Bacterial microcompartments (BMCs) are proteinaceous organelles involved in both autotrophic and heterotrophic metabolism. All BMCs share homologous shell proteins but differ in their complement of enzymes; these are typically encoded adjacent to shell protein genes in genetic loci, or operons. To enable the identification and prediction of functional (sub)types of BMCs, we developed LoClass, an algorithm that finds putative BMC loci and inventories, weights, and compares their constituent pfam domains to construct a locus similarity network and predict locus (sub)types. In addition to using LoClass to analyze sequences in the Non-redundant Protein Database, we compared predicted BMC loci found in seven candidate bacterial phyla (six from single-cell genomic studies) to the LoClass taxonomy. Together, these analyses resulted in the identification of 23 different types of BMCs encoded in 30 distinct locus (sub)types found in 23 bacterial phyla. These include the two carboxysome types and a divergent set of metabolosomes, BMCs that share a common catalytic core and process distinct substrates via specific signature enzymes. Furthermore, many Candidate BMCs were found that lack one or more core metabolosome components, including one that is predicted to represent an entirely new paradigm for BMC-associated metabolism, joining the carboxysome and metabolosome. By placing these results in a phylogenetic context, we provide a framework for understanding the horizontal transfer of these loci, a starting point for studies aimed at understanding the evolution of BMCs. This comprehensive taxonomy of BMC loci, based on their constituent protein domains, foregrounds the functional diversity of BMCs and provides a reference for interpreting the role of BMC gene clusters encoded in isolate, single cell, and metagenomic data. Many loci encode ancillary functions such as transporters or genes for cofactor assembly; this expanded vocabulary of BMC-related functions should be useful for design of genetic modules for introducing BMCs in bioengineering applications. 2 fixation, pathogenesis, and niche colonization. While the encapsulated enzymes differ among functionally distinct BMCs, the shell architecture is conserved. This enables their detection computationally, as genes for shell proteins are typically nearby genes for the encapsulated enzymes. We developed a novel algorithm to comprehensively identify and categorize BMCs in sequenced bacterial genomes. We show that BMCs are often encoded adjacent to genes that play supporting roles to the organelle's function. Our results provide the first glimpse of the extent of BMC metabolic diversity and will inform design of genetic modules encoding BMCs for introduction of new metabolic functions in a plug-and-play approach.Some enzymatic transformations have undesirable side reactions, produce toxic or volatile intermediates, or are inefficient; these shortcomings can be alleviated through their sequestration with their substrates in a confined space, as in the membrane-bound organelles of eukaryotes. Recently, it was discovered that bacteria also form organelles–bacterial microcompartments (BMCs)–composed of a protein shell that surrounds functionally related enzymes. BMCs long evaded detection because they typically form only in the presence of the substrate they metabolize, and they can only be visualized by electron microscopy. A few BMCs have been experimentally characterized; they have diverse functions in CO Membrane-bound organelles for compartmentalization of specific functions are the hallmark feature of all eukaryotic cells. Bacteria also have organelles, but they are not ubiquitous throughout the domain; instead they are sporadically distributed and frequently provide functions that are key to niche specialization. For example, anammoxosomes are lipid-bound compartments that enable certain planctomycetes to obtain energy from anaerobic ammonium oxidation (reviewed in 12-dependent propanediol (PDU) and ethanolamine utilization (EUT) in Salmonella enterica were sequenced and found to encode genes for BMC-H, BMC-T, and BMC-P proteins Carboxysomes were thought to be a peculiarity confined to some autotrophic organisms until operons involved in vitamin B2 is generated by carbonic anhydrase (CA) and then fixed within the compartment by RuBisCO; the shell helps to confine the CO2 near RuBisCO Whether anabolic or catabolic, experimentally characterized BMCs share a common functional theme: the shell sequesters enzymes and provides a diffusion barrier for volatile and/or toxic reaction intermediates. For example, in carboxysomes, CO+ and CoA-dependent aldehyde dehydrogenase (AldDH), forming NADH and an acyl∼CoA product +, an alcohol dehydrogenase (AlcDH) reduces a second aldehyde, forming an alcohol Recently, comparison of experimentally characterized catabolic BMCs led to the identification of their common core biochemistry  [25]. ThIn experimentally characterized alpha-carboxysome and metabolosome loci, the essential BMC components , to surveying and classifying BMC loci with an emphasis on discovery of novel BMC locus types and variants of the paradigmatic types. It is a novel locus-centric method for predicting and sorting loci through the generation of a locus similarity network. To compare loci, we approximate the functional attributes of a locus by representing it as the set of pfam domains encoded by genes in the locus. While the use of pfams to visualize the functionality in a locus is relatively low-resolution, in that one pfam may correspond to a variety of homologous sequences of different functions, this coarse resolution allows for the recognition of pfams corresponding to the functionally similar biochemical cores. Moreover, the lost resolution is regained through the multiplicity of pfams conferring organelle-supporting functions present in any given locus; the diversity among these pfams aids in distinguishing different BMC locus types and sub-types. By focusing on the regions flanking BMC shell protein genes instead of being confined to presumed operons, we are able to circumvent the lack of transcriptional data for the majority of sequenced genomes that contain BMCs. LoClass also captures the genomic neighborhood of BMC shell protein genes, revealing that genes encoding the organelles are frequently situated in the context of other genes that provide ancillary functions, such as regulation, co-factor synthesis, or transport for BMC substrates. Recognition of these gene products and their roles in supporting BMC function will be useful both in functionally characterizing diverse BMCs and in the design of BMC locus modules that are ready for “plug and play” applications. Finally, LoClass is able to perform direct comparisons and classification of loci, granting high sensitivity to rare locus types. The result is a comprehensive taxonomy of BMC locus (sub)types and the identification of several novel putative BMC locus types, including one that we predict extends BMC functions beyond the paradigmatic metabolosomes and carboxysomes.hmmsearch in the hmmerSynechococcus elongatus PCC 7942 (Syn7942) was not included in this analysis due to the formatting irregularities of its sequence records. The closely related organism Synechococcus elongatus PCC 6301 (Syn6301), whose BMC proteins are identical to those of Syn7942, was included. We examined the similarity of these genomes using NUCmer in the MUMmer 3.1 The standard PF00936 hidden markov model (HMM) is often not robust for the identification of N-terminal cryptic BMC domains of CsoS1D/CcmP-type BMC-T proteins, which have only weak sequence similarity to other BMC domains Halothiobacillus neapolitanus) contain a gap greater than 10 kb between BMC-related genes and the nearest BMC shell protein gene. This resulted in several characterized loci being incomplete. Prospective BMC Loci were then redefined as the region 10 kb upstream and downstream of BMC shell protein genes . Variousd represents the minimum number of open reading frames between the envelope and a gene that contains the pfam, then a distance weight can be applied to account for the decreasing likelihood that genes in a locus are related to the function of the BMC the further they are from the envelope (R(p) represents the proportion of BMC loci that do not contain the pfam p, the weight approaches but never reaches 1 for very rare pfams and 0 for very common pfams.If envelope . This diFinally, when a pfam is only found in one of the loci, it is necessary to consider whether that pfam is frequently found within loci that also contain the set of pfams that the two loci share in common. This aids in down-weighting secondary pfams that are not necessary for the core function of a BMC, are not always localized in the BMC loci of a given type, or are only occasionally found near the periphery of a BMC locus while not actually being related to the BMC function. This co-occurring pfam weight can be represented byAnalysis and clustering of the locus similarity network was complicated by the presence of what we term satellite loci, loci that encode an insufficient complement of BMC shell protein genes  to form a BMC; genomes containing satellite loci putatively encode additional shell proteins and possibly other BMC components for a Candidate/Confirmed BMC in another locus. We define satellite BMC loci as those that meet three conditions: the locus contains an insufficient shell protein gene complement, all the shell protein genes in the locus are contiguous to each other, and there is at least one other non-satellite BMC locus in the genome. In order to prevent false positives, predicted satellite loci were manually inspected for the presence of genes with common BMC-associated pfams immediately nearby the BMC shell protein genes. Where such genes were found or where the locus met all requirements except that there was more than one locus in the genome, these loci were labeled as satellite-like loci. Where a signature enzyme was present or a putative function had already been assigned to the locus in the literature, as with the putative ethanol utilization locus, type names were assigned to these loci (see below) clusterMakerThe locus similarity network of BMC loci was visualized in Cytoscape 2.8.3 Resulting clusters were then assigned numbers . WheneveAt each level of clustering, the loci were manually compared with previously studied or predicted BMC loci. Where all of the loci in a large cluster appeared to be of the same type as a previously studied locus (e.g. EUT), those loci were first assigned that type name as a super-type. Where that cluster was further sub-clustered once or twice, the loci in those clusters were additionally assigned a numeral followed by an alphabetic character . In our taxonomy, all loci with a different numeral are deemed different types, while those whose type names are differentiated only by the alphabetic character  are deemed different sub-types. Where a locus (sub)type clustered with loci of a known type but either was lacking the signature enzyme in the pathway or bore significant differences from the type, that locus was designated to be “like” that BMC type (e.g. PDU-like). Where a locus type had not previously been named in the literature, if all genomes containing that type came from a distinct set of taxa that do not contain any other types, we assigned that type a three-letter name