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The cryptic β-glucoside GFB (bglGFB) operon in Escherichia coli (E. coli) can be activated by mutations arising under starvation conditions in the presence of an aromatic β-glucoside. This may involve the insertion of an insertion sequence (IS) element into a “stress-induced DNA duplex destabilization” (SIDD) region upstream of the operon promoter, although other types of mutations can also activate the bgl operon. Here, we show that increased expression of the bglG gene, encoding a well-characterized transcriptional antiterminator, dramatically increases the frequency of both IS-mediated and IS-independent Bgl+ mutations occurring on salicin- and arbutin-containing agar plates. Both mutation rates increased with increasing levels of bglG expression but IS-mediated mutations were more prevalent at lower BglG levels. Mutations depended on the presence of both BglG and an aromatic β-glucoside, and bglG expression did not influence IS insertion in other IS-activated operons tested. The N-terminal mRNA-binding domain of BglG was essential for mutational activation, and alteration of BglG’s binding site in the mRNA nearly abolished Bgl+ mutant appearances. Increased bglG expression promoted residual bgl operon expression in parallel with the increases in mutation rates. Possible mechanisms are proposed explaining how BglG enhances the frequencies of bgl operon activating mutations.

Tracking the evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through genomic surveillance programs is undoubtedly one of the key priorities in the current pandemic situation. Although the genome of SARS-CoV-2 acquires mutations at a slower rate compared with other RNA viruses, evolutionary pressures derived from the widespread circulation of SARS-CoV-2 in the human population have progressively favored the global emergence, though natural selection, of several variants of concern that carry multiple non-synonymous mutations in the spike glycoprotein. These are often placed in key sites within major antibody epitopes and may therefore confer resistance to neutralizing antibodies, leading to partial immune escape, or otherwise compensate infectivity deficits associated with other non-synonymous substitutions. As previously shown by other authors, several emerging variants carry recurrent deletion regions (RDRs) that display a partial overlap with antibody epitopes located in the spike N-terminal domain (NTD). Comparatively, very little attention has been directed towards spike insertion mutations prior to the emergence of the B.1.1.529 (omicron) lineage. This manuscript describes a single recurrent insertion region (RIR1) in the N-terminal domain of SARS-CoV-2 spike protein, characterized by at least 41 independent acquisitions of 1-8 additional codons between Val213 and Leu216 in different viral lineages. Even though RIR1 is unlikely to confer antibody escape, its association with two distinct formerly widespread lineages (A.2.5 and B.1.214.2), with the quickly spreading omicron and with other VOCs and VOIs warrants further investigation concerning its effects on spike structure and viral infectivity.

Optimizing microbial hosts for the large-scale production of valuable metabolites often requires multiple mutations and modifications to the host’s genome. We describe a three-round screen for increased L-DOPA production in S. cerevisiae using FACS enrichment of an enzyme-coupled biosensor for L-DOPA. Multiple rounds of screening were enabled by a single build of a barcoded in vitro transposon-mediated disruption library. New background strains for screening were built for each iteration using results from previous iterations. The same in vitro transposon-mediated disruption library was integrated by homologous recombination into new background strains in each round of screening. Compared with creating new transposon insertions in each round, this method takes less time and saves the cost of additional sequencing to characterize transposon insertion sites. In the first two rounds of screening, we identified deletions that improved biosensor compartmentalization and, consequently, improved our ability to screen for L-DOPA production. In a final round, we discovered that deletion of heme oxygenase (HMX1) increases total heme concentration and increases L-DOPA production, using dopamine measurement as a proxy. We further demonstrated that deleting HMX1 may represent a general strategy for P450 function improvement by improving activity of a second P450 enzyme, BM3, which performs a distinct reaction.

