACS analysis with Annexin V-FITC demonstrated that following treatment of K562 cells with Imatinib 1 μM, annexin-positive cells appeared 12 h after treatment (17% of treated cells). The percentage of apoptotic cells reached its peak 24 h after treatment (22.3%), while the conclusion of the apoptotic process apparently occurred within 72 h Figure , Panel A5 Figure , Panel C5 Figure , Panel C5 Figure , Panel D5 Figure , Panel Breatment   as a tool to identify new cancer genes; (ii) to profile cancers for diagnostic purposes; and finally, (iii) to pinpoint new targets for pharmacological therapy. Our data suggest that the potential involvement of AM genes is heterogeneous in different cancer models, both for number and type of chromosomal breakpoints as for frequency of genomic mutations, suggesting heterogeneity of pathogenesis  was already assembled . Interestingly, a sizeable fraction of the genes expressed during the cell cycle do not have orthologs in all the organisms analyzed: this suggests that each species may also synthesize a different and specific set of cell cycle proteins [original sin hypothesis) [Few AM peripheral proteins are expressed in Prokaryotes and unicellular Eukaryotes, possibly with biological roles unrelated to metazoan apoptosis . At the d Figure . With thccretion ,89  ,98. AM gothesis) . Most AMothesis) . Furtherothesis) ,102. AM othesis) . Our datothesis) . Howeverothesis) . Our datThe analysis of the AM transcriptome profile showed that the transcriptional involvement of this apparatus is heterogeneous in different cancer models for number and type of AM genes involved. These data suggest that AM could be involved in different tumours through tumour-specific mechanisms of transcriptional activation or repression ,106. Accscenario (the three dimensions of space plus time) that would allow one to detect the relevance and the functional influence of all the elements of the machinery within the organism. The AM network shows a very interesting structure. Namely, approximately 50% of its core proteins (effectors and executor of death) have at least one death domain : this demonstrates a very efficient strategy of modular signal transduction . We are encouraged to believe that this approach, if also applied to other cell machineries as CCDRA and DRA, will eventually allow the identification of the molecular signals critically involved in not just the normal functioning of an organism, but also in its differentiation from the zygote. Time could now be ripe for the emergence of a new research field, that under the name of Signal Biology could focus on the search for the determinants of specific molecular interactions within organisms and their cells. Such a field would rely on the vast amount of biomolecular data already available and amenable to manipulation by the wide set of tools devised by the Computational Biology and Experimental BioMedicine communities. Data from these integrated studies should be critically important to deepen our knowledge of development and differentiation, as well as of biological complexity and its relationship to the phenotype. They should also help to intelligently devise strategies for therapeutic interventions and individualized Medicine, and eventually for fulfilling the promises of Synthetic Biology and Bionics ,133.(AM): Apoptotic Machinery; (CCDRA): Cell Cycle and DNA Replication Apparatus; (CML): Chronic Myelogenous Leukemia; (DRA): DNA Repair Apparatus; (GO): Gene Ontology; (HT): High Throughput; (MIRs): MicroRNAs; (NUPs): Natively Unfolded Proteins; (PTMs): Post-Translational Modifications.The authors declare that they have no competing interests.MP conceived and coordinated the project; MP, CD, MR, SL, SS, FD, BM designed experiments, the other Researchers performed them; all contributed to the critical revision of the data; MP, CD, MR, BM wrote the paper. All Authors contributed to its revision.The pre-publication history for this paper can be accessed here:Gene symbol, name and localization of AM protein-coding genes and AM MIR-coding genesClick here for fileGO classification of AM genes. Panel A. General Function of AM protein – encoding genes. Panel B. Biological Processes. Panel C. Molecular Functions. Panel D. Subcellular Localization. Panel E. General Function of AM genes targets of MIRs.Click here for fileExperimentally verified MIRs targeting AM genes.Click here for fileMIR host genes in AM.Click here for fileAM genomics. Panel A. Genome Map of AM Genes. Panel B. Comparison of the genome distribution of AM genes with respect to the other human genes.Click here for fileGenome clusters of AM genes in human, chimpanzee, mouse.Click here for fileProteomic features of AM. Panel A. Molecular weight distribution of 548 AM proteins. Panel B. Percentage of post-translational modifications of AM proteins. Panel C. Percentage of metal ions within the tertiary structure of AM proteins.Click here for fileDistribution of protein motifs and domains in AM.Click here for fileAM genes positional candidates in cancers (data from array-CGH).Click here for fileAM transcriptome data.Click here for fileAM genes localized in loss- or gain-regions and down or up regulated, respectively.Click here for fileFeatures of AM hubs.Click here for file


A key challenge in chemical biology is the design and synthesis of compound libraries spanning large tracts of biologically relevant chemical space.–3] Libr Libr3] LDiversity-oriented synthesis (DOS) involves the preparation of compound libraries with high substitutional, stereochemical, and/or scaffold diversity. Varying 9 could be attached to the linker (1 or 2) using either a Fukuyama–Mitsunobu reaction The  The 8] T reaction (→3) or al alkene of the “9, 12, 13, 14, 17, and 18 were metathesized, and a range of products with alternative scaffolds were obtained. The most promising results were fed into the design of our synthetic scheme.The building blocks were prepared on a multi-gram scale. Chiral building blocks were generally prepared in enantiomerically-enriched form, often using enzymatic desymmetrisation to induc9, 9′, (±)-10, 11, 12, 13, 15, and 16 were attached in good to excellent yield to the fluorous-tagged sulfonamide 1 using a Fukuyama–Mitsunobu reaction.,, In this The vast majority of the 96 products prepared have a distinct molecular scaffold. However, the scaffold diversity of the library is even more striking at higher levels of the hierarchy. This scaffold tree, like that of natural products, is dominOur synthetic approach allowed the systematic exploration of chemical space defined by scores of different molecular scaffolds. Each final compound was prepared from the building blocks in either four or five steps. Column chromatography was avoided at all intermediate stages, facilitating the application of the approach in parallel format. Remarkably, our approach used combinations of just six reaction types: the Mitsunobu reaction, silaketal formation, esterification, deacetylation, metathesis, and desilylation. The ethos of DOS was firmly retained since compounds with >80 scaffolds were prepared using a small number of optimised procedures.Excitingly, the compounds prepared had many of the broad structural features that are reminiscent of, and have evolved to constrain, natural products: isolated, fused, and spirocyclic ring systems, intramolecular hydrogen bonding motifs, unsaturation, and dense substitution. In this sense, many of the compounds prepared are natural product-like. As with polyketide biosynthesis, a whollyThe key to our approach was the extraordinary scope of ring-closing metathesis, which alExperimental procedures, characterisation data, and NMR spectra for all novel compounds are provided in the Supporting Information.