based on those taxa (e.g. RMM1). Where multiple loci of unknown function did not meet any of these prior criteria, they were designated Metabolosome of Unknown Function (MUF), Metabolosome with an Incomplete Core (MIC), or BMC of Unknown Function (BUF) type, depending on whether they contained all core metabolosome genes, only the AldDH but not a complete core, or none of the metabolosome core enzymes, respectively. Where multiple MIC loci clustered together, an additional numeral was assigned to the name to designate that specific type (MIC1).For each locus (sub)type, a representative locus that best captures the pfam diversity of all loci of that (sub)type was chosen . For eacIn order to identify BMC loci in genomes from candidate phyla not archived in NR, we examined the single-cell genomes from the recent GEBA-MDM project Shigella sonnei  and Roseburia inulinivorans (EEG94445.1) were significantly shorter than the rest of the sequences and were also removed  of total BMC loci in one genome . SubtracClostridium saccharolyticum WM1  contain the metabolosome core genes  for AldDSalmonella entericacob operon, which encodes the accessory function of synthesizing cobalamin  of the diol dehydratase, and the core AldDH and AlcDH enzymes , it is instead more closely related to EUT2A loci  of S. entericaL. monocytogenesS. enterica, suggesting that other factors may have driven a fusion event in L. monocytogenes, such as evolutionary pressures for a reduced genome; L. monocytogenes has a genome of 3 Mbp, while S. enterica has a genome of almost 5 Mbp. Similar evolutionary forces may have been at play to fuse the PDU and EUT loci in the non-pathogenic strains.Several selective pressures may potentially have driven the repeated fusion events of these two particular classes of loci, such as the benefit of coregulation with the cobalamin biosynthesis genes found in the merged locus, since both PDU and EUT require vitamin BLoClass identified five distinct types of loci that contain the metabolosome core enzymes and a glycyl radical enzyme , which, Olsenella uli, a member of the Actinobacteria (The GRM1 locus (Cluster 2.2) is mainly found in the Firmicutes but also in some species of the Deltaproteobacteria and bacteria . This lobacteria , suggestbacteria . Surprisbacteria ; this se12, an essential cofactor for propionaldehyde dehydrogenase 12-dependent enzymes present in the locus. PduS has an iron sulfur cluster-binding domain, as does PduT (a BMC-T shell protein) and the pair of proteins has been proposed to be involved in electron transport across the shell Apart from the signature and core enzymes, other notable ancillary proteins in the GRM1 loci include homologs to PduS, PduV/EutP, EutQ, EutJ, a multidrug resistance (MDR) efflux transporter, and a predicted transcription factor . In the The GRM2 locus (Cluster 2.3) is only found in the Gammaproteobacteria and mainly in pathogens. The only ancillary genes encoded in this locus are three distinct transcription factors  and two multi-drug resistance proteins . Due to Clostridium, Desulfosporosinus, and Oscillibacter genera of the Firmicutes, as well as in various alpha- and gammaproteobacteria (12 in the PDU BMC The GRM3 locus (Cluster 2.4) is found in both innocuous and pathogenic species of the bacteria . Ancillabacteria . The potbacteria , and theShewanella species (S. enterica), as well as a protein containing the PduO component domain DUF336  is restricted to two  species . Without species . In thisRuminococcus genus, Roseburia inulinivorans, and Clostridium phytofermentans (The GRM5 locus (Cluster 4.2) is only found in firmicutes of the rmentans . Ancillarmentans . Transcr+One BMC locus (Cluster 4.1) is almost exclusively restricted to species in the phyla Planctomycetes and Verrucomicrobia . The metCandidatus Solibacter usitatus, and the ignavibacterium Melioribacter roseus  are compositionally distinct yet cluster with the PVM loci . The PVMr roseus . A classr roseus . The comproteins . One locrganelle . Given tClostridium kluyveri species, forms a distinct subgroup of Cluster 2  loci. Two more locus types were observed in phylogenetically distinct organisms; these are designated Metabolosomes of Unknown Function (MUF) or Metabolosomes with an Incomplete Core (MIC).In addition to Candidate/Confirmed Loci that we could classify and for which we could infer putative BMC-related reactions, we also identified five locus types for which we could not easily predict a function. Four of these are distinctly different from metabolosomes in that they do not encode a complete biochemical core. Two of these locus types are found exclusively in members of the phylum Actinobacteria – specifically in several species of the genus s jostii ; accordi+ and CoA used in this step must be regenerated somehow within the compartment, and the core metabolosome genes to do so are not obviously present. Perhaps the aforementioned ancillary enzymes form a circuit that recycles the required cofactors. For example, a hydrolase could regenerate CoA to form lactate. Alternatively, it may be that some BMC shells allow cofactors across, or that the AldDH may not be acylating, at least not requiring CoA to be recycled.The major difference between the two types of RMM loci is the presence  or absence  of diol dehydratase genes . Putativ+ or CoA. The subset of genes in the locus spanning from the permease to the transcription factor are highly syntenic between RMM1 and RMM2  is present in aska E43  and inclaska E43 . This loaska E43 . These oThe MIC1 (Cluster 7) metabolosome locus contains a dehydrogenase in addition to AldDH and two putative AlcDHs, as well as a hydrolase, a phosphatase, and an MreB-like protein , but lacVerrucosispora maris AB-18-032 and the firmicute Mahella australiensis 50-1 BON, each of which encodes a class II aldolase, which could serve as the signature enzyme  , which catalyzes the reversible conversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. When acted on by an aldolase, sugar phosphates can be metabolized to either or both of these products, in some cases resulting in another product such as an aldehyde, which could be metabolized according to the canonical metabolosome model. Thus, based on the presence of TIM and a DeoC-like aldolase, we name these BMCs putative sugar phosphate utilizing (SPU) metabolosomes with an incomplete core; most of the enzymes encoded in the SPU loci fit into the deoxyribulose/deoxyribose 5-phosphate degradation pathway for DNA catabolism.The The second Candidate BMC Locus type that appears in single-cell genomes is found in two representatives from the candidate phylum Latescibacteria. This locus is similar to the SPU type, containing genes encoding an AldDH, PduL, PduS, an RpiB-like protein, and an aldolase , so we rCaldithrix abyssi contains a locus which encodes AldDH, a pta type PTAC, and PduS. However, the absence of an apparent signature enzyme necessitates the designation of this locus as a MIC. Each sequenced locus from Poribacteria and BRC1 meets all of the criteria of a satellite-like locus ; this may indicate that this additional locus has a distinct function. The GRM3 outliers near the PDU/EUT sequences are secondary AldDH genes in their respective loci . The GRM3 AldDH near the ETU branch belongs to Clostridium novyi; its proximity to ETU may indicate an evolutionary link between GRM3 and ETU. In addition, some GRM3 taxa were found nested within the large GRM1 clade, which may indicate that they share common origin with GRM1 loci. Thus, apart from these few outlier taxa, we observed congruence between the molecular phylogeny of one gene (AldDH) and our clustering results based on total pfam complement of a locus  and ParA (PF01656) . MreB, aC. phytofermentansIn addition to expanding the vocabulary of pfams associated with bacterial organelle function, LoClass unveiled BMC-associated puzzles, pfams for which we could not readily infer a reason for their conservation in various locus types. For example, GRM1, GRM3, GRM4, GRM5, MUF, SPU, and SPU-like loci, which do not encode cobalamin-dependent enzymes, encode various genes that have been associated with cobalamin synthesis, such as PduS and PduO (both full length and a single DUF336 domain). These may be relics of the evolutionary history of these BMC loci or, more likely, these genes have been co-opted to perform different functions useful to the BMC metabolism, as has been predicted in the GRM5 locus in 3/NDH-14 CO2 transport gene cluster, which is conserved across cyanobacterial carboxysome loci of both types.LoClass also highlighted the diversity within numerically abundant locus types ; this haSeveral striking observations from our classification enlarge our view of the diversity of BMCs. New locus types identified in this study are the PDU/EUT and PDU/GRM fusions, GRM2, GRM3, GRM4, PVM-like, RMM2, MUF, MIC1, SPU, SPU-like, and BUF locus types. While many of these BMC loci adhere to the complete biochemical model of the metabolosome paradigm combinations of groups of domains that constitute loci and contribute to BMC function and evolution. Such a comprehensive view of the requisite building blocks for diverse BMC functions can likewise inform the design of BMC loci for the introduction of genetic and metabolic modules for applications in synthetic biology.Dataset S1Gene information for all BMC loci in NR analyzed with LoClass.(XLSX)Click here for additional data file.Dataset S2Gene information for all BMC loci from candidate phyla examined in IMG.(XLSX)Click here for additional data file.Dataset S3Seed alignment for modified PF00936 domain.(FASTA)Click here for additional data file.Dataset S4Modified PF00936 domain HMM.(HMM)Click here for additional data file.Dataset S5Cytoscape session file for BMC locus similarity network.(CYS)Click here for additional data file.Dataset S6Aldehyde dehydrogenase alignment.(FASTA)Click here for additional data file.Figure S1Similarity network of BMC loci at all clustering levels. All stages of clustering beyond the first stage are represented in this figure, where above each cluster is written its number. Clustering was accomplished by setting score (S) cut-offs and modifying the inflation value (I), a property of MCL (TIF)Click here for additional data file.Figure S2Heatmap of common pfam occurrence in BMC locus (sub)types. The 75 pfams present in the highest number of Candidate/Confirmed BMC Loci from NR and IMG are sorted by percentage of these loci that contained them. For each locus (sub)type, the proportion of loci of that given (sub)type that contain a specific pfam is represented by the darkness of the corresponding square, where black indicates that all of the loci contain that pfam and white indicates that none of the loci contain the pfam. Only locus (sub)types with more than one locus are shown. * Many of these loci are incomplete, and percent abundances may not reflect the percent of loci in these organisms that contain the corresponding pfam.(TIF)Click here for additional data file.Figure S3Similarity network of BMC loci colored by phylum.(TIF)Click here for additional data file.Table S1Phyla containing BMC loci analyzed with LoClass.(DOC)Click here for additional data file.Table S2Genomes containing BMC loci analyzed with LoClass.(DOC)Click here for additional data file.Text S1Genes and pfams associated with the PDU and EUT loci.(DOCX)Click here for additional data file.