The lack of predictive preclinical models is a fundamental barrier to translating knowledge about the molecular pathogenesis of cancer into improved therapies. Insertional mutagenesis (IM) in mice is a robust strategy for generating malignancies that recapitulate the extensive inter- and intra-tumoral genetic heterogeneity found in advanced human cancers. While the central role of "driver" viral insertions in IM models that aberrantly increase the expression of proto-oncogenes or disrupt tumor suppressors has been appreciated for many years, the contributions of cooperating somatic mutations and large chromosomal alterations to tumorigenesis are largely unknown. Integrated genomic studies of T lineage acute lymphoblastic leukemias (T-ALLs) generated by IM in wild-type (WT) and Kras mutant mice reveal frequent point mutations and other recurrent non-insertional genetic alterations that also occur in human T-ALL. These somatic mutations are sensitive and specific markers for defining clonal dynamics and identifying candidate resistance mechanisms in leukemias that relapse after an initial therapeutic response. Primary cancers initiated by IM and resistant clones that emerge during in vivo treatment close key gaps in existing preclinical models, and are robust platforms for investigating the efficacy of new therapies and for elucidating how drug exposure shapes tumor evolution and patterns of resistance., Author summary A lack of predictive cancer models is a major bottleneck for prioritizing new anti-cancer drugs for clinical trials. We comprehensively profiled a panel of primary mouse T lineage leukemias initiated by insertional mutagenesis and found remarkable similarities with human T-ALL in regard to overall mutational burden, the occurrence of specific somatic mutations and large chromosomal alterations, and concordant gene expression signatures. We observed frequent duplication of the Kras oncogene with loss of the normal allele, which has potential therapeutic implications that merit further investigation in human leukemia and in other preclinical models. Mutations identified in mouse leukemias that relapsed after in vivo treatment with signal transduction inhibitors were also observed in relapsed human T-ALL, indicating that this model system can be utilized to investigate strategies for overcoming intrinsic and acquired drug resistance. Finally, preclinical models similar to the one described here that are characterized by a normal endogenous tumor microenvironment and intact immune system will become increasingly important for testing immunotherapy approaches for human cancer.

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelityin vitrofor the prototypicalBordetellabacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observedin vivo. We identified RNAcDNA molecules to be branched and most plausibly linked through 2′-5′ phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream ofTR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsX and ΔenvC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsX and ΔenvC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated., IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsX and ΔenvC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen.

Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-RasG60_A66dup and K-RasE62_A66dup proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications., Amino acid substitutions in K-Ras that constitutively activate the protein are common in cancer. Here, the authors describe mutations in the K-Ras Switch 2 domain and show that the mutant proteins accumulate in the active conformation, exhibit defective binding to PI3 kinase, and are hypersensitive to MEK inhibitors.

The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to the inherent ability of Actinomyces to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about trans-acting factors contributing to these processes. We report here a large-scale Tn5 transposon screen for mutants defective in Actinomyces oris coaggregation with Streptococcus oralis. We obtained 33 independent clones, 13 of which completely failed to aggregate with S. oralis, and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn5 insertions in fimA, cafA, or srtC2, as expected; the latter were mapped to genes coding for uncharacterized proteins and various nuo genes encoding the NADH dehydrogenase subunits. Electron microscopy and biochemical analyses of mutants with nonpolar deletions of nuo genes and ubiE, a menaquinone C-methyltransferase-encoding gene downstream of the nuo locus, confirmed the pilus and coaggregation defects. Both nuoA and ubiE mutants were defective in oxidation of MdbA, the major oxidoreductase required for oxidative folding of pilus proteins. Furthermore, supplementation of the ubiE mutant with exogenous menaquinone-4 rescued the cell growth and pilus defects. Altogether, we propose that the A. oris electron transport chain is biochemically linked to pilus assembly via oxidative protein folding., IMPORTANCE The Gram-positive actinobacterium A. oris expresses adhesive pili, or fimbriae, that are essential to biofilm formation and Actinomyces interactions with other bacteria, termed coaggregation. While the critical role of the conserved sortase machine in pilus assembly and the disulfide bond-forming catalyst MdbA in oxidative folding of pilins has been established, little is known about other trans-acting factors involved in these processes. Using a Tn5 transposon screen for mutants defective in coaggregation with Streptococcus oralis, we found that genetic disruption of the NADH dehydrogenase and menaquinone biosynthesis detrimentally alters pilus assembly. Further biochemical characterizations determined that menaquinone is important for reactivation of MdbA. This study supports the notion that the electron transport chain is biochemically linked to pilus assembly in A. oris via oxidative folding of pilin precursors.