Hepatitis C virus isolates have been classified into six main genotypes and a variable number of subtypes within each genotype, mainly based on phylogenetic analysis. Analyses of the genetic relationship among genotypes and subtypes are more reliable when complete genome sequences (or at least the full coding region) are used; however, so far 31 of 80 confirmed or proposed subtypes have at least one complete genome available. Of these, 20 correspond to confirmed subtypes of epidemic interest.We present and analyse the first complete genome sequence of a HCV subtype 1g isolate. Phylogenetic and genetic distance analyses reveal that HCV-1g is the most divergent subtype among the HCV-1 confirmed subtypes. Potential genomic recombination events between genotypes or subtype 1 genomes were ruled out. We demonstrate phylogenetic congruence of previously deposited partial sequences of HCV-1g with respect to our sequence.In light of this, we propose changing the current status of its subtype-specific designation from provisional to confirmed. Flaviviridae family, is the leading etiologic agent of chronic liver disease. According to WHO, about 180 million people, an estimated 3% of the world population, are infected with HCV , ten sequences representing subtype 1a , twelve sequences for subtype 1b  and one sequence that corresponds to an unassigned subtype 1 [EMBL: AJ851228].Estimates of mean distances between subtypes of HCV genotype 1 and between these and the new subtype 1g sequence were obtained with the maximum likelihood distance (see above) with PAUP*4.0b10 . For thiThe authors declare that they have no competing interests.MAB, VS, and EM co-conceived, designed and coordinated the study, participated in the molecular studies, sequence alignment, phylogenetic and genetic analyses, interpreted data, and co-drafted the manuscript; AB and VA performed the clinical work, recruitment of the patient, procurement of specimens and participated in proofreading of the manuscript; FG-C coordinated the study, interpreted data, co-performed phylogenetic and genetic analyses and participated in proofreading of the manuscript. All authors read and approved the final manuscriptOligonucleotide primers used for amplification and sequencing. List of oligonucleotide primers including name, sequence, position and sense.Click here for file


Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome, is a hereditary condition characterized by a wide range of developmental abnormalities and a predisposition to neoplasms.The estimated prevalence varies from 1/57,000 to 1/256,000, with a male-to-female ratio of 1:1.Main clinical manifestations include multiple basal cell carcinomas (BCCs), odontogenic keratocysts of the jaws, hyperkeratosis of palms and soles, skeletal abnormalities, intracranial ectopic calcifications, and facial dysmorphism . Intellectual deficit is present in up to 5% of cases. BCCs  are most commonly located on the face, back and chest. The number of BBCs varies from a few to several thousand. Recurrent jaw cysts occur in 90% of patients. Skeletal abnormalities  are frequent. Ocular, genitourinary and cardiovascular disorders may occur. About 5–10% of NBCCS patients develop the brain malignancy medulloblastoma, which may be a potential cause of early death.PTCH1 gene and is transmitted as an autosomal dominant trait with complete penetrance and variable expressivity.NBCCS is caused by mutations in the Clinical diagnosis relies on specific criteria. Gene mutation analysis confirms the diagnosis. Genetic counseling is mandatory. Antenatal diagnosis is feasible by means of ultrasound scans and analysis of DNA extracted from fetal cells (obtained by amniocentesis or chorionic villus sampling). Main differential diagnoses include Bazex syndrome, trichoepithelioma papulosum multiplex and Torre's syndrome (Muir-Torre's syndrome).Management requires a multidisciplinary approach. Keratocysts are treated by surgical removal. Surgery for BBCs is indicated when the number of lesions is limited; other treatments include laser ablation, photodynamic therapy and topical chemotherapy. Radiotherapy should be avoided. Vitamin A analogs may play a preventive role against development of new BCCs.Life expectancy in NBCCS is not significantly altered but morbidity from complications can be substantial. Regular follow-up by a multi-specialist team  should be offered. Patients with NBCCS should strictly avoid an excessive sun exposure. Nevoid basal cell carcinoma syndrome (NBCCS)Gorlin syndromeGorlin-Goltz syndromeBasal cell nevus syndrome, BCNS (OMIM 109400)Fifth phacomatosisThe syndrome, delineated by Gorlin and Goltz in 1960 ,3 and ha• Gorlin syndrome• Gorlin-Goltz syndrome• Nevoid basal cell carcinoma syndrome (NBCCS)• Multiple nevoid basal-cell carcinoma syndrome (MNBCCS)• Multiple basal-cell carcinoma syndrome• Basal cell nevus syndrome (BCNS)• Multiple basalioma syndrome• Fifth phacomatosis• Jaw cysts-basal cell tumors-skeletal anomalies syndrome• Odontogenic keratocytosis-skeletal anomalies syndrome• Multiple nevoid basal-cell epithelioma-jaw cysts-bifid rib syndrome• Hereditary cutaneomandibular polyoncosis• Epitheliomatose multiple generaliseePTCH1 gene, is called the nevoid basal cell carcinoma syndrome (NBCCS), as Professor Gorlin suggested. However, patients and their relatives prefer to use the name "Gorlin syndrome", since it does not contain the word "carcinoma", even if about 50% of white patients will develop a significant number of skin cancers. Skin cancers do not generally occur in black patients [Currently, this disorder, resulting from mutations in the patients .et al. in the Journal of Medical Genetics in 1993 [et al. in 1997 [Diagnostic criteria, based on the most frequent and/or specific features of the syndrome, were defined by Evans  in 1993 , and mod in 1997 .et al. estimated that the minimum prevalence is 1 per 57,000 [This syndrome existed during Dynastic Egyptian times as shown by a constellation of findings compatible with the syndrome in mummies dating back to 1,000 b.c. . In 1992r 57,000 ,9. An alr 57,000 . A studyr 57,000  of the dr 57,000  and in tr 57,000 . Recentlr 57,000 , and in r 57,000 .Many different signs and symptoms may be associated with NBCCS Table . ClinicaBasal cell carcinomas may arise in various stages of the syndrome: most often they appear between puberty and 35 years of age [polypoid basal cell carcinomas (PBCC) in the perineum, leading to the recommendation that the perineum, perianal, and genital areas should be included in the routine examination of patients with NBCCS [BMP) gene [s of age , but cass of age ; in genes of age , as alres of age ,19. BCCss of age . BCCs ins of age ,21, sincs of age . Evidencs of age ,23. If lth NBCCS . The histh NBCCS . The tumth NBCCS . At the th NBCCS . Commonlth NBCCS . PerhapsMP) gene .palmar-plantar pits by the age of 10 years, this percentage rising to 80% by the age of 15 [Between 30% and 65% of patients with the syndrome have small asymmetric ge of 15 ,25. Pitsge of 15 , with thge of 15 .Multiple nevi are present in 30–50% of patients under 20 years of age, rising to 70% in patients over the age of 20 years. The nevi are flesh colored, reddish brown or pearly, and the groups resemble moles, skin tags, an ordinary nevus, cell nevi or hemangiomas. Some of them grow rapidly for a few days (to a few weeks), then mostly remain static.nodular or patch lesions, and benign dermal cysts. Small keratin-filled cysts (milia) can be found on the face in 30% of cases, most commonly in the area below the eyes, but they can also occur on the forehead.Other skin signs are Epidermoid cysts occur on the limbs and the trunk in over 50% of cases. They are usually 1–2 cm in diameter and are particularly common around the knee. Skin tags are especially common around the neck; histopathology reveals the typical features of a basal cell carcinoma, but the skin tags do not generally change in size or shape.et al. reported the occurrence of hairy skin patches  on the skin of three patients affected by Gorlin syndrome from two unrelated families with confirmed heterozygous mutations in the PTCH1 gene, suggesting that the patches may represent a sign associated with the syndrome [Recently, Wilson syndrome .keratocysts as they are characterized by a thin external fibrous capsule and an internal lining of keratinized stratified squamous epithelium. Keratocysts appear in the tooth-bearing areas of the jaws, and are believed to arise from the dental lamina [i.e., from retention of epithelial islands and/or satellite microcysts, which occur with great frequency in the connective tissue capsule, or from proliferation of the basal layer of the epithelium [Recurrent jaw cysts are the main oral sign, being present in 90% of patients . These cl lamina . Cysts ml lamina ,18. Odonl lamina ,12: 13% l lamina ,29. In Nl lamina . Most noithelium . There hithelium  or, rareithelium . Microscithelium . Buddingithelium .Other defects of the stomatologic system are malocclusion, impacted teeth, mandibular prognathism, dental ectopy or heterotopy, and dental agenesis. Cleft palate and lip are rare (5%) ,35. Recelamellar calcification of the falx cerebri (70–85%) [calcification of the tentorium cerebelli (20–40%) [the petroclinoid ligament (20%) and the diaphragma sellae (60–80%) [complete or partial bony bridging