The individual variance in the efficiency of repair of damage induced by genotoxic therapies may be an important factor in the assessment of eligibility for different anticancer treatments, the outcomes of various treatments and the therapy-associated complications, including acute and delayed toxicity and acquired drug resistance. The second part of this paper analyses the currently available information about the possibilities of using experimentally obtained knowledge about individual repair capacity for the purposes of personalised medicine and healthcare. CCNHcyclin HGGRglobal genome repairHER2human epidermal growth factor receptor 2HNPCChereditary non-polyposis colorectal carcinomaMSHMutS homologueMLHMutL homologueNERnucleotide excision repairNHEJnon-homologous end joiningTP53tumour protein 53 (p53)Remedium ante venenum non valet.(Never take the antidote before the poison)Latin proverbAs most of the currently used anticancer therapies work by induction of DNA damage, individual capacity for DNA repair of DNA may be an important factor in the assessment of eligibility for genotoxic therapies. At present, only the most common types of tumours and the most commonly used basic therapeutic regimens have been studied with regards to eligibility for certain types of therapy, but the field is currently in development.TP53 gene, which is usually associated with poorer prognosis for the patient TP53 gene (or the surrounding genomic region) may be deleted or inactivated in another manner and different segments of the gene may be altered, deleted or rearranged in order to ensure cell survival even in the presence of unrepaired damage and/or genome instability. If wild-type p53 is preserved, its up-regulation may cause mass apoptosis in cancer cells, and several anticancer agents operate on this principle. For example, the histone deacetylase agent CG200745 works by stimulating the acetylation of p53 on selected lysine residues, inducing the accumulation of p53 and the subsequent transactivation of pro-apoptotic genes TP53 locus. The patients without deletions of 17p are eligible for genotoxic treatments , which may produce long-lasting remissions. Patients with 17p deletions, however, may benefit more from treatments other than therapies based induction of the p53-dependent pathways, such as antibodies , immunomodulators , CDK inhibitors (flavopiridol) and steroids One of the basic factors in the assessment whether the patient is eligible for treatment with certain type of anticancer therapy is the p53 status of the tumour – specifically, whether the tumour expresses p53 or not; if it does, whether the p53 is wild-type or a cancer-specific isoform. About 50% of human tumours carry alterations in the ERCC1 expression may assist in the identification of patients eligible for therapy with platinum derivatives ERCC1 gene is associated with lower levels of ERCC1 mRNA and protein, and is likely to be a factor in eligibility for treatment with genotoxic agents. In a large study from 2008, among 25 DNA polymorphisms in genes coding for proteins of DNA repair, maintenance of genome integrity, and progression through cell cycle, several were associated with predictably poorer response in patients with advanced lung cancer treated with platinum derivatives: rs1800975 polymorphism in the 5′-untranslated region of the XPA gene; XPCins83; XPD Lys751Gln; CCNH (cyclin H) Val270Ala (rs2266690); RAD23B Ala249Val (rs1805329); and ERCC1 C8092A polymorphisms Determination of levels of TP53, XPA, XPC, XPD, XPG and the ERCC1 genes.Polymorphisms in genes coding for products functioning in maintenance of genome integrity, DNA repair and/or induction of apoptosis may significantly affect response to treatment and patient survival in cancer. Better response to treatment does not always translates directly to longer patient survival, as factors other than progression of the cancer (e.g. toxic effects from the therapy) may shorten the survival. Generally, lower-than-normal capacity for DNA repair in patients with cancer is believed to be associated with better response to genotoxic treatments. It results in increased levels of therapy-induced damage in tumour cells, causing them to slow down or stop the progression in the cell cycle or reroute to apoptosis, while cells with near-normal repair capacity would repair the damage quickly, then continue proliferating. The association, however, is not that straightforward, and the field is currently in development. At present, best studied with regard to response to genotoxic therapies and patient survival are polymorphisms in the TP53 status (presence/absence of wild-type TP53 gene copies) may be a significant factor of survival in patients with chronic lymphocytic leukemia. In 20–30% of all patients CLL may present as indolent disease, with a prolonged clinical course (up to 10–20 years) and requiring specific treatment only in the late stages or not at all. In patients with 17p deletions, however, the clinical course may be typical of an aggressive tumour, resistant to chemotherapy. Deletion of the TP53 locus is associated with more aggressive course and, respectively, with shorter survival, in multiple myeloma As it was already mentioned, TP53 gene as a survival-modifying factor in various cancers has been extensively studied, but the results so far have been, at best, contradictory. As the 72Arg allele of the TP53 gene is associated with increased propensity to apoptosis, it could be expected that the carriers of Arg alleles  would be at selective advantage in cancer, as the cancer cells with Arg/Arg genotype would presumably be routed more easily to apoptosis. Indeed, patients with the Pro/Pro homozygous genotype and hereditary predisposition to nonpolyposis colon cancer exhibited earlier age of onset and a generally poorer prognosis than the carriers of Arg alleles, specifically the Arg/Arg homozygotes TP53 has recently been found to be associated with longer survival after conventional chemotherapy in patients with sarcoma that have retained the wildtype TP53 gene TP53 turned out to be a preferred target for conversion into cancer-specific TP53 variants in HPV-induced head and neck cancers, while the Pro allele was selectively inactivated or deleted The role of the common Pro72Arg polymorphism in the TP53 gene may be associated with poorer prognosis in patients with non-small-cell lung cancer Carriership of the duplication variant in intron 3 of the XPA gene variant rs1800975 in patients with advanced lung cancer treated with platinum-based regimens is associated with shorter survival Homozygous carriership of the XPC gene may be associated with poorer prognosis in patients with advanced lung cancer treated with platinum derivatives XPC Lys939Gln polymorphism was recently associated with shorter survival in esophageal squamous cell carcinoma Genotypes containing at least one deletion allele by the intron 9 polymorphism in the XPD gene is generally reported as not associated with significant differences in the response to chemotherapy with platinum derivatives, or patient survival (for review see [41]), albeit earlier studies indicated for shorter survival in carriers of the 751Gln allele XPD gene and the 399Gln allele in the XRCC1 may be associated with shorter survival in patients with non-small-cell lung cancer treated with platinum agents Analysis of the available data indicates that the Lys751Gln polymorphism in the XPD genotype made of Asn at codon 312 and Gln at codon 751 of the XPD gene may be associated with poorer response to chemotherapy and decreased overall survival in advanced non-small-cell lung cancer A composite XPD Asp312Asn and the XRCC1 Arg399Gln polymorphisms was found to be associated with higher rate of occurrence of TP53 mutations in non-small-cell lung cancer TP53 gene mutations may also be observed in breast cancer, where the XPC 939 Gln/Gln, XRCC1 399 Gln/Gln and XPC 499 Ala/Ala homozygous genotypes were associated with increased risk for mutations in TP53Sometimes, polymorphism in one of the genes coding for products acting in DNA repair may affect the status of another repair gene. For example, carriership of the variant alleles of the ERCC1 T19007C polymorphism may be associated with better response  was repeatedly reported to be associated with better response to chemotherapy with platinum derivatives and increased overall survival ERCC1 gene  was found to be associated with longer overall survival and better response to therapy in patients with refractory multiple myeloma treated with thalidomide Another single-nucleotide polymorphism in the 3’- UTR of the ERCC1 gene, also associated with differential mRNA levels, conferred longer survival in patients with advanced colorectal carcinoma treated with platinum derivatives ERCC1 Asn118Asn polymorphism compared to patients with C/T and C/C genotypes The C allele of the synonymous (Asn-Asn) C-to-T substitution at codon 118 of the XPD Lys751Gln, XPC Ala499Val and XPC Lys939Gln polymorphisms were associated with higher risk for relapse and shorter survival in patients with acute myeloid leukemia that had been placed by pre-treatment cytogenetics into the ‘intermediate’ risk group, where risk of relapse was difficult to evaluate by other methods The XRCC1 Arg194Trp polymorphism may indicate better response to treatment with platinum derivatives in patients with advanced non-small-cell lung cancer Carriership of variant allele of the ERCC1 (C8092A), XRCC1 (Arg399Gln) and XPD (Lys751Gln), may confer lower risk for development of resistance to platinum-based therapy in some tumours  High levels of mRNA and protein of the factors of NER ERCC1 and XPD were reported to be associated with resistance to cisplatin therapy in some tumours  TP53 copies in multiple myeloma is a predictor for resistance to genotoxic therapy with 6-mercaptopurine  or melphalan Deletion of both somatic BRCA1 gene show that some tumours that initially were sensitive to anticancer therapy  may be capable of restoring the functionality of BRCA1BRCA2 allele to a functional allele, conferring resistance to a genotoxic drug to which the tumour was initially sensitive, was described to have occurred in vivo, in a tumour from a patient with Fanconi anemia complementation group D1 and acute myelogenous leukemia The genome of a cancer cell is changeable, and some traits may be lost, while others may be newly acquired. Resistance to an anticancer drug usually develops in the course of treatment with the drug. So far there have been several experimental proofs that some cancer cells may restore the activity of previously inactive