Background There is no consensus regarding the treatment of Achilles insertional tendinopathies. Eccentric training remains the main choice in the conservative treatment of this illness however, the good results in the management of non-insertional Achilles tendinopathy were not replicated in the insertional condition. Low energy shock wave therapy has been described as an alternative to these patients, but has yet to be empirically tested. Hypothesis Shock wave therapy, adjunctive to the eccentric strengthening protocol, will improve measures of pain and function. Design Double blind, placebo-controlled, parallel groups, randomised clinical trial. Materials and methods 93 patients with a diagnosis of chronic insertional tendinopathy, referred from primary or secondary healthcare services, will be assessed and enrolled in this study. They will be divided into two groups (randomised by sequentially numbered identical envelopes, which will be administered serially to participants), one containing the combination of low energy shock wave and eccentric exercises, as treatment and the other comprehending the exercises and the placebo treatment (an apparatus placed in the therapeutic head). The assessments will occur in 2, 4, 6, 12 and 24weeks. Patients will be evaluated primarily by the Victorian Institute of Sport Assessment-Achilles questionnaire and secondarily by the visual analogue scale, Algometry, the American Orthopedic Foot and Ankle Society scale, the Foot and Ankle Outcome Score and the 12-item Short Form Health Survey. We will use comparison of two proportions via relative frequency analysis, the Pearson Correlation the (2) test and the analysis of variance for statistical analyses. Discussion This study intends to demonstrate if the association of the eccentric exercise programme with the shock wave therapy can produce good results regarding the treatment of the Achilles insertional tendinopathy. In an attempt to prevent the high costs and complications associated with the surgical intervention, we will try to prove this combination as a viable therapeutic option in the conservative management of this prevalent condition. The strengths of the study are the design and the novelty of the combination of methods. The main limitation is the short follow-up course. Ethics and dissemination The study is registered in the Clinical Trials database (protocol number: 8094833648737701) and was approved by the University Ethics Committee (number: 1373481). Trial registration number 8094833648737701 (NCT02757664) Pre-results. Univ Fed Sao Paulo, Dept Orthopaed, Sao Paulo, SP, Brazil Fed Univ Sao Paulo UNIFESP EPM, Orthoped & Traumatol Div Hand Surg & Upper Limb, Sao Paulo, SP, Brazil Department of Orthopaedics, Universidade Federal de São Paulo (UNIFESP), Sao Paulo, SP, Brazil Orthopedics and Traumatology—Division of Hand Surgery and Upper Limb, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil Web of Science

In many taxa, sex chromosomes are heteromorphic and largely non-recombining. Evolutionary models predict that spread of recombination suppression on the Y chromosome is fueled by the accumulation of sexually antagonistic alleles in close linkage to the sex determination region. However, empirical evidence for the existence of sexually antagonistic alleles is scarce. In the mosquito Aedes aegypti, the sex-determining chromosomes are homomorphic. The region of suppressed recombination, which surrounds the male-specific sex-determining gene, remains very small, despite ancient origin of the sex chromosomes in the Aedes lineage. We conducted a genetic analysis of the A. aegypti chromosome region tightly linked to the sex locus. We used a strain with an enhanced green fluorescent protein (EGFP)-tagged transgene inserted near the male-determining gene to monitor crossing-over events close to the boundary of the sex-determining region (SDR), and to trace the inheritance pattern of the transgene in relation to sex. In a series of crossing experiments involving individuals with a recombinant sex chromosome we found developmental abnormalities leading to 1:2 sex biases, caused by lethality of half of the male or female progeny. Our results suggest that various factors causing sex-specific lethal effects are clustered within the neighborhood of the SDR, which in the affected sex are likely lost or gained through recombination, leading to death. These may include genes that are recessive lethal, vital for development and/or sexually antagonistic. The sex chromosome fragment in question represents a fascinating test case for the analysis of processes that shape stable boundaries of a non-recombining region.

Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes.

West Nile virus (WNV) is an important agent of human encephalitis that has quickly become endemic across much of the United States since its identification in North America in 1999. While the majority (∼75%) of infections are subclinical, neurologic disease can occur in a subset of cases, with outcomes including permanent neurologic damage and death. Currently, there are no WNV vaccines approved for use in humans. This study introduces a novel vaccine platform for WNV to reduce viral replication in the central nervous system while maintaining peripheral replication to elicit strong neutralizing antibody titers. Vaccine candidates were engineered to incorporate microRNA (miRNA) target sequences for a cognate miRNA expressed only in neurons, allowing the host miRNAs to target viral transcription through endogenous RNA silencing. To maintain stability, these targets were incorporated in multiple locations within the 3'-untranslated region, flanking sequences essential for viral replication without affecting the viral open reading frame. All candidates replicated comparably to wild type WNV in vitro within cells that did not express the cognate miRNA. Insertional control viruses were also capable of neuroinvasion and neurovirulence in vivo in CD-1 mice. Vaccine viruses were safe at all doses tested and did not demonstrate mutations associated with a reversion to virulence when serially passaged in mice. All vaccine constructs were protective from lethal challenge in mice, producing 93-100% protection at the highest dose tested. Overall, this is a safe and effective attenuation strategy with broad potential application for vaccine development.

There have been major changes since the incidents of leukemia development in X-SCID patients after the treatments using retroviral gene therapy. Due to the risk of oncogenesis caused by retroviral insertional activation of host genes, most of the efforts focused on the lentiviral therapies. However, a relative clonal dominance was detected in a patient with β-thalassemia Major, two years after the subject received genetically modified hematopoietic stem cells using lentiviral vectors. This disappointing result of the recent clinical trial using lentiviral vector tells us that the current and most advanced vector systems does not have enough safety. In this review, various safety features that have been tried for the retroviral gene therapy are introduced and the possible new ways of improvements are discussed. Additional feature of chromatin insulators, co-transduction of a suicidal gene under the control of an inducible promoter, conditional expression of the transgene only in appropriate target cells, targeted transduction, cell type-specific expression, targeted local administration, splitting of the viral genome, and site specific insertion of retroviral vector are discussed here.

We describe the use of DNA transposons as tools for carrying out functional screenings in murine embryonic stem (ES) cell-derived neural stem (NS) cells. NS cells are a new type of stem cells featuring radial glial properties, that undergoes symmetric cell division for an indefinite number of passages, expanding as a monolayer. In this model, the previously unreported Sleeping Beauty transposase M3A achieves an optimal blend of clone generation efficiency and low redundancy of integrations per clone, compared to the SB100X Sleeping Beauty variant and to the piggyBac transposon. The technology described here makes it possible to randomly trap genes in the NS cell genome and modify their expression or tag them with fluorescent markers and selectable genes, allowing recombinant cells to be isolated and expanded clonally. This approach will facilitate the identification of novel determinants of stem cell biology and neural cell fate specification in NS cells.

© 2015 Schmidt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. CYP4B1 belongs to the cytochrome P450 family 4, one of the oldest P450 families whose members have been highly conserved throughout evolution. The CYP4 monooxygenases typically oxidize fatty acids to both inactive and active lipid mediators, although the endogenous ligand(s) is largely unknown. During evolution, at the transition of great apes to humanoids, the CYP4B1 protein acquired a serine instead of a proline at the canonical position 427 in the meander region. Although this alteration impairs P450 function related to the processing of naturally occurring lung toxins, a study in transgenic mice suggested that an additional serine insertion at position 207 in human CYP4B1 can rescue the enzyme stability and activity. Here, we report that the genomic insertion of a CAG triplet at the intron 5-exon 6 boundary in human CYP4B1 introduced an additional splice acceptor site in frame. During evolution, this change occurred presumably at the stage of Hominoidae and leads to two major isoforms of the CYP4B1 enzymes of humans and great apes, either with or without a serine 207 insertion (insSer207). We further demonstrated that the CYP4B1 enzyme with insSer207 is the dominant isoform (76%) in humans. Importantly, this amino acid insertion did not affect the 4-ipomeanol metabolizing activities or stabilities of the native rabbit or human CYP4B1 enzymes, when introduced as transgenes in human primary cells and cell lines. In our 3D modeling, this functional neutrality of insSer207 is compatible with its predicted location on the exterior surface of CYP4B1 in a flexible side chain. Therefore, the Ser207 insertion does not rescue the P450 functional activity of human CYP4B1 that has been lost during evolution.

© 2015 S. Karger AG, Basel. The phosphoenolpyruvate:sugar phosphotransferase system (PTS) has been shown to control transport, cell metabolism and gene expression. We here present results supporting the novel suggestion that in certain instances it also regulates the mutation rate. Directed mutations are defined as mutations that occur at higher frequencies when beneficial than when neutral or detrimental. To date, the occurrence of directed point mutations has not been documented and confirmed, but a few examples of transposon-mediated directed mutations have been reported. Here we focus on the first and best-studied example of directed mutation, which involves the regulation of insertion sequence-5 hopping into a specific site upstream of the glpFK glycerol utilization operon in Escherichia coli. This insertional event specifically activates expression of the glpFK operon, allowing the growth of wild-type cells with glycerol as a carbon source in the presence of nonmetabolizable glucose analogues which normally block glycerol utilization. The sugar-transporting PTS controls this process by regulating levels of cytoplasmic glycerol-3-phosphate and cyclic (c)AMP as established in previous publications. Direct involvement of the glycerol repressor, GlpR, and the cAMP receptor protein, Crp, in the regulation of transposon-mediated directed mutation has been demonstrated.

Disruption of the SC3 gene in the basidiomycete Schizophyllum commune affected not only formation of aerial hyphae but also attachment to hydrophobic surfaces. However, these processes were not completely abolished, indicating involvement of other molecules. We here show that the SC15 protein mediates formation of aerial hyphae and attachment in the absence of SC3. SC15 is a secreted protein of 191 aa with a hydrophilic N-terminal half and a highly hydrophobic C-terminal half. It is not a hydrophobin as it lacks the eight conserved cysteine residues found in these proteins. Besides being secreted into the medium, SC15 was localized in the cell wall and the mucilage that binds aerial hyphae together. In a strain in which the SC15 gene was deleted (DeltaSC15) formation of aerial hyphae and attachment were not affected. However, these processes were almost completely abolished when the SC15 gene was deleted in the DeltaSC3 background. The absence of aerial hyphae in the DeltaSC