of the sella turcica (25%) [Ectopic calcifications of the central nervous system have been reported: (70–85%) , calcifi20–40%) , the pet(60–80%) , and comca (25%) . Spottedca (25%) , "empty"ca (25%) , communi% , the pca (25%) , meningica (25%) ,45, cystca (25%)  and meduca (25%) ,47-55 (sca (25%) ,57. Seizca (25%) . Mental th centile has been reported in 50% of cases [et al., the spina bifida occulta occurred in only 20% of patients [i.e., they are hamartomas [Skeletal manifestations are also present in NBCCS . The sigof cases ,59. As aof cases . Rib abnof cases . Howeverpatients . Bifid wpatients ; usuallypatients . Histolomartomas .NBCCS patients may have various ocular problems that occur with a frequency higher than that in the normal population (20%). These include hypertelorism (70%), exophthalmus, rotatory nystagmus, internal strabismus, congenital cataracts, orbital cysts, coloboma of the iris, choroid, and optic nerve, microphthalmia, and chalazions ,11,21,63Ovarian cysts and fibromas are present in 25–50% of affected women  and primSingle or multiple chylous or lymphatic mesenteric cysts have been documented ,66-68. Ti.e., arrhythmias) may also be present due to involvement of the intraventricular septum [Fibroma of the heart has been described ,46,69-74r septum . The tumPTCH1 [Medulloblastoma is frequent [i.e., from the nape to base of the spine) [Tumors in patients with NBCCS include basal cell carcinoma, medulloblastoma, fibroma and rhabdomyosarcoma (RMS) -78. RMSsPTCH1 . Medullofrequent ,47-55; Hfrequent . The tumfrequent . There ifrequent ,82, withe spine) .astrocystoma, craniopharyngioma, and oligodendroglioma, are infrequent, while meningioma is slightly more common. These tumors may well be secondary to radiation therapy.Other brain tumors, such as et al. reported fetal rhabdomyoma [In 1968, Schweisguth bdomyoma . This tubdomyoma ,78,85-89There are several reports that support an increased incidence of other neoplasms or hamartomas, such as leiomyomas of bowel and mesentery, leiomyosarcoma, adrenal cystic lymphangioma, thyroid adenoma, lymphangiomyoma, melanoma, ameloblastoma, craniopharyngioma, mesenchymoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, seminoma, paratesticular pseudotumor, schwannoma, pleiomorphic adenoma of parotid, adenoid cystic carcinoma of salivary gland, and adrenal tumors -92.PTCH1 gene and is transmitted in an autosomal dominant manner with high penetrance and variable expressivity [NBCCS is a hereditary condition caused by mutations in the essivity ,94. Theressivity .PTCH1 gene has recently been mapped to the long arm of chromosome 9 (q22.3-q31) with no apparent heterogeneity [PTCH1 gene, simultaneously in Australia and in the USA [The ogeneity ,96. Approgeneity . Data suogeneity ,99. Furtogeneity . This ge the USA ,102.PTCH1 gene in the fruit fly is important for body segmentation. If the gene is faulty, abnormal segmentation occurs. Several mutations of the PTCH1 gene have been identified in patients with NBCCS [The th NBCCS -118, andth NBCCS ,120.PTCH1 and the second loop is not formed, SHH cannot bind.The gene consists of 23 exons, which span 34 kb. It encodes a transmembrane glycoprotein composed of 1447 amino acids, with 12 transmembrane domains and two large hydrophilic extracellular loops where Sonic Hedgehog (SHH) ligand binding occurs. If a mutation occurs in PTCH1 gene product acts as membrane receptor [PTCH1, these genes are no longer repressed.Thus, the receptor  for the receptor . In the PTCH1 locus has been observed in almost 90% of hereditary BCCs. Various physical anomalies  apparently need only one hit. An interesting aspect of this syndrome is that some of the developmental defects (e.g. jaw cysts) appear to have two genetic hits but without developing into a true malignancy. In some patients, the lining of these odontogenic keratocysts has lost the normal gene for NBCCS, while retaining the mutant allele. Agaram et al. found that 70% of jaw cysts showed loss of heterozygosity of one or more of the several common tumor suppressor genes [The molecular biology of cancers in NBCCS is similar to that of retinoblastoma, the prototypical pediatric cancer syndrome, which is characterized by mutations in a recessive oncogene. Data from patients with retinoblastoma support the Knudson two-hit hypothesis; this theory of oncogenesis states that normal cells require two mutagenic hits (two distinct episodes of DNA damage) to produce a cancer. Patients with retinoblastoma, NBCCS, and other similar syndromes have a constitutional (germline) defect in the DNA sequence in one of the two copies of a tumor suppressor gene. A defect in one of the two copies is insufficient to cause a cancer. If a second DNA injury or if loss of the normal remaining allele occurs at the same locus (the second hit), the cell may become malignant. In NBCCS, various tumors and hamartomas  exhibit loss of heterozygosity ,124, butor genes . This fiPTCH1 mutations are rearrangements. Over 80% of these mutations seem to result in a truncation of the coded protein [PTCH1 mutations have been reported as a result of errors in the recombinational repair process [Analysis of gene mutations in the syndrome has identified deletions, insertions, splice site alterations, nonsense and missense mutations, with no hot spots identified for mutations. About 70% of germ-line  protein , which s protein ; recentl process .et al. analyzed 284 mutations and 48 single nucleotide polymorphisms (SNPs) located in the PTCH1 gene, compiled from a PTCH1 mutation database [PTCH1 mutations were mainly clustered into the two large extracellular loops of the corresponding protein and its large intracellular loop [PTCH1 domains [PTCH1 gene harbors mutational hot spot residues and regions, including a slippage-sensitive sequence in the N-terminus [In 2006 Lindström database . They folar loop . The SNPlar loop . NBCCS c domains . Moreove domains . Finallyterminus .PTCH1 gene in North American [i.e., 1686C>T, 2199A>G, 3944T>C, IVS10-51G>C, IVS10-8T>C) occur with a high frequency. A recent study has reported that certain haplotypes of PTCH1 polymorphisms may mediate the susceptibility to basal cell carcinomas [PTCH1 mutations [PTCH1 mutations [Several studies reported polymorphisms for the American , EuropeaAmerican ,129, ChiAmerican , and JapAmerican  populatircinomas . Pedigreutations . Pedigreutations .PTCH2, highly homologous to PTCH1, has been mapped to chromosome 1p32.1-p32.3 [PTCH2 comprises 22 coding exons and spans approximately 15 kb [i.e., a single occurrence of the disease in a family) of medulloblastoma and one simplex case of BCC [PTCH2 mutations were found in 11 simplex cases of NBCCS or in 11 individuals with familial cases of NBCCS who did not have identifiable PTCH1 mutations. In many cases, medulloblastoma arising from perturbations of PTCH1 function leads to a concomitant up-regulation of the highly similar gene PTCH2. Lee et al. investigated the role of PTCH2 in tumor suppression by generating PTCH2-deficient mice [PTCH1-/- mice, PTCH2-/- animals were born alive and showed no obvious defects and were not cancer prone [PTCH1+/-PTCH2-/- and PTCH1+/-PTCH2+/- animals showed a higher incidence of tumors and a broader spectrum of tumor types compared with PTCH1+/- animals [PTCH2 modulates tumorigenesis associated with PTCH1 haploinsufficiency [.1-p32.3 . PTCH2 cly 15 kb . It encoe of BCC . No PTCHent mice . In strier prone . However animals . Therefoficiency .Diagnosis of NBCCS can be made in the presence of two major criteria or one major and two minor criteria :1. Multiple (>2) BCCs or one under 20 years2. Odontogenic keratocysts of the jaws proven by histopathology3. Palmar or plantar pits (3 or more)4. Bilamellar calcification of the falx cerebri5. Bifid, fused or markedly splayed ribs6. First degree relatives with NBCCS1. Macrocephaly determined after adjustment for height2. Congenital malformation: cleft lip or palate, frontal bossing, "coarse face", moderate of severe hypertelorism3. Other skeletal abnormalities: sprengel deformity, marked pectus deformity, marked syndactyly of the digits4. Radiological abnormalities: bridging of the sella turcica, vertebral anomalies such as hemivertebrae, fusion or elongation of the vertebral bodies, modeling defects of the hands and feet, or flame-shaped lucencies of the hands or feet5. Ovarian fibroma6. Medulloblastoma.The presence of the most frequent and/or specific features of the syndrome make the diagnosis easier; however, diagnosis in a child with a 50% risk of having inherited the condition may not be so easy because of the extreme variation in expression, both within and between families. Some children may have only rib anomalies, whilst others have the "typical face" without other signs. Gene tracking and mutation analysis may be helpful for pre-symptomatic diagnosis.For apparently isolated cases, detailed examination and X-ray investigation of the relatives should be undertaken before concluding that a child's condition is the result of a new mutation. If an adult has no sign, no pertinent history and normal radiology, it is unlikely that he or she has Gorlin syndrome. Direct mutation analysis of the gene may be helpful. It is particularly helpful to follow a specific clinical protocol in the examination of these subjects Table .In case of a family history of the disease, at birth or immediately after, a detailed examination should look for the presence of a large head, frontal and temporal bossing, cleft palate , eye anoA panoramic radiograph of the jaws once a year from the age of 8 years onward is also suggested . The appet al. described the radiologic findings that are most helpful in diagnosing NBCCS [In children at risk, diagnosis can often be achieved by radiographic means . Depending on the situation, imaging studies may include magnetic resonance imaging (MRI) of the brain, echocardiography, abdominal ultrasonography, dental radiography, and skeletal survey. Kimonis ng NBCCS . As pating NBCCS ,136.No specific laboratory studies are indicated; however, several laboratories offer commercial testing for Gorlin syndrome and testing is certainly indicated in children with desmoplastic medulloblastoma, where radiation is contemplated. Patients with suspected Gorlin syndrome should undergo biopsy with samples obtained from several suspicious skin lesions. The histologic features of BCCs in Gorlin syndrome are usually identical to those observed in sporadic BCCs. Medulloblastomas in Gorlin syndrome usually have the desmoplastic phenotype, which has been linked, in some studies, with an improved prognosis . If an iPrecise differential diagnosis needs exclusion of some rare dermatological syndromes, such as Bazex syndrome , trichoepithelioma papulosum multiplex , or Torre's syndrome (Muir-Torre's syndrome).PTCH1 gene pair contains a fault; this means that every child of a person with the syndrome has a 1 in 2 (50:50) chance of inheriting the faulty gene. The risk to family members is various, about 70–80% of individuals diagnosed with NBCCS have an affected parent and about 20–30% of probands have a de novo mutation. The risk to a sib of a proband depends on the genetic status of the parents: if a parent of the proband is affected, the risk to the sibs is 50%; when the parents are clinically unaffected, the risk to the sibs of a proband appears to be low; if the disease-causing mutation cannot be detected in the DNA of the parent, the risk to sibs is low, but greater than that of the general population because of the possibility of somatic mosaicism or germline mosaicism exists. The offspring of an individual with mild NBCCS caused by somatic mosaicism may have a risk of less than 50% of inheriting the disease-causing mutation. The risk to other family members depends upon the genetic status of the proband's parents; if a parent is affected, his or her family members are at risk.The syndrome is a hereditary condition, thus referral to a geneticist for counseling is mandatory. NBCC syndrome is caused when one copy of the PTCH1 are not common [DNA tests, by looking directly at the mutation in the gene, may confirm that the patient is affected by this syndrome. In clinical situations suggesting genetic predisposition to BCC, germline mutations of t common . Genetict common .It is recommended to involve families in regular screening. It may be possible to prove whether someone in a family has inherited the condition or not by DNA tests, either by tracking the chromosome 9 containing the faulty gene, or by direct analysis of the mutation causing the disease in a particular family.PTCH1 gene has occurred for the first time in that family. It is a "new mutation" [In some families, only one person appears to be affected. This event is the result of two conditions: 1) the mother or the father have the syndrome, but the effects are so mild that it has never been diagnosed; 2) the fault in the utation" . If a faPTCH1 gene, although this is labor-intensive as there are 24 exons. Recently Fujii et al. used newly designed high-resolution oligonucleotide microarrays with a median distance between the probes of 776 bp  to detect gene deletions in NBCCS patients. This probe interval was chosen because small submicroscopic genomic deletions and duplications constitute up to 15% of all mutations underlying human monogenic diseases. Two out of three deletions could not be detected by conventional chromosomal analysis. A submicroscopic deletion as small as 165 kb was detected affecting only PTCH1, whereas the other two deletions were much larger (5 and 11 Mb) [The definitive diagnostic test is to demonstrate a mutation in the d 11 Mb) .Antenatal diagnosis may be useful to prevent complications. Ultrasound scans during pregnancy may be helpful in detecting serious developmental malformations, even if they are rare. Some fetuses with NBCCS have large heads and so may need assistance in delivery either by forceps or by Cesarian section. Very rarely, fibroma of the heart may be detected. Prenatal diagnosis for pregnancies at increased risk is possible by analysis of DNA extracted from fetal cells obtained by amniocentesis  or by chorionic villus sampling (CVS) at about 10–12 weeks of gestation. The disease-causing allele of an affected family member must be identified or linkage established in the family before prenatal testing can be performed. Requests for prenatal testing for conditions such as NBCCS that do not affect intellect, have variable expression and for which some treatments are available, are not common.Patients affected by Gorlin syndrome must be treated by several specialists. Keratocysts can be treated by surgical removal. This requires exposure of the lesion by making an osteotomy in the jawbone under local or general anesthesia, finding the wall of the cyst and removing this completely. Cysts that recur after such a treatment are removed with a bone box including a layer of bone at the margin of the cyst. This is intended to ablate any daughter cysts; only in extreme cases it is necessary to remove an entire section of the affected jawbone.As regards BCC, only a small fraction of these tumors become invasive and the very rare cases of death result from spread to the brain or lung. Results from several epidemiologic studies have indicated that the risks of BCCs show a strong positive correlation with exposure to UV radiation. Thus, these patients need to avoid excess sun exposure. They must use 100% UV protective sunglasses as the skin surrounding the eyes (similar to that of the nose/ears) is vulnerable to BCCs. Sunshine during the mid-day hours should be avoided if possible. High factor sunscreens (SPF30+) should be applied before going outside and re-applied every 2–3 hours, or more frequently, if perspiring or swimming.Radiotherapy should be avoided if possible ,21. TumoManagement of superficial BCCs without follicular involvement can be accomplished by total body application of topical 0.1% tretinoin cream . PatientRecent studies indicated that imiquimod 5% cream appears to be an effective treatment for nodular basal cell carcinoma alone -145, or Surgical excision is the typical approach for a patient with BCC if the number of lesions is limited. Other treatments that may be effective and more efficient than surgical excision in patients with multiple lesions include laser ablation, photodynamic therapy, and topical chemotherapy .Electrodessication and curettage is a very common procedure used as an alternative to topical chemotherapy in the treatment of small BCCs located in low recurrence areas of the body  . The areSurgical excision by scalpel allows removal of the cancerous tissue plus surrounding normal skin, with minimal discomfort during treatment and in the post-operative period. The cosmetic result is good, but it depends on the size and location of the tumor. The overall chance of a cure with surgical excision may range from 94–98%. Long term side effects include scarring, and rarely, nerve damage. The scalpel excision gives the advantage of being able to check the margins of the specimen microscopically by a pathologist.Another therapeutic possibility is photodynamic therapy (PDT), a cancer treatment involving use of a photosensitizing dye  that preferentially accumulates within malignant cells . BCCs arInterferon has also been proposed in experimental studies for the treatment of BCCs. This substance is injected directly into the cancer three times each week, for three weeks. Some reports have shown complete resolution of treated tumors, but other studies demonstrated that this treatment was less effective . Side efPatients with Gorlin syndrome can present with medulloblastoma, a malignant tumor of the posterior fossa . Intensii.e. radiation therapy for medulloblastoma that may cause multiple BCCs. For this reason, nonstandard treatments should be considered in children with Gorlin syndrome and medulloblastoma. Chemoprevention may also be used to avoid skin lesions. Vitamin A analogs, such as retinoids, including isotretinoin, may play an important role in preventing or slowing the development of new BCCs [The most common lesions accounting for NBCCS are usually not life-threatening. Medulloblastoma may be a potential cause of early death, even if NBCCS patients may have better outcome than patients with sporadic medulloblastoma . Therefore, this could be one argument to limit therapy to young children with Gorlin syndrome and desmoplastic medulloblastoma. Problems can derive from some techniques used for the therapy of specific lesions: new BCCs ,160-164.A list of groups that support patients, families, and clinicians caring for patients with NBCCS is supplied in Additional file NBCCS: Nevoid basal cell carcinoma syndrome; MNBCCS: Multiple nevoid basal-cell carcinoma syndrome; BCNS: Basal cell nevus syndrome; BCCs: Basal cell carcinomas; OKCs: Odontogenic keratocysts; PBCC: Polypoid basal cell carcinomas; RMS: Rhabdomyosarcoma; SHH: Sonic Hedgehog; SNPs: Single nucleotide polymorphisms; MRI: Magnetic resonance imaging; PDT: Photodynamic therapy.The author declares that they have no competing interests.A list of groups that support patients, families, and clinicians caring for patients with NBCCS.Click here for file


Vibrio cholerae, Pseudomonas aeruginosa and Burkholderia mallei. A total of 18 genes have been identified to be belonging to this secretion system in V. cholerae. Here we attempt to identify presence of T6SS in other bacterial genomes. This includes identification of orthologous sequences, conserved motifs, domains, families, 3D folds, genomic islands containing T6SS components, phylogenetic profiles and protein-protein association of these components. Our analysis indicates presence of T6SS in 42 bacteria and its absence in most of their non-pathogenic species, suggesting the role of T6SS in imparting pathogenicity to an organism. Analysis of genomic regions containing T6SS components, phylogenetic profiles and protein-protein association of T6SS components indicate few additional genes which could be involved in this secretion system. Based on our studies, functional annotations were assigned to most of the components. Except one of the genes, we could group all the other genes of T6SS into those belonging to the puncturing device, and those located in the outer membrane, transmembrane and inner membrane. Based on our analysis, we have proposed a model of T6SS and have compared the same with the other bacterial secretion systems.A new secretion system, called the Type VI Secretion system (T6SS), was recently reported in  Pathogenicity of bacteria is often critically dependent upon machineries which mediate the transport of toxic molecules out of their cells into the environment or into the host cells Vibrio cholerae, Pseudomonas aeruginosa and Burkholderia malleiV. cholerae, 4 are thought to be a part of the structural components, 2 are effector molecules and 1 is a chaperone P. aeruginosa, a triggering mechanism of the T6SS has been proposed where a serine-threonine phosphorylation event has been considered to be essential in bringing about the activation of the T6SS system B. mallei, it has been observed that there are three T6SS clusters and the regulation of these is carried out by virAG, a two component regulatory system Since many of the secreted proteins are virulence factors, a complete understanding of the mechanism underlying bacterial protein secretion is crucial for understanding the host-pathogen association. The mechanistic details of T6SS are less well understood due to its recent discovery. T6SS has been reported in V. cholerae, P. aeruginosa and B. mallei and show a conserved pattern in terms of sequence similarity, the mechanistic details of the machinery are not very clear. In order to understand the functioning of T6SS as well as to predict this system in other bacterial species, we have carried out an analysis using informatics approaches and results are presented in this paper.Although the genes which play a central role in carrying out secretion by T6SS pathway are known in V. cholerae, P. aeruginosa and B. mallei. The bit scores and E-values of all the orthologs obtained are listed in the V. cholerae, genes comprising of the Virulence Associated (VAS) cluster, namely, vasH, vasK, vasF and vasA have been suggested to be of structural importance and critical for the T6SS machinery V. cholerae, P. aeruginosa and B. mallei showed that in case of Shigella, Shigella sonnei showed the presence of T6SS orthologs. However, orthologs in Shigella dysentriae and Shigella flexneri were detected only when a blast of T6SS orthologs of S. sonnei was done against all other Shigella species.The analysis of Blast search using the sequences of Escherichia coli, the strain K12 did not show orthologs of any of the T6SS proteins, whereas all the other virulent strains, namely, O157, B171, 536, UTI89, APEC and CFT073, showed orthologs of all the T6SS components, with identities ranging from 90 to 99%. Similarly, in Vibrio, Vibrio fischeri and Vibrio harvey, which are avirluent, did not show any orthologs of the T6SS components whereas the virulent V. cholerae, Vibrio parahemolyticus and Vibrio fulvinicus showed orthologs of all the 18 components of T6SS. In Burkholderia too, the avirluent Burkholderia thailandensis did not show any T6SS orthologs whereas the virulent species had orthologs of 14 of the T6SS components. In Shigella it was seen that the virulent species S. sonnei, S. flexneri and S. dysentriae contained 18, 13 and 13 T6SS orthologs respectively, whereas the avirulent species Shigella boydii did not show any orthologs. The plant pathogen Xanthomonas also followed similar trend, where avirulent Xanthomonas populi and Xanthomonas codiaii lacked any orthologs. These results suggested strongly that T6SS could play a crucial role in imparting pathogenicity to an organism.Analysis of completely sequenced genomes of various species and strains of each of the organisms appeared to suggest that the avirulent species lacked T6SS orthologs in most of the organisms . For exaS. flexneri and S. dysentriae, each of which had 13 out of 18 orthologs. Similarly, E. coli CFT073 with 15 orthologs and all the species of Burkholderia and Xanthomonas, with 14 components each, also lacked these 4 genes.Species specific study  also indMesorhizobium loti, belonging to the delta and the alpha Proteobacteria respectively, possessed T6SS. Thus, representatives of all the proteobacteria sub-groups  showed T6SS components; gamma proteobacteriaceae being the most widely represented one.All the bacterial species identified to have T6SS components belonged to the proteobacteria group of the gram negative pathogens . Among tOn analyzing the phylogenetic profiles of T6SS proteins , three sCluster 1: VasA, VasF, VasH and VgrG–All these proteins were present across all the organisms.Cluster 2: VCA0107, VCA0108 and VCA0111-These proteins were present across all organisms except in Shewanella.Cluster 3: VCA0113, VasK, VCA0114–These were absent only in Geobacter.Cluster 1 differed from clusters 2 and 3 by one bit whereas clusters 2 and 3 differed from each other by two bits. Some of the proteins, namely ClpB, Hcp, VCA0109, and VCA0112 did not cluster with any other proteins. However they were seen to be located in the neighborhood space  and the UCP (UnCharacterized Protein) domains. However some of the others had domains like the transmembrane domain, porin outer membrane domain, ImpA related domain, etc. For instance, ClpB exhibited an ATP domain and a chaperonin domain, indicating its role as an ATPase as well as a chaperone for the T6SS proteins. Similarly, VasH belonged to sigma-54 domain containing protein which is implicated in the transcriptional regulation of the T6SS gene cluster. VgrG had three major domains, namely, a peptidoglycan binding domain, a gp-5 domain and an Actin Cross linking Domain (ACD). Another protein called Hcp, contained a domain corresponding to the hemolysin-coregulated protein. Likewise, while VasF had a transmembrane domain, VasK was seen to be a IcmF like protein having a transmembrane domain. The hypothetical proteins, VCA0109 and VCA0112 contained a gp-25 domain acting as a lysozyme and a SMAD_FHA domain containing protein respectively. Similarly hypothetical proteins VCA0119 and VCA0121 had ImpA domains. All the information regarding the domains for each of the other T6SS components is given in Family and superfamily searches also revealed information about some of the proteins which included ClpB, VasH, VgrG, VasK, VCA0109, VCA0112 and VCA0121. ClpB belonged to a ATPase family associated with various cellular activities (AAA). Hcp belonged to a hemolysin coregulated-protein family of virulence factors for secretion apparatus. VasH belonged to the P-loop containing superfamily having a ATP/GTP binding motif and was also indicated to be a part of a homeodomain-like superfamily, which are implied in activation of transcriptional regulation. Similarly, VgrG, was seen to be a part of phage fibre protein superfamily, containing a gp-5 lysozyme at its C-terminal end and a peptidoglycan binding domain. VasK was also seen to be a part of the P-loop containing superfamily and a G-proteins family, suggesting its role in signal transduction. The hypothetical proteins VCA0109 and VCA0112 belonged to a Gene25-like lysozyme family and a SMAD/FHA domain containing superfamily respectively. The SMAD/FHA domain containing superfamily is known to carry a nuclear signaling domain which interacts with serine-threonine kinase receptors. Similarly the hypothetical protein, VCA0121 belonged to an ImpA related N terminal family which is known to have inner membrane proteins participating in intracellular protein transport.The search for motifs could not detect any known motifs in most of the T6SS proteins. Only ClpB and VasH had the motif specific for ATPase and Sigma54 respectively, suggesting their roles as a ATPase and a transcriptional regulator respectively.et al.All the orthologs of the effector molecules of the T6SS lacked the cannonical N-terminal signal peptide, as predicted by SignalP Proteins which could be annotated based on their 3D folds included ClpB, Hcp, VgrG, VasF, VCA0109, VCA0112, VCA0119 and VCA0121. The three dimensional fold of ClpB matched with the folds of ClpA ATP-dependent Clp protease and to chaperone Hsp104 and related ATP-dependent Clp proteases. VasH showed remote homology to the fold of various transcriptional regulatory proteins, including that of two component system. VgrG protein showed homology to a vgr_GE Rhs element Vgr protein and also to the N-terminal domain of the tail-associated lysozyme, gp5. Similarly, the folds of hypothetical proteins, VCA0121 and VCA0119 matched with the VHS domain known to have a role in general membrane targeting and recognition as well as in protein trafficking. In addition, VCA0119 had a TPR like repeat domain which is known to be involved in protein-protein interactions and is known to mediate assembly of multiprotein complexes. While, the hypothetical protein VCA0112 showed similarity to a phosphoprotein/serine-threonine kinase binding domain, VCA0109 had a remote homology to the phage base plate assembly protein. The three dimensional fold of VCA0108 and VCA0107 showed some similarity to phage tail sheath protein and a protein ImpB implicated in protein secretion respectively. VCA0111 showed remote homology to a phage tail protein domain. Coiled coil structures were predicted for VasK and the hypothetical protein VCA0118. These two proteins were also predicted to be transmembrane proteins from our domain and family searches.Analysis of the associating partners of T6SS components revealed that a search against any one of the T6SS genes (in each organism) picked up all the 18 genes of the putative T6SS cluster of that organism, as its functional interacting partners. The prediction suggested that these proteins participate in a common pathway and function together. There were two additional proteins, VCA0893 and OmpA, which were not a part of the T6SS cluster but were seen to be associated with the T6SS genes. While VCA0893 is a chemotaxis protein, OmpA is a structural protein of the outer membrane which connects the outer membrane to the periplasmic peptidoglycan via its periplasmic domain.E.coli BEN2908 AGI-1 AY395687 and Photorhabdus luminiscens-W-14 Mt Locus AY144117.Secretion systems have often been found to be located in the Pathogenicity Islands of the organisms . Apart from the identified T6SS genes, a number of genes were found to be located in these regions  as identified by the centroid method  regions . These iV. fulvinicus and the various species of Pseudomonas, Salmonella and Burkholderia. Based on the Pfam family information, this protein is known to interact with and manipulate eukaryotic cells, although the exact mechanism is not known.ImpE family proteins are known to be involved in a temperature dependent secretion of various organisms. This gene was present within the genomic island regions of 17 organisms which included E. coli, Yersinia, Pseudomonas, Burkholderia, Xanthomonas, Ralstonia, Hahella and Mesorhizobium. OmpA is known to be responsible for the translocation of effector molecules OmpA, an outer membrane protein, was seen in the genomic islands of all species of P. aeruginosa, which requires both a serine-threonine kinase and a protein phosphatase along with an FHA containing protein A Serine threonine kinase protein and a phosphoprotein phosphatase were seen to be present in the compositionally distinct regions in a few organisms which included species of Pseudomonas, Vibrio, Yersinia, Burkholderia, Xanthomonas, Hahella, Geobacter and Mesorhizobium. Literature survey shows that a serine threonine phosphorylation event is required for the initial activation of the T6SS in All the organisms having orthologs of at least 10 of the known T6SS components were considered to have T6SS machinery. Our analysis indicated presence of T6SS in 42 bacterial species, all belonging to the proteobacteriaceae family. However, some of the pathogenic organisms belonging to the proteobacteriaceae family were not predicted to have T6SS. Examples of these included pathogens like Legionella, Klebsiella and Enterobacteria, which had orthologs of only 4, 8 and 9 T6SS components respectively. This implies that the pathogenicity of these organisms is dependent on some other factors. For example, the type III secretion system in Klebsiella, type IV secretion system in Legionella and types II and III secretion systems in Enterobacter, might be responsible for the pathogenicity of these organisms. The discordant distribution of T6SS genes across proteobacteriaceae family clearly indicates that there must have been gene(s) loss in certain organisms.Based on the function, T6SS components were characterized as structural, regulatory, effector and chaperon proteins. Except for VasH, which is known to be a Sigma 54 activating protein and a transcriptional regulator for the T6SS gene cluster VgrG is known to be a Rhs element containing protein and is a cytotoxic effector molecule VCA0109, which was annotated as hypothetical protein, belonged to the lysozyme family protein that contained a gp25 domain which is known to hydrolyse NAG and NAM of peptidoglycan residues of the cell wall . A similar domain gp27 is known to participate in the assembly of the needle apparatus in T4 tail spike protein ClpB is known to have ATPase domains and clp chaperonin domain, and is known to hydrolyze ATP to provide energy VCA0113, a hypothetical protein, had a lipoprotein domain and was involved with the exported proteins. It is involved in the intracellular trafficking, secretion and transport. This was identified to be the only outer membrane T6SS protein.VasF and VasK, possessing transmembrane domains and having coiled-coil structures, are known to be similar to IcmH and IcmF proteins. It is known that the proteins IcmF and IcmH help in translocating a pore in the host cell and help in intracellular cytotoxicity 3D fold search of the hypothetical protein, VCA0118 indicated it to be a coiled-coiled structure. In addition, this protein had a signal peptide domain as well as a transmembrane domain and thus could be a transmembrane protein, although the exact functioning could not be predicted.VasA was predicted to be an inner membrane protein and thought to participate in intracellular activities.Similarly, VCA0111, annotated as hypothetical protein, belonged to transposase family and had a cytoplasmic domain and thus predicted to be an inner membrane protein and thought to participate in inner membrane activities.Domain information of the hypothetical protein VCA0112 showed presence of FHA domain. Proteins having FHA domains are known to interact with serine-threonine kinase, which in turn activates T6SS in Pseudomonas Hypothetical proteins, VCA0119 and VCA0121 belonged to the ImpA family. Since ImpA related proteins are known to be inner membrane proteins which are associated with the exported proteins, VCA0119 and VCA0121 could be though as inner membrane protein associated with effector proteins of T6SS.From the literature as well as from the present study, it can be inferred that the T6SS machinery is similar to all the 3 types of sec-independent pathways, namely T1SS, T3SS and T4SS. For instance, all the four secretion systems  transport proteins that lack an N-terminal signal sequence and use a sec independent transport for translocation of proteins. Also, all these systems extend from the inner membrane, through the periplasmic space and upto the outer membrane. Similarly, while YscN functions as the ATPase in T3SS, ClpB performs this job in T6SS, since both share similar domains and belong to a P-loop containing nucleoside triphosphate hydrolases superfamily. In the same way, while ClpB is the chaperone of the T6SS, InvB (CesD) acts as the T3SS chaperone. In terms of mechanism, T3SS is known to function by entering the host cell by puncturing through its needle formed by YscF. In a similar fashion, T6SS functions by puncturing the host cell with the help of its protein VgrG which is similar to T4 phage tail spike protein Combining all the information from the different studies performed, following model of T6SS has been predicted and is schematically shown in The initial activation of the system depends on environmental factors, which are not known. After appropriate signals are sensed by the system, VCA0112, having a FHA domain brings about the initial activation event on interaction with a serine threonine kinase. Once activated, the assembly of the T6SS machinery occurs, where the translocation channel is formed by Hcp, VCA0109 and VgrG. While Hcp forms a hexameric translocation channel in the transmembrane region, VgrG forms a trimeric needle like structure on the bacterial cell surface that reaches the host cell and acts as a puncturing device. VCA0109 forms a channel from the bacterial outer membrane to the host membrane and interacts with the gp5 domain of each monomer of VgrG. Assembly of all the inner membrane proteins, namely, VCA0107, VCA0108, VCA0111, VCA0114, VCA0119, VCA0121 and VCA0122, also occur through an unknown mechanism. After the assembly of all the components, ClpB, which acts as as a chaperone and ATPase, forms a hexameric structure which interacts with Hcp to hydrolyse ATP in order to translocate of effector molecules. Accessory proteins VasK and VasF (icmF family proteins) bring about cell surface reorganization, resulting in increased adherence to host cells. VasK also helps in recognition of the transport signal, thereby increasing the efficiency of translocation. The effector proteins , with the help of the outer membrane associated lipoprotein VCA0113, pass through the translocation pore formed by the protein OmpA. All The inner membrane proteins mentioned above assist in stabilizing the complex and in bringing about other intracellular activities essential for the functioning of the T6SS. The transcriptional control of the T6SS machinery is probably brought about by VasH, a sigma-54 family protein. The system can be thought to be involved in a temperature dependent secretion, as it has proteins belonging to the imp family, which is known to be associated in a temperature dependent secretion.Comparison of V. cholerae, P. aeruginoasa and B. malleihttp://www.ncbi.nlm.nih.gov/). These comprise of the 18, 16 and 14 genes in the T6SS cluster of V. cholerae, P. aeruginosa and B. mallei, respectively. The protein sequences corresponding to all the genes which have been identified experimentally to be components of type VI secretion system in V. cholerae, P. aeruginoasa and B. mallei using the online NCBI BLAST search (http://www.ncbi.nlm.nih.gov/BLAST). Only those hits having homology with its corresponding T6SS gene in at least one of the above three organisms were considered and the orthologs were identified using reverse-BLAST. Genes belonging to the previously known secretion systems (Type I through type V) have been seen to be conserved across all bacterial species possessing them In order to identify genes which were widely represented across all organisms, a phylogenetic profile, which is matrix of the presence/absence of genes across all organisms, was created . While aet al, 1999 Since the proteins which participate in the same pathway tend to show a similar pattern of inheritance and co-evolution and hence a similar phylogenetic profile http://wdcm.nig.ac.jp/RDP/html/index.html) and a 16S rRNA phylogenetic tree was constructed , CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and ProDOM (http://prodom.prabi.fr/prodom/current/html/form.php) databases were used for searching domains, Pfam database (http://pfam.sanger.ac.uk/) was used to search for protein families. We also looked for Superfamily (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/) information for each protein.Since the GO annotation is not available for the Type VI secretion system, we searched for the protein family and domain annotations of the T6SS genes for their functional classification. While InterPro (http://gibk26.bse.kyutech.ac.jp/jouhou/HOMOLOGY/dbsearch/pdb/pdb_seq.html). Known patterns and motifs in these components were searched against the Prosite (http://www.expasy.ch/tools/scanprosite/), Block (http://blocks.fhcrc.org/) and Prints (http://www.bioinf.manchester.ac.uk/dbbrowser/PRINTS/) databases.Presence of known motifs in the T6SS components was searched using the Motif Finder  in all organisms were checked using the software SignalP http://toolkit.tuebingen.mpg.de/hhpred) based on HMM-HMM comparison was used.Proteins lacking significant sequence similarity sometimes have similar three dimensional structures, thereby suggesting similar functions. In order to identify the three dimensional folds of the T6SS components, a tool Hhpred (http://string.embl.de) which uses co-occurrence, gene neighborhood, gene fusion and text mining for predicting functionally related proteins, was used To look for association partners among the components of the type VI secretion system, the web based tool called STRING Click here for additional data file.Table S2Binary matrix showing the phylogenetic profile of each T6SS orthologs across all the organisms. The gene names are the corresponding names in V. cholerae T6SS components.(0.15 MB DOC)Click here for additional data file.Table S3T6SS orthologs in various strains for which its complete genome sequence is available.(0.15 MB DOC)Click here for additional data file.Table S4Conserved domains and families of the T6SS components.(0.12 MB DOC)Click here for additional data file.


Alcohol dependence is a worldwide public health problem, and involves both environmental and genetic vulnerability factors. The heritability of alcohol dependence is rather high, ranging between 50% and 60%, although alcohol dependence is a polygenic, complex disorder.Genome-wide scans on large cohorts of multiplex families, including the collaborative study on genetics of alcoholism (COGA), emphasized the role of many chromosome regions and some candidate genes. The genes encoding the alcohol-metabolizing enzymes, or those involved in brain reward pathways, have been involved. Since dopamine is the main neurotransmitter in the reward circuit, genes involved in the dopaminergic pathway represent candidates of interest. Furthermore, gamma-amino-butyric acid (GABA) neurotransmitter mediates the acute actions of alcohol and is involved in withdrawal symptomatology. Numerous studies showed an association between variants within GABA receptors genes and the risk of alcohol dependence.In accordance with the complexity of the “alcohol dependence” phenotype, another field of research, related to the concept of endophenotypes, received more recent attention. The role of vulnerability genes in alcohol dependence is therefore re-assessed focusing on different phenotypes and endophenotypes. The latter include brain oscillations, EEG alpha and beta variants and alpha power, and amplitude of P300 amplitude elicited from a visual oddball task.Recent enhancement on global characterizations of the genome by high-throughput approach for genotyping of polymorphisms and studies of transcriptomics and proteomics in alcohol dependence is also reviewed. Alcohol dependence is a complex addictive disorder, affecting 5.4% of the general population during lifetime . AlcoholThis paper focuses on current studies on the genetics of alcohol dependence. After an overview of genetic approaches, including genome-wide scans and candidate genes analyses, we will review post gene methods, such as expression profile studies of genes differentially expressed in phenotypes relevant for alcohol dependence.We will particularly discuss the results from two emerging fields, namely genomic and proteomic approaches of alcohol dependence. et al. [et al. [et al. [The identification of genes involved in alcohol initiation, use, abuse and dependence has been investigated by two complementary genetic methods, namely (i) genome wide scan analyses, with no a priori assumptions about which genes might be involved, and (ii) the genotyping of candidate genes of interest. About fourteen-hundreds papers came out from the PubMed/NCBI database with the key words “alcohol, dependence, gene”. This review is not an exhaustive view of linkage studies on alcohol dependence, neither a cover of all genes possibly affecting the risk for alcohol dependence. We have rather decided to focus on particularly relevant linkage and candidate genes studies. For a more complete review refers to Goldman et al.  and Ramo [et al. . We also [et al. .Recently, linkage studies have identified several chromosomal regions, supporting the involvement of loci on chromosomes 1, 2, 4, 5, 6, 7, 12, 14, 16 and 17 in alcohol dependence , 4, 6. MhTAS2R16 gene, located on the 7q chromosomal region, shows significant association with alcohol dependence and with a related phenotype, the maximum number of drinks ever consumed in 24 hours in the COGA sample, especially in African Americans sub-sample [hTAS2R16 gene codes for a bitter receptor and this SNP is associated with a diminution of the sensitivity to bitter-taste stimuli. These findings are consistent with previous investigations of the relationship between taste perception and alcohol dependence. Indeed, in animal models, the knock-out of several genes involved in taste transduction is consistently associated with a reduction in alcohol intake [CHRM2 gene. It codes for a Cholinergic muscarinic 2 receptor and shows significant association not only with alcohol dependence and the maximum number of drinks ever consumed [A non-synonymous coding single nucleotide polymorphism in the b-sample . The hTAl intake . Anotherconsumed , cogniticonsumed , 19 or econsumed .2B receptor located on 2q, which was previously associated with drug abuse [Furthermore, in a subset of 1364 individuals N=123 families), significant linkage was recently found for quantitative traits related to alcohol dependence, on two novel chromosome regions 2q and 10p , with a  familiesug abuse .Studies upon genetically isolated population, such as Native American populations, are very promising due to the high homogeneity of the genetic background, and, in this case, a high prevalence of alcohol dependence . LinkageADH7-ADH1C-ADH1B-ADH1A-ADH6-ADH4-ADH5). However, only two exhibit functional polymorphisms, ADH1B*2 and ADH1C*2 alleles, and confer an increase in alcohol degradation rate [ADH1A, ADH1B and ADH1C) and the class II enzyme (encoded by ADH4) are significant contributors to ethanol metabolism, at least in vitro [ADH1B*2 is relatively common among Asian population [ADH1C*2 has protective effects on liver cirrhosis and alcohol chronic pancreatitis [ADH1B and ADH1C are in linkage disequilibrium[ADH1B and ADH1C are separated by a distance of only 15Kb in the cluster of ADH genes. Contrasting with their major effect in vitro, ADH1C and ADH1B genes have minor effects in vivo, suggesting a possible role of other genes of the ADH cluster. Indeed, the effect of the quantitative trait loci (QTL) linked to the ADH region gene account for 64% of the genetic variance in alcohol metabolism [ADH4 contribute to 30% of the total ethanol oxidizing capacity of the liver [The chromosome 4q region surrounding the alcohol dehydrogenase (ADH) gene was linked in a genome-wide scan with the maximum numbers of drinks ever consumed in 24 hours, a phenotype associated with alcohol dependence . The rolion rate , 30. Thepulation , as wellpulation . It is apulation . ADH1C*2reatitis , an is auilibrium. Indeed,tabolism . For exahe liver , as wellhe liver , althoughe liver , and is he liver , drug dehe liver  as well he liver .ALDH) genes encodes a protein that acts as a tetramer to oxidize acetaldehyde [ALDH2*2 variant, coding for an inactive enzyme. The deficient variant allele acts dominantly, thus ALDH2*1/ALDH2*2 heterozygous carriers have reduced enzyme activity, but the flushing reaction is more severe and immediate in ALDH2*2 homozygous carriers [ALDH2*2 is associated with a reduced rate of alcohol dependence in Asian population [Acetaldehyde dehydrogenase  and represents a promising candidate gene in the dopaminergic system. This transporter is mainly located at the synapse of the presynaptic neuron and is responsible for dopamine reuptake. Human genetic studies report associations between variants of the DAT1 gene and alcohol dependence with both positive [DAT1 is associated with more severe withdrawal symptomatology [The positive , 59 and positive , 61 resumatology , and commatology . More spmatology .A receptor, particularly receptors containing the α4 or α6 subunits in combination with β2 or 3 subunits and the δ subunit [GABA is the major inhibitory neurotransmitter in the brain. The GABA receptor is a pentameric protein containing two α, two β, and one γ or δ subunit among 19 isoforms. This arrangement allows considerable diversity of the GABA receptor, but their observed number is in fact limited. Alcohol is an agonist of the GABA subunit . The GAB subunit .GABRA6 variants were associated with alcohol dependence in different populations, including a Scottish [GABRA2 were associated with alcohol dependence [Scottish , a GermaScottish , a Finn Scottish . Variatipendence  as well pendence  in the Cpendence .Since almost a decade now, the traditional tools used in human genetic studies to identify alcohol dependence genes, genome-wide scans and candidate gene associations, have been helped by the high-throughput characterization of the expression patterns of the genes (transcriptome studies) and the proteins (proteome studies) from tissue samples. This tissue can be extracted from specific brain regions, of patients compared to controls, as well as from animal used as models of alcohol dependence, from the yeast to the murine models. Indeed, although there are several studies describing global gene expression changes associated with alcohol dependence, changes in the expression levels of proteins has not been characterized until recently. Characterization of the liver, brain and serum proteome is the next step toward the investigation of alcohol-dependent patients to discover new biological markers as well as prognostic tools.S-transferase activity), S100 class calcium-binding, and cytokine/proinflammatory activity (IL-1). Using microarray analyses of cerebellar tissue from mutant and wild-type mice exposed to alcohol, 109 genes were associated with either the development or the resistance to tolerance, or with the effects of chronic alcohol administration [Twik-1, coding for a potassium channel, was associated with tolerance, and JunB and Nur77, members of an immediate early gene family and of the nuclear receptor family respectively, had an increased expression that persisted a long time after alcohol ingestion [in vitro cell lines and in vivo animal models, especially in mice and rats [Whole-brain gene expression databases of five genetic mouse models with different alcohol consumption were examined . A meta-stration . Specifingestion . These tand rats -77. It iand rats , 76, 77.hNP22/TAGLN3, over-expressed in the superior cortex of patients [Due to the difficulty to have access to human post-mortem brain samples, the number of differential display and transcriptome studies on alcohol dependence, as well as the sample size of their studies, are modest. Nevertheless, several works have been published in the last years on independent samples of different brain regions, including temporal cortex, frontal cortex and nucleus accumbens of alcohol dependent patients and controls -82. One patients . Signifipatients . Genes epatients . Nucleuspatients . Anotherpatients . This stThe different pathways identified by the modifications of gene expression should help in the understanding of the molecular mechanisms of alcohol dependence, in particular by allowing the characterization of protein levels to reveal new biological markers, and therefore increase the chance to further understand the pathophysiology of this psychiatric disorder.Different methods in proteomics, such as liquid chromatography, mass spectrometry (MALDI-TOF and SELDI-TOF), and two-dimensional gel electrophoresis, have been recently carried out to identify and quantify proteins in the field of addiction. They have been used on cell lines, animal models, as well as on post-mortem tissues, in particular brain, of patients to characterize changes in protein expression in response to alcohol consumption, as presented in recent reviews , 83, 84.vice versa, which reinforce their probable implication in alcohol dependence. Finally, the last presented study point out the fact that the clinical features and phenotype of the patients need to be the most well documented to perform studies on comparable patients and homogenous groups. This lead to the novel strategy followed in genetic studies looking for susceptibility to psychiatric disorder which consists to identify specific traits that help to increase the homogeneity of the group of patients, when the disorder has a broad spectrum phenotype, such as alcohol dependence.Thus, a key area for proteomics consists in biomarker discovery that is the identification of protein expression which correlates with disease, treatment outcome or global prognosis, thus allowing patient-tailored therapy. In that view, the results presented above showed promising results. Furthermore, some biological markers were also found by gene profiling, and An endophenotype is a biological or a psychological characteristic transmitted with a disease, with a strong biological substrate and a higher heritability than the disease itself . The reqEEG features are partially heritable and relatively stable among lifespan . The alpThe P300 amplitude reflects the allocation of attention, with the larger amplitude, the more attention allocated. It is measured during an oddball paradigm where patients are asked to detect target stimuli, and to ignore standard and non-target rare events. It is a complex wave, with at least two distinct components or ERPs, namely the P3a and the P3b waves. The P3a, the early component, is elicited by novel and unexpected stimuli, whereas the later P3b wave is elicited when context updating is necessary. The P300 is a highly heritable phenotype, since the estimated heritability ranges between 0.30 and 0.81 depending on the studies , and washangover gene was found as required for normal development of ethanol tolerance in the fruit flies [HANGOVER/ZNF699 gene polymorphisms were associated in a large population of Irish alcohol dependant patients compared to matched controls [Our current knowledge on the neurobiological substrate of alcohol dependence leads to propose a high number of candidate genes. Association and linkage studies of the genes encoding proteins involved in alcohol metabolism  and in the rewarding circuits, including dopamine  and GABA  neurotransmitter biological pathways, are the most replicated findings. It is important to note that novel candidate genes for alcohol dependence were identified from animal models. Thus, the drosophila it flies . Human Hcontrols . The gencontrols . Convergcontrols . Recentlcontrols , 103. ThGenomic functional, including gene and protein expression profiling studies, on cell lines, animal models and human post-mortem samples confirmed that several biological pathways are involved in the vulnerability to alcohol preference, and opened avenues for patient-tailored treatment, in both pharmacogenomic and clinical proteomic. This genomic functional approach has to be combined with genetic analyses, and have to be performed on large number of patients, assessing a large set of clinical features and phenotypic information, also including endophenotypes to narrow the associated gene, or biomarker, to a specific step in the pathway to alcohol dependence. Finally, pharmacogenomics is the next issue in the comprehension of the molecular biology of alcohol dependence.