or weakly active repair proteins by mutagenesis, resulting in development of resistance to a drug to which the tumour was initially sensitive. Experiments with mouse models of breast cancer carrying frameshift mutations in the ATM gene may be associated with high toxicity in patients treated with ionising radiation ATM gene – 126713G-to-A, 111G-to-A (rs189037) and G5557A, were found to be associated with increased risk for severe radiation pneumonitis in patients with lung cancer treated with radiotherapy XPC gene; Lys751Gln and Asp312Asn polymorphisms in the XPD gene; Arg194Trp, Arg280His and Arg399Gln polymorphisms in the XRCC1 gene; and the Asp148Glu polymorphism in the gene coding for the apurinic/apyrimidinic endonuclease APE1, an enzyme functioning in base excision repair and mismatch repair XRCC1 gene and the Thr241Met polymorphism in the XRCC3 gene may predispose to radiation-induced subcutaneous fibrosis and telangiectasias after radiotherapy Heterozygous carriership of mutations associated with the phenotype of ataxia-telangiectasia or ‘neutral’ polymorphisms in the MSH6 Gly39Glu; nucleotide excision repair: the rs1800975 polymorphism in the 5′-untranslated region of the XPA gene; XPC Lys939Gln; XPD Lys751Gly; and the more rare synonymous substitution XPD Asp711Asp; ERCC1 C8092A; specifically for transcription-coupled repair: ERCC6 (CSB) Met1097Val; base excision repair: XRCC1 Arg399Gln; control of the progression in the cell cycle: CCNH Val270Ala; repair of double-strand breaks: XRCC2 5′-untranslated region polymorphism rs6464268 (homologous recombination) and the synonymous Asp568Asp polymorphism in the gene LIG4 (non-homologous end joining); damage-related signaling: BRCA1 Pro871Leu (rs799719). The highest odds ratio (>8) was identified for the 5’-UTR rs6464268 in the XRCC2 gene. This was not unexpected, as double-strand breaks are among the major types of damage caused by ionising radiation. As XRCC2 protein functions in repair of double-strand breaks by homologous recombination, it could be expected that even subtle deficiencies in this repair pathway would have significant impact on repair of therapy-inflicted damage XPD Asp312Asn, RAD23B Ala249Val, APE1 Asp148Glu (rs1130409) and PARP1 Val762Ala (rs1136410), the association with risk of radiation-induced toxicity was estimated to be weak.Studies of the effects of multiple (more than 4–5 per study) polymorphisms on the risk of an associated disease or condition, response to therapy and/or associated adverse effects are still rare in the specialized literature. The results from one extensive study for association of carriership of DNA polymorphisms and therapy-associated toxicity in patients with esophageal cancer treated with ionising radiation were published in 2011. Significant association with severe (grade 3-4) toxicity (myelosuppression/dysphagia) was identified for 12 out of 21 polymorphisms in genes coding for products functioning in the maintenance of genome integrity and/or DNA repair and the control of the progression in the cell cycle LIG4 gene and Asp711Asp in the XPD gene, the 5′-untranslated region polymorphism in XRCC3 and the Val219Ile polymorphism in the coding sequence of the MLH1 gene were found to be associated with late rectal or bladder toxicity in patients treated with radiotherapy for prostate cancer The synonymous polymorphisms Asp568Asp in the ERCC1 gene (associated with lower ERCC1 transcript stability and lower protein levels), but in carriers of the A allele It would be logical to assume that polymorphic gene variants associated with superior survival after genotoxic treatments may also be factors in the constitution of the risk for treatment-associated toxicity. Tumour cells with lower capacity for DNA repair rapidly accumulate unrepaired DNA damage, resulting in cell cycle arrest and/or apoptosis. The healthy cells in the patient, however, carry the same genotype, and have the same lower-than-normal capacity for DNA repair, therefore, they would also suffer more damage from the genotoxic treatments. The relationship is more complex, however, and alleles that confer lower repair capacity are not always associated with severe toxic effects. For example, among patients with non-small-cell lung cancer treated with platinum derivatives, increased risk for high-grade gastrointestinal toxicity was not seen in carriers of the C allele of the Modern biomedical science develops rapidly, and more and newer therapies are being developed every day, providing if not a cure, then at least a considerable improvement in patient survival and quality of life. This is especially valid in cancer, a very common disease that was not that long ago considered incurable and uniformly fatal. Cancer is currently believed to result from accumulation of unrepaired damage in DNA, and treatment of cancer is very often based on inflicting DNA damage in order to reduce the proliferative potential of tumour cells. Natural variance in the individual capacity for repair of damage in DNA may play significant role in the risk for development of cancer. When cancer has already developed, individual repair capacity may modulate various aspects of its course, such as whether the patient would benefit from this or that therapy; the uneventful and overall survival of the patient; whether there are modifiable environmental factors that may increase the chances for better therapeutic response and/or lower treatment-related toxicity; the risk for development of resistance to antitumour drugs in the course of treatment; and the possible adverse effects of genotoxic therapies. Knowledge about the individual specificities of capacity for repair of DNA damage provides a base for making informed decisions about potential changes in lifestyle, selection of treatments best suited to the particular patient, prognostication about survival and anticipation and management of potential complications.


PCa represents a complex organ-like multicellular structure maintained by the dynamic interaction of tumoral cells with parenchymal stroma, endothelial and immune cells, and components of the extracellular matrix (ECM). The lack of PCa models that recapitulate this intricate system has hampered progress toward understanding disease progression and lackluster therapeutic responses. Tissue slices, monolayer cultures and genetically engineered mouse models (GEMM) fail to mimic the complexities of the PCa microenvironment or reproduce the diverse mechanisms of therapy resistance. Moreover, patient derived xenografts (PDXs) are expensive, time consuming, difficult to establish for prostate cancer, lack immune cell-tumor regulation, and often tumors undergo selective engraftments. Here, we describe an interdisciplinary approach using primary PCa and tumor initiating cells (TICs), three-dimensional (3D) tissue engineering, genetic and morphometric profiling, and humanized mice to generate patient-derived organoids for examining personalized therapeutic responses in vitro and in mice co-engrafted with a human immune system (HIS), employing adaptive T-cell- and chimeric antigen receptor- (CAR) immunotherapy. The development of patient specific therapies targeting the vulnerabilities of cancer, when combined with antiproliferative and immunotherapy approaches could help to achieve the full transformative power of cancer precision medicine.Prostate cancer (PCa) is the most common malignancy and the second most common cause of cancer death in Western men. Despite its prevalence, PCa has proven very difficult to propagate  With more knowledge gained, new approaches have been developed, for growing both normal and diseased tissues, allowing the creation in a dish of organ-specific mini 3D structures termed “organoids”  and PCa foci with regions of normal glands, PIN and PCa in the patient's H&E stained and mapped section Figures  along wis Figure .hiCD29hiCD44hi cell subpopulation (TICs) that has stem-like features and in vivo tumorigenic potentials  have organoid forming potential. Using 3D chambers of Matrigel, we have generated prostate organoids that have survived for 3–6 months and could be re-derived to generate serial cultures -mediated immunotherapy approaches have shown recent successes, but there are no platforms to examine these immunotherapy strategies with standard therapy  trial that included 512 metastatic castration resistant PCa patients showed that Sipuleucel-T improved the overall survival (OS) by ~4 month and reduced the risk to die by only 22%  mice, NOD SCID IL2Rαnull (NSG) or Balb/C Rag2−∕− IL2Rαnull (BRG), that support better human hematopoietic cell engraftment  that give rise to various lineages of human blood cells throughout the life of the mouse , and lack absolute efficiency of a memory-based HIS in mice. In the future, advances in the regenerative medicine field would allow for utilizing artificial human organoids and/or cells with multilineage engraftment potentials derived from tissue-restricted/induced progenitors or induced pluripotent stem (iPS) cells for generating ideal multicellular “humanized” models.One of the important objectives of biomedical sciences is to decipher the understanding of the fundamental biological principles in order to improve clinical-pathological outcomes. It is clear that no single model is likely to recapitulate all aspects of the complex genetics and biology of human cancers; therefore, understanding the strength and limitation of each model is necessary to maximally leverage these complimentary engineered model systems to facilitate the development of improved therapeutic approaches. 3D cultures enable the identification of cellular interactions and molecular signals capable of promoting tissue regeneration and disease progression. Primary human cells or tissues can be used to guide the choice of personalized interventions, to test therapeutic schemes or to grow replacement tissues from the cells of a given patient.The success of this approach will involve the expertise of an interdisciplinary team of scientists, clinicians, and bioengineers and require an extraordinary level of standardization; nevertheless, given the vast applications, we believe that 3D cultures represent the new frontiers of precision medicine.MB designed research, performed research, analyzed data, and wrote the manuscript. AF designed research and analyzed data. AP designed research, performed research and analyzed data. MY designed research and analyzed data. HS designed research, performed research, analyzed data, wrote the manuscript, and supervised the study.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.


Protein phosphorylation has long been recognized as an essential mechanism to regulate many important processes of plant life. However, studies on phosphorylation mediated signaling events in plants are challenged with low stoichiometry and dynamic nature of phosphorylated proteins. Significant advances in mass spectrometry based phosphoproteomics have taken place in recent decade, including phosphoprotein/phosphopeptide enrichment, detection and quantification, and phosphorylation site localization. This review describes a variety of separation and enrichment methods for phosphoproteins and phosphopeptides, the applications of technological innovations in plant phosphoproteomics, and highlights significant achievement of phosphoproteomics in the areas of plant signal transduction, growth and development. Phosphorylation is one of the most important post-translational modifications (PTMs) of proteins  analysis. Commonly used enrichment techniques were summarized in Table The most widely used enrichment method for phosphopeptides takes advantage of the affinity binding between negatively charged phosphate and positively charge metal ions  and zirconium dioxides (ZrO2) are gaining popularity for phosphopeptide enrichment. A comparison of the performance of TiO2 and ZrO2 performed with α-casein and β-casein as standard proteins showed that TiO2 tends to enrich multiply phosphorylated peptides, and ZrO2 tends to enrich singly phosphorylated peptides. A serial enrichment procedure with both TiO2 and ZrO2 can significantly increase the efficiency of capturing phosphopeptides in biological samples  resulted in identification of 2305 phosphopeptides belonging to 964 proteins in wheat  has been a widely used technology that resolves thousands of proteins by isoelectric point and molecular weight. Pro-Q Diamond is a fluorescent stain that provides a convenient method for selectively staining phosphoproteins in acrylamide gels. The result shows a global map of the modified proteins and their relative abundances compared to non-phosphorylated counterparts when a total protein staining is used after Pro-Q Diamond staining. Differentially phosphorylated proteins in wild-type and 15N in Arabidopsis suspension cells  and tandem mass tags (TMT). The tags are designed to be isobaric during MS and fragment to reveal differential low mass ion reporters during MS/MS. Due to its capability of multiplexing up to 10 samples in a single experiment and the enrichment effect for low abundance proteins, iTRAQ/TMT labeling has become popular in plant phosphoproteomics . The first SILAC in plants was done by introducing in vivo and in vitro label methods are limited by the number of samples, label free approaches enable quantitative phosphoproteomics of unlimited number of samples. There are two main methods in label free quantitation. The first is based on precursor ion peak intensity/area, and the second is based on the number of MS/MS spectra acquired for a given peptide . Both methods were used in plant phosphoproteomics  has been used for quantification of targeted phosphopeptides  have been identified. For example, time for coffee (TIC) and non-phototropic hypocotyl 3 (NPH3), which are involved in circadian clock and phototropism, were found to be MPK3/6 substrates. The result suggests that plant circadian rhythm and phototropism may be regulated by the MAPK signaling network.The mitogen-activated protein kinases (MAPKs) constitute one of the most important signaling mechanisms in plants, and they play essential roles in linking the perception of different stimuli with cellular adaptive responses. The MAPK signal transduction pathways are evolutionarily conserved in all eukaryotic organisms such as plants, yeast, fungi, insects, nematodes, and mammals   is a phytohormone that plays an important role in many aspects of plant life. For example, ABA is essential for regulating seed maturation and stomatal closure under abiotic and biotic stresses , and on transcription factors, chromatin remodeling proteins, and spliceosome components. Surprisingly, many novel phosphopeptides from proteins involved in vesicle trafficking such as components of the exocyst complex  were identified. How phosphorylation of these SEC proteins plays a role in vesicle trafficking is intriguing. Recently, phosphorylation of Sec31 by a casein kinase 2 was found to control the duration of COPII vesicle formation, decrease its association with ER and promote ER-to-Golgi trafficking  and STN8 kinases are light regulated and participate in phosphorylation of thylakoid membrane proteins and stroma proteins. Ingelsson and Vener  performeSucrose non-fermenting 1 related kinase (SnRK1) acts as a sensor of energy levels in plant development, and regulates plant growth by maintaining energy homeostasis during stress conditions  defect as revealed by Northern blot analysis. To test the role of phospho-motif-4 in translation termination efficiency, a well-established dual luciferase assay was used. The deletion-mutant lacking phospho-motif-4 was not able to rescue this defect, indicating that this motif has a role in translation termination efficiency. To dissect the sequences within phospho-motif-4 required for NMD activity, PCR-mediated mutagenesis was used to generate three additional deletion mutants . The results revealed that deletion of residues 736–745 reduced NMD activity as measured by Northern blot analysis. To test the functional role of Y738 and Y742, site-directed mutagenesis was used to create phosphorylation mimic mutants. Interestingly, the Y738F and Y742F fully rescued NMD activity of a chromosomal UPF1 deletion-mutant strain, indicating that they are not compromised in their ability to function in NMD. These results provided strong evidence that UPF1's ability to promote translational termination fidelity is depended on the conserved C-terminal phosphorylation motif, which is important for its NMD activity.A large phosphoprotein, the RNA surveillance protein UP-frameshift 1 (Upf1) in e et al.  used tan4+)-based IMAC and the reverse phase (RP)-strong cation exchange (RP-SCX) biphasic trap column-based online RPLC is a great example of the advancements (Bian et al., in vitro labeling and SILAC in vivo labeling have shown to be successful in combination with IMAC and MS (Isner et al., Phosphopeptide enrichment and MS have been essential tools for studying protein phosphorylation. It is challenging to directly detect phosphoproteins in biological samples due to the low abundance and low stoichiometry of phosphorylation in different biological processes. The enrichment methods of phosphoproteins/phosphopeptides from complex mixtures have greatly improved over the years. For instance, combining the titanium (TiThe 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.


AbstractThe study of ENT cancer allows the implementation of molecular biology methods in diagnosis, predicting the evolution of the disease and suggesting a certain treatment.  MMPs are proteolytic enzymes, zinc dependent endopeptidases, secreted by tissues and proinflammatory cells that play a role in the clearance of cell surface receptors. They are expressed as zymogens (inactive forms). Proteolytic enzymes cleave zymogens generating active forms.  They are involved in cell proliferation, adhesion, differentiation, migration, angiogenesis, apoptosis and host defense. There are studies about 26 human MMPs, named according to their structure: Matrix metalloproteinases (MMPs) are proteolytic enzymes that contribute to tissue growth and remodeling and also to tumor expansion by degrading components of the extracellular matrix  . Considering their connection with cell membrane, MMPs are classified into 8 types: transmembrane type I and II, (contains an intracellular or transmembranar domain), gelatin-binding, simple hemopexin, furin activated, vitronectin-like, minimal domain and GPI-linked [3], gelatin zymography and Western Immunoblot analysis [4]. MMPs expression and activity is determined by in situ zymography [5]. Extracellular proteolysis is a complex process that involves the degradation of the extracellular matrix and the weakening of intracellular connections, but also has a role in the synthesis of bioactive molecules [6] and also regulated by hormones, growth factor, cytokines. MMPs activity is modulated by tissue inhibitors of metalloproteinases (TIMP) [6]. In a pathophysiological way, the regulation of pericellular proteolysis contributes to the clinical aspects of the diseases, with both diagnostic and therapeutic significance [7]. Reports revealed a direct involvement of matrix metalloproteinase (MMPs) overexpression in development and progression of ENT squamous cell carcinoma [8], p53 status and activity of the iNOS pathway in ENT squamous cell carcinoma. Imbalance between MMP and TIMP is a prognostic indicator of the pathological process, tumor aggressiveness and cancer progression . A correlation between MMP9 expression and angiogenesis has been documented [11]. Overexpression of this kinase has demonstrated to be an early event in squamous cell carcinoma and associated with metastases in cervical lymph nodes. FAK increases cell motility and MMP2 production. The level of expression of MMPs is useful for treatment planning and prognosis, being related to clinical/histological features of the tumor and lymph nodes [12]. Cancer progression can also involve focal adhesion kinase (FAK) . The in vivo interaction of MMPs with cell surface receptors and stimulating or inhibiting apoptosis has been demonstrated. MMP2 and MMP9 increase apoptosis during tissue remodeling and neoangiogenesis. In cell survival and proliferation, MMPs play an important role by augmenting "survival signals" . TGFβ induce the secretion of MMPs by endothelial cells and is also a good chemotactic for inflammatory cells and recruits pericytes for the new vessels, to complete their maturation. Angiogenic factors  are secreted by inflammatory or tumor cells. They bind on the surface of the endothelial cells that begin to secret MMPs, change their expression of integrins and undergo proliferation [15]. MMPs in extracellular matrix release proangiogenic factors  [10]. Also, MMPs facilitate endothelial cell migration by removing adhesion sites and cleaving cell-cell and cell-matrix receptors [ In conclusion, MMPs are involved in tumoral processes by allowing the tumor cells invade the surrounding stroma , breaking blood or lymphatic vessels’ basal membrane, generating intravasation and extravasation. They also initiate and sustain the growth of tumor cells (by angiogenesis to metastatic site). MMPs activate mutation of proto-oncogenes k-ras/H-ras in tumor cells (up regulate expression of VEGF and inhibit trombospondine) and erb B2 (stimulate the proangiogenic factor and inhibit trombospondine). MMPs inhibitors can be natural or synthetic. They have been documented as cytostatic and antiangiogenic agents. Unfortunately, they have short half-life in vitro. MMPs inhibitors were developed from a peptide sequence recognized by the targeted protease that interacts with the zinc of the active site. Synthetic MMP inhibitors are, for example: marimastat, batimastat. They are low molecular weight inhibitors. Natural MMP inhibitors are tetracyclines, neovastat, nicotiamide, rifampicin. The development of MMPs inhibitors was a way to improve the selectivity of newly developed drugs toward a certain MMPs, optimizing the interaction with MMPs in ENT cancer patients. An important step in cancer treatment was to understand the mechanism by which drugs elicit their antitumoral and antimetastatic effect.


Panulirus argus) when subject to the socially transmitted PaV1 virus, appears to have prevented an epizootic over a large seascape. We capitalized on a "natural experiment" in which a die-off of sponges in the Florida Keys (USA) resulted in a loss of shelters for juvenile lobsters over a ~2500km2 region. Lobsters were thus concentrated in the few remaining shelters, presumably increasing their exposure to the contagious virus. Despite this spatial reorganization of the population, viral prevalence in lobsters remained unchanged after the sponge die-off and for years thereafter. A field experiment in which we introduced either a healthy or PaV1-infected lobster into lobster aggregations in natural dens confirmed that spiny lobsters practice behavioral immunity. Healthy lobsters vacated dens occupied by PaV1-infected lobsters despite the scarcity of alternative shelters and the higher risk of predation they faced when searching for a new den. Simulations from a spatially-explicit, individual-based model confirmed our empirical results, demonstrating the efficacy of behavioral immunity in preventing epizootics in this system.Sociality has evolved in a wide range of animal taxa but infectious diseases spread rapidly in populations of aggregated individuals, potentially negating the advantages of their social interactions. To disengage from the coevolutionary struggle with pathogens, some hosts have evolved various forms of “behavioral immunity”; yet, the effectiveness of such behaviors in controlling epizootics in the wild is untested. Here we show how one form of behavioral immunity  practiced by Caribbean spiny lobsters ( Sociality confers certain advantages to animals and has nurtured the evolution of complex social systems, but it has evolved with costs –3. SociaPanulirus argus), a social species that shares and defends dens with conspecifics. However, healthy spiny lobsters are repelled by odors produced by diseased lobsters [Panulirus argus virus 1; PaV1) [Some of the most sophisticated behaviors that serve to minimize the spread of pathogens and parasites evolved in eusocial animals. For example, some species of ants and bees when infected exhibit "altruistic suicide" by abandoning their colonies to prevent further transmission –18. Othelobsters ,23 and a1; PaV1) .Spiny lobsters are an iconic and abundant species in the Caribbean where they support one of the most valuable commercial and recreational fisheries in the region, whose estimated value exceeds $500M US annually . Spiny lPaV1 (Panulirus argus virus 1) is an undefined icosahedral dsDNA virus first described in 2000 from infected lobsters in the Florida Keys, Florida (USA) . Since t2) of the Florida Keys was decimated by the occurrence of a persistent bloom of cyanobacteria (Synechococcus spp.), similar to circumstances observed two decades earlier [In August-September 2007, nearly the entire sponge community in a large area  conduct a field test of competition for shelter between healthy versus diseased lobsters in the affected area, (2) model the effect of this massive social reorganization of lobsters to estimate potential changes in the prevalence of PaV1 infection, and (3) evaluate model predictions with empirical estimates of disease prevalence before and after the sponge die-off. Our findings indicate that behavioral immunity is operating in this natural host-pathogen system and appears to limit the development of epizootic outbreaks.No protected, endangered, or vertebrate species were used in this investigation. Most lobsters used in this study were simply observed; if collected, most were released unharmed after acquisition of a blood sample through non-lethal sampling or after use in the tethering experiment. Only naturally PaV1-infected lobsters collected near the study area were used in field experiments and no lobsters infected with PaV1 were released into the wild. Lobsters collected for histological examination of tissues were anesthetized on ice prior to dissection. This research was conducted under permits granted by the Everglades National Park  and Florida Fish and Wildlife Conservation Commission (SAL-06SRR-582).We tested whether aggregations of healthy lobsters in natural dens differed in their response to the introduction into their den of an additional healthy lobster or a PaV1-infected lobster in a field experiment  conducted on sites that had experienced the sponge die-off and thus where lobsters were highly aggregated. Lobsters (16–56 mm CL) were first collected by divers from natural habitats in the middle Florida Keys region, but not at the study site. The presence or absence of PaV1 in collected lobsters was assayed using PCR and all diseased lobsters used in the experiment were visibly diseased . Lobsters were tethered by tying a loop of monofilament (8 kg test) around their carapace (between the second and third periopods) with a snap swivel tied on the dorsal side of the carapace and the knot sealed with cyanoacrylate superglue. The snap swivel was clipped to a brick by a 50 cm long monofilament line. This arrangement permitted tethered lobsters to move freely and naturally within the limits of the tether, but they could not escape. Divers introduced either a single healthy or single visibly diseased juvenile lobster into separate, haphazardly selected natural aggregations of lobsters found under coral heads or within rocky holes. Dens were individually marked with numbered surface buoys and their GPS positions recorded so they could be relocated. We counted and estimated the sizes of all lobsters within each den prior to the introduction of the tethered lobster and then again 24 hrs later; the size of the lobster aggregation did not differ significantly between treatments  and lobsters in aggregations were similar in size in both treatments . Healthy tethered lobsters were released after the study, whereas diseased lobsters were returned to the laboratory and euthanized to prevent further spread of the PaV1 virus. Lobsters were tethered in a total of 46 dens  that initially contained 177 lobsters with aggregations ranging from 2–15 lobsters per den (mean & sd of aggregation size: 4 ± 2). These tethering data were analyzed using a 2-way log-linear contingency table analysis.Earlier versions of our individual-based spatially-explicit model were developed to describe lobster recruitment dynamics in response to habitat and climate changes –31. The 2 square habitat cells  that correspond with true habitat locations determined from GIS data. Hard-bottom cells contained realistic densities of several types of benthic structures, including several species of large sponges, whose density and size were previously determined from diver-based field surveys at more than 300 sites. The density of sponges and other lobster shelters at unsampled locations in the model's seascape were generated with ordinary kriging and each shelter was randomly assigned a lobster carrying capacity as determined from the maximum lobster group sizes for each shelter type recorded by divers in repeated surveys.The model’s spatial domain consisted of a spatially-explicit seascape "map" of the primary juvenile lobster habitat in the Florida Keys and consisted of 2792 contiguous 1 kmThe model operated on a daily time step composed of a sequence of processes that mimic the daily dynamics of real lobsters, including the settlement of postlarvae followed by the movement, shelter selection, growth, and mortality of juvenile lobsters . The modNew lobsters entered the model every 28 days in the 10 yr simulation, which mimics the natural, pulsed lunar arrival of postlarvae to the coastal environment. Monthly variation in the number of postlarvae arriving in the model was based on a 10 yr empirical time-series collected and provided by the Florida Fish and Wildlife Conservation Commission. The number of new postlarval recruits entering the model each month was obtained by scaling the monthly deviation from the ten-year average to an average of 114,500 new individuals/mo in the model. The postlarvae entering the model domain each month were distributed uniformly among cells of similar habitat without regard to their geographic location. Based on experiments involving habitat preferences at settlement , we distInitial sizes  were randomly assigned to individual early benthic stage lobsters in the model based on a truncated normal distribution with a mean of 6.3 mm CL and a standard deviation of 0.3 based on empirical data . Growth Shelter selection depended on lobster body size, lobster density, and the habitat cell type . The type of shelter utilized affected lobster mortality and the movement of lobsters among cells, which was represented in other subroutines. Shelter selection rules were represented separately for algal-stage juveniles (< 12 mm CL) and postalgal-stage juveniles (25 to 50 mm CL). Transitional juveniles (13 to 25 mm CL) sheltered both in macroalgae, as did algal-stage juveniles, and in crevice shelters, as did postalgal juveniles. Shelter selection rules were formulated based on field data and laboratory investigations of habitat choice –36. All The daily probability of mortality for a lobster was computed as a weighted sum of day, night, and twilight mortality probabilities with each dependent on lobster size and shelter use. Daytime was considered to be 12 hrs, twilight was 2 hrs, and nighttime was 10 hrs. We determined the probability of mortality for each of the daytime, twilight, and nighttime periods based on the shelter inhabited by the lobster for each of the periods, and then computed a total daily probability by weighting the three probabilities by the hours of each period. Mortality probabilities were determined through least-squares fitting of equations to size-specific and shelter-specific mortality data derived from field tethering and mark-recapture studies –39.2 field sites within hard-bottom habitat [The probability of movement of lobsters among habitat cells was a function of lobster size and the availability of shelter in their habitat cell. The functions that described the size-specific probability of movement for lobsters in different types of shelter are based on mark-recapture records for over 500 individual lobsters in two dozen 2500 m habitat . Small j habitat . Thus, sDetailed descriptions, algorithms, and data sources for the PaV1 disease dynamics in the model are published elsewhere , but an We simulated sponge-die offs in the area of the model corresponding to areas of Florida Bay that experienced the cyanobacteria bloom and resultant sponge die-off in 2007. Sponge populations declined within each model cell based on the number of days of exposure to the harmful algal bloom and species-specific mortality functions. No regrowth of the sponge community was permitted during each simulation as little recovery of large sponges suitable for lobster occupancy could have occurred over that time period.We ran three simulations. In two simulations we modeled disease dynamics with and without the occurrence of sponge die-offs, and those simulations included the avoidance of PaV1 infected lobsters by uninfected lobsters. A third simulation was run with sponge die-off conditions but we “turned off” behavioral immunity in lobsters so that uninfected lobsters no longer detected and avoided infected lobsters. This third simulation provided a hypothetical baseline against which we could examine the effectiveness of behavioral immunity in constraining the spread of the PaV1 virus. Each model simulation ran for 10 yrs and 10 replicates per simulation were run, which exceeded the minimum sample size necessary to detect a 1% difference from the mean prevalence at the α = 0.05 level as estimated from a Visual Jackknife technique. Each simulation replicate tracked as many as 750,000 individual lobsters in the model at one time and generated tens of billions of unique individuals over each 10 yr simulation. The potential output of individual-based simulations like these is nearly limitless, but we focused on two metrics: (a) the abundance of juvenile lobsters in the 45–50 mm CL size range  and (b) the prevalence of PaV1 in this same group of juvenile lobsters.The “acid-test” of our simulation model predictions came from our field studies of PaV1 prevalence before and after the sponge die-off. Since 2000, we had been monitoring PaV1 infection in juvenile lobster populations at 10 nursery sites in the same region of the Florida Keys as depicted in our simulation model. This time-series of data allowed us to evaluate the prevalence of PaV1 in lobsters just before the sponge die-off (May-June 2007), eight months after the sponge die-off (May-June 2008), and then annually for five years after the sponge die-off (2009–2013). During each 30-min survey, two divers collected every juvenile lobster that they encountered on each site, and each lobster was then assessed for PaV1 infection by one or more of three commonly used methods: visual inspection for clinical signs of disease , histoloThe normal nightly reshuffling of lobsters among dens, along with emigration from dens that might be caused by diver disturbance, resulted in the departure of some lobsters from nearly all dens. However, significantly more lobsters vacated dens when a PaV1-infected lobster was added to their aggregation as compared to the addition of an uninfected lobster . Annual surveys of PaV1 prevalence at those same 10 sites for another five years following the sponge die-off confirmed that viral prevalence remained relatively stable over time  revealed that PaV1 prevalence in lobsters was unchanged eight months after the sponge die-off when lobsters were more aggregated in dens as compared to just a few months before  regardlever time , consistWe report results from a large-scale "natural experiment" in which sponges used as dens by juvenile spiny lobsters disappeared after a bloom of cyanobacteria decimated the sponge community across a large portion of the central Florida Keys. In turn, the loss of sponge shelters across the region forced lobsters into unusually large aggregations within the remaining dens, setting in place conditions ripe for the spread of the pathogenic PaV1 virus over the Florida Keys seascape. However, field surveys of PaV1 prevalence in lobsters immediately after the sponge die-off and over the next six years, both within and outside the affected region, showed that PaV1 prevalence instead remained unchanged in the host population. Experimental manipulation of the presence of infected and uninfected lobsters within naturally occurring lobster aggregations demonstrated that even when shelters were scarce, healthy lobsters still vacated dens into which a diseased lobster was introduced, thus preferring an increased risk of predation to an increased risk of infection. Modeling results from an individual-based, spatially-explicit simulation of lobster-PaV1 disease dynamics corroborated the results and mechanisms deduced from our field studies. Specifically, behavioral immunity conferred by the avoidance of infected lobsters by healthy conspecifics appears to have suppressed what otherwise would have been a viral epizootic caused by increased lobster aggregation and viral transmission following the sponge die-off.PaV1, perhaps in conjunction with similarly influential but unknown pathogens, has been a powerful selective force driving the evolution of flexible social behavior in Caribbean spiny lobsters, including those that confer behavioral immunity against contagious pathogens. The aversion that lobsters display toward infected conspecifics is so strong that they choose to risk predation and search for another den, rather than cohabit a den with an infected conspecific. Indeed, we have previously shown that lobsters will even select a den with the scent of an octopus—a formidable predator whose odor they normally avoid—rather than a shelter with the smell of a diseased lobster ,46. The Predatory culling of infected lobsters might minimize the spread of PaV1 among lobsters, and thus contribute to population resilience. We showed in an earlier study  that PaVDisentangling the relative importance of multiple, often interacting factors that contribute to a population's resistance to pathogens is a daunting task, but models that explicitly incorporate complex host-pathogen spatial dynamics offer useful platforms for evaluating alternative, mechanistic hypotheses. The modeling of marine pathogen-host dynamics is still in its infancy, and there is concern that traditional, terrestrial-based epidemiological models may not apply in the sea where host and pathogen life history and modes of pathogen dispersal are fundamentally different , 56. MorWe do not know how common behavioral immunity is in nature, but when present , 57 it f


Endothelial dysfunction may play a crucial role in initiation of the pathogenesis of vascular disease and atherosclerosis. The identification and quantification of circulating endothelial cells (CEC) have been developed as a novel marker of endothelial function. We describe, in great detail, mechanisms of endothelial dysfunction and CEC detachment. We also review the relationship between numbers of CEC and disease severity and response to treatment. In addition, we describe the possible clinical use of CEC in chronic kidney disease (CKD) and kidney transplantation. In summary, CEC have been developed as a novel approach to assess the endothelial damage. Measurement of the CEC level would provide an important diagnostic and prognostic value on the endothelium status and the long-term outcome of vascular dysfunction. Atherosclerosis is well known to be advanced in patients with chronic kidney disease (CKD). Cardiovascular disease (CVD) is one of the major causes of morbidity and mortality in this population, 10 to 20 times higher than in general population .Recent evidence demonstrated that endothelial dysfunction may play a crucial role in initiation of atherosclerosis  and is, Endothelial damage can be assessed in many ways –11. CircIn this review we cover the possible contents of (1) introduction, (2) endothelial dysfunction in association with chronic kidney disease, (3) circulating endothelial cells, (4) measurement of circulating endothelial cells, (5) circulating endothelial cells—as a biomarker of endothelial dysfunction, (6) potential value of circulating endothelial cells in chronic kidney disease, (7) circulating endothelial cells in transplantation, and (8) conclusions.13 endothelial cells [The endothelium, the largest organ in the body, comprises more than 10al cells  and servEndothelial dysfunction is an early event in arteriosclerosis and is observed even as early as in stage 1 CKD patients . IncreasAmong the insults to the vessel wall in CKD patients, uremia is regarded to be an elicitor of endothelial dysfunction. In uremic environment, there is an impairment of vascular endothelial nitric oxide synthase activity, and an increase in endothelial adhesion molecules such as von Willebrand factor, thrombomodulin, and circulating endothelial microparticles, which are stimulated by different uremic toxins –25. EvidVascular calcification is common in patients with chronic kidney disease as a result of inflammation, uraemia, and mineral metabolic disturbance. In disorders with high activation of bone morphogenetic protein, the endothelium is a source of osteoprogenitor cells in vascular calcification . At the Circulating endothelial cells, a subpopulation of endothelial cells, are mature endothelial cells thought to originate from blood vessel walls and are released into the circulation in response to endothelial damage , 31. LikBouvier and Hladovec were the first to describe the presence of CEC of possible endothelial origin in blood , 33. Theα, macrophage inflammatory protein-1α, and IL-8 and induced elevated neutrophil migration. They concluded that these cells might be an additional inflammatory cell type that may play an important role in the pathogenesis of GPA [Increased numbers of CEC detached from vessel wall due to a variety of factors related to endothelial damage, such as oxidative stress, infectious agents, cytokines, proteases , antiends of GPA .The turnover time of the normal tissue endothelium is estimated to be 47 to 23000 days , and endCirculating endothelial cells are identified by the presence of endothelial cell specific/selective antigens . A numbeTo date, standardization of methods for quantification of CEC has not been well established. Improved detection of these rare cells has been achieved by combining cell enrichment techniques with labeling of CEC with certain selective surface antigens. Most studies isolate CEC either by immunomagnetic separation or by flow cytometry from blood samples , 51–54. Endothelial dysfunction can be assessed in several ways, for example, by physiological techniques as flow mediated dilatation  and by tMore recently, emerging evidence suggest that CEC have been developed as a sensitive and specific marker for assessing endothelial damage in both renal , 61 and In certain disease conditions, CEC detached from affected vascular wall could provide useful information for studying vascular injury. Increased levels of CEC have been observed in several diseases with widespread vascular damage, as recently demonstrated in patients with sickle cell anemia , 64, infIncreased CEC numbers are present in various forms of kidney diseases. As a marker of endothelial damage, the measurement of CEC could provide important value in the clinical setting.In maintenance hemodialysis (MHD) patients, Koç et al. reported that the number of CEC was increased compared with healthy controls, and even higher level of prehemodialysis CEC was observed in patients with active atherosclerotic cardiovascular disease (ACVD) compared with patients with stable ACVD and no ACVD . In the In systemic autoimmune diseases, such as systemic lupus erythematosus (SLE) and ANCA-associated vasculitis, endothelial cells are targets of antibody or immune complex-mediated injury. Increased numbers of CEC also have a good relationship with the severity of these diseases. In systemic lupus erythematosus, patients with active disease expressed higher level of CEC in peripheral blood compared with patients with inactive disease or healthy subjects . Yao et Data in glomerular diseases are limited. Even in the normal aging process, it has been shown that the loss of glomerular endothelium is associated with progressive renal impairment. Futrakul et al. demonstrated that enhanced CEC level, elevated transforming growth factor beta, and depleted vascular endothelial growth factor were observed in patients with focal segmental glomerulosclerosis (FSGS), and they presumed that the increased endothelial cell loss may be due to the elevated transforming growth factor beta, which can induce apoptosis of podocyte as well as tubular epithelium . But ele2) [In CKD patients, increased CEC level was found in patients with moderately to severely impaired renal function (creatinine clearance less than 60 mL/min/1.73 m2) , even in2) . Rodrígu2) .In patients with kidney transplantation, the risk of cardiovascular disease is higher than in general population, although cardiovascular mortality is lower compared with those receiving dialysis .Recently, the level of CEC has been identified as a useful marker of endothelial damage and potential vascular rejection in renal transplant recipients. More and more studies showed that CEC numbers are elevated in patients after kidney transplantation , 61, 80.Taken together, the above accumulative evidence indicates that endothelial dysfunction contributes to the development of cardiovascular and renal disease. Circulating endothelial cells have been developed as a novel approach to assess the endothelial damage in various disorders including chronic kidney disease. Evaluating CEC directly detached from vessel wall may provide direct insights on the status of the endothelium, as well as providing mechanisms underlying endothelial dysfunction. Increased CEC level is closely related to clinical development of vascular disease in patients with chronic kidney disease. Treatment of the underlying disease may recover endothelial function and decreased the CEC levels. In particular, CEC are easily obtainable from blood samples. So, measurement of the CEC level would provide an important diagnostic and prognostic value on the endothelium status and long-term outcome of vascular dysfunction.


The angiotensin converting enzyme inhibitors (ACEIs) are a group of pharmaceuticals that are used primarily in treatment of hypertension and congestive heart failure, in some cases as the drugs of first choice. The renin-angiotensin system is activated in response to hypotension, decreased sodium concentration in the distal tubule, decreased blood volume and in renal sympathetic nerve stimulation. This study examines the effects of angiotensin converting enzyme inhibitor (Lisinopril) on blood pressure (BP) 131±2.4 and proteinuria 0.198±0.005 in Kurd hypertensive patients, mean arterial blood pressure and proteinuria excretion were measured weekly along the period of 12 weeks. Lisinopril significantly reduced mean arterial blood pressure, and attenuated proteinuria level in patients subjected to this study in lisinopril 10mg dose dependent manner . In conclusion, lisinopril is of beneficial of renoprotection and in lowering BP Elevated blood pressure and severe proteinuria are important predictions of progressive renal injury . How proThe excretion of protein in the urine (proteinuria) is generally thought to be an indicator of deteriorating kidney function. While drugs which lower high blood pressure (hypertension) may all contribute to the preservation of kidney function, experiments in diabetic rats with hypertension have shown that the drugs which function by inhibiting angiotensin-converting enzyme are more effective in reducing proteinuria than other antihypertensive drugs .Urinary excretion of albumin is a sign of mild kidney disease . There iIntervention in the renin-angiotensin system (RAS) with angiotensin-converting enzyme inhibitors (ACEIs) is the therapy of choice for proteinuric renal disease, since these drugs lowered blood pressure (BP) and proteinuria and preserve renal function in the long term . It is gThis research was performed on randomly chosen hypertensive patients , the treatment period lasted three months, during which 24 patients; 11 males and 23 females were taking lisinopril 10mg/day. Baseline measurements of BP, is taken, and at 1, 3, 5, 7, 9, and 11 weeks of treatment. Urine samples were collected from patients on day 1 prior to first dosing with antihypertensive therapy (baseline) and at 2, 4, 6, 8, 10, 12 weeks of the treatment period of time. Serum creatinine level was measured before treatment and after one week of it, the result was within the normal range: patients on (ACEÍs) or (ARBs), their serum creatinine and after one week of starting treatment should not be exceeding more than 30% of their baseline value, otherwise it may precipitate unilateral and/or bilateral artery stenosis which lead to its exclusion .BP was determined according to Riva Rocci , by two 24-hr urine samples were collected by spontaneous voiding urineprotein excretion was determined by spectrophotometer , Autoanalyzer t-test for dependent samples.Statistical Analysis was performed using the ANOVA with respect to mean arterial pressure (MAP), and proteinuria. Differences between two measurements within one group were tested by First part of the results is revealing the effect of lisinopril on MAP in patients with essential hypertension. It is evident from the The second part is the effect of lisinopril on proteinuria in patients with essential hypertension. Residual proteinuria is a strong modifiable risk factor for renal failure progression . HyperteThe remaining risk of progression of hypertension kidney disease to end-stage renal disease (ESRD) is still high despite introduction of diverse categories of new drugs . The preThe role of blockade of the rennin-angiotensin system has been explored in many clinical contexts with positive results . A numbeBy contrast, positive effects were observed in a trial of four weeks duration in normotensive subjects with nephropathy .We evaluated the antihypertensive and renoprotective effects as reflected by short-term changes in blood pressure and in proteinuria. Across past two decades ACEIs expressed powerful effects in lowering blood pressure by inhibiting production of angiotensin II and impeding breakdown of bradykinin to which the cough is attributed after administration of lisinopril .The results in this study show that inhibition of the rennin-angiotensin system is particularly effective in lowering mean arterial pressure to about normal values 97±0.9 in Kurdish patients after one week of the treatment. This work provides evidence of an important role for lisinopril therapy in Kurd patients with hypertension as this remedy is effective at reducing mean arterial blood pressure and has beneficial effect on proteinuria in Kurd patients after 8, 10, and 12 weeks of medication, a result which is parallel to that documented by many other researchers . This st


Gene therapy is a promising approach for treating a spectrum of neurological and neurodegenerative disorders by delivering corrective genes to the central nervous system (CNS). In particular, adeno-associated viruses (AAVs) have emerged as promising tools for clinical gene transfer in a broad range of genetic disorders with neurological manifestations. In the current review, we have attempted to bridge our understanding of the biology of different AAV strains with their transduction profiles, cellular tropisms, and transport mechanisms within the CNS. Continued efforts to dissect AAV-host interactions within the brain are likely to aid in the development of improved vectors for CNS-directed gene transfer applications in the clinic. Numerous congenital disorders exhibit distinct manifestations in the central nervous system (CNS). Loss of functionality in affected cell types within the brain can often be attributed to defects in single genes. For instance, a range of neurological disorders arise from the inability of cells in the CNS to break down metabolic end products . One such example of LSDs with fatal manifestations includes globoid-cell leukodystrophy (GLD) or Krabbe disease in which mutations in galactosylceramidase leads to accumulation of the toxin “psychosine” in the CNS . This diAnother broad category is neurodegenerative disorders, where functionally distinct neuronal subpopulations are lost due to genetic predispositions or environmental toxins. Commonly known examples of this category include dopaminergic (DA) neuronal loss in Parkinson’s disease and GABAergic neuronal loss in Huntington’s disease . A commocis-acting molecular signature for successful packaging of a transgene within an AAV capsid  on the 5′ and 3′ ends . The wilV capsid . These sV capsid . It is nin vivo tropism and receptor interactions based on their capsid structures  ratructures ; and (c)ructures . We discN-linked sialic acid (SA), whereas AAV4 is the only natural AAV isolate that binds O-linked SA moieties on mammalian cell surfaces . Traditionally, these injections have been performed by exposing the subarachnoid space at the suboccipital cisterna magna region or the intravertebral space at lumbar region. In general, applications requiring enhanced transduction at the motor, sensory and nociceptive neuronal subpopulations  utilize lumbar punctures. AAV serotypes 1, 5, 8, and 9 have shown extensive transduction in the spinal cord and DRG neurons from IT injections at the intravertebral lumbar region . In an iN-linked SA binding AAV1 and AAV5 are superior to AAV2 in terms of spread of transduction from a single parenchymal microinjection into the hippocampus, substantia nigra, globus pallidus, striatum, and spinal cord. Widespread transgene expression was also achieved by parenchymal injections of AAV7, 8 and 9 in rodents  injections adds value to clinical administration of AAV vectors via the bloodstream. Two major roadblocks currently impede our ability to utilize this technique for therapeutic gene transfer of the CNS. The first major concern is the broad biodistribution of AAV vector particles into off-target tissues such as the liver, spleen and kidneys during IV administration of AAVs. For instance, IV injections of AAV9 achieves exceptional transduction of neurons and glia in rodents and NHPs, but also leads to enrichment of viral genomes (∼10 fold or more) in the liver and spleen as compared to the brain . CarefulSambucus nigra lectin that enhanced SA depositions in the MPS VII affected mouse CNS might be detrimental for AAV9-mediated CNS transduction . Successful, but differential levels of CNS transduction were reported from all tested vectors . Only a few of these trials are aimed at treating diseases with CNS manifestations. In the current review, we have attempted to provide an overview of various parameters that might play a role in determining the success of AAV mediated therapeutic gene transfer to the CNS. Interactions of AAV vectors with different primary receptors, directional transport and cellular tropisms following different routes of administration are summarized in 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.


Reprogramming of somatic cells to pluripotency by transient expression of transcription factors was achieved a few years ago. Induced pluripotent stem cells (iPSC) from both healthy individuals and patients suffering from debilitating, life-threatening neurological diseases have been differentiated into several specific neuronal subtypes. An alternative emerging approach is the direct conversion of somatic cells  into neuron-like cells. However, to what extent neuronal direct conversion of diseased somatic cells can be achieved remains an open question. Optimization of current expansion and differentiation approaches is highly demanded to increase the differentiation efficiency of specific phenotypes of functional neurons from iPSCs or through somatic cell direct conversion. The realization of the full potential of iPSCs relies on the ability to precisely modify specific genome sequences. Genome editing technologies including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat/CAS9 RNA-guided nucleases have progressed very fast over the last years. The combination of genome-editing strategies and patient-specific iPSC biology will offer a unique platform for in vitro generation of diseased and corrected neural derivatives for personalized therapies, disease modeling and drug screening. S Pluripotency is defined by the ability to produce from a single undifferentiated cell type, cell derivatives representing the three germ layers: meso-, endo-, and ectoderm. Pluripotent stem cells (PSCs) of human origin were first isolated from teratocarcinomas, giving rise to embryonal carcinoma cell lines Successful reprogramming of somatic cells to a pluripotent state by transient expression of four transcription factors  was achieved for the first time with mouse cells in 2006 However, the generation of iPSCs is still inefficient, yielding a low proportion of reprogrammed cells Several protocols for neuronal differentiation of human PSCs, including iPSCs, have been reported. Neural commitment of PSCs can be achieved through formation of embryoid bodies and treatment with retinoic acid, a neuroectoderm inducer, or by pharmacological inhibition of transforming growth factor-β and bone morphogenic protein pathways . The resulting neural stem/progenitor cells can be expanded either attached to a substrate or as floating neurospheres to be further exposed to growth factors inducing specific neuronal subtypes  Induced PSCs have been generated from biopsies of patients harboring mutations associated to amyotrophic lateral sclerosis Collections of patient/disease-specific hiPSCs, including neurological conditions After the seminal reprogramming work of Yamanaka and coworkers, other investigators started testing the hypothesis that expression of transcriptional regulators might act as neural cell determinants to non-neural differentiated somatic cells. Indeed, ectopic expression of ASCL1, BRN2A, and MYT1L turned fibroblasts directly (no pluripotent stage involved) into induced neurons (iNs) ASCL1, BRN2A, and MYT1L are converted into neurons within the rodent brain after doxycycline addition. Interestingly, endogenous brain astrocytes were also converted into iNs highlighting a specie-independent direct conversion Direct conversion of non-neuronal cells into neurons is also possible in vivo. Human fibroblasts or astrocytes engineered to express doxycycline-inducible hiPSCs are widely being used in developmental biology and disease modeling. However, they are envisioned to become a unique tool for disease-specific drug screening, and possibly, a patient-specific cell replacement approach SMN1 gene which results in a nonfunctional protein causing motor neuron degeneration. As a compensatory mechanism, these patients rely on the expression of SMN2, which is highly homologous to SMN1 but has a change in a nucleotide that alters its splicing, with a consequent reduction in the amount of SMN produced. Using single-stranded DNA sequences, editing of the SMN2 was performed, resulting in a SMN1-like protein that includes the exon 7. This change was shown to be stable and correlated with increased motor neuron survival in repaired cells Huntington disease is caused by expansion of a poly-glutamine motif in HUNTINGTIN. Through homologous recombination in iPSCs, the number of glutamines in this protein was reduced, resulting in decreased apoptosis and improvement in oxygen consumption rate in corrected neural progenitors Over the last decade we have witnessed significant progress in both our understanding of cell fate determinants and also in the possibilities to use reprogramming in the preclinical setting. The generation of human neurons by these methodologies will definitively allow the analysis of the mechanisms causing familial and sporadic cases of neurological conditions. Patients therefore can benefit from in vitro differentiated neurons and specific drugs identified by high-throughput screening. If genome editing turns out to be safe and effective, we envision a future prospective production of clinically relevant neuronal types within the brain of affected people. Whether or not the synaptic communication will be re-established properly in the diseased brain is a different question, but repopulation of the affected neuronal phenotype, either by transplantation of differentiated neurons or by direct conversion of resident glial cells is a very encouraging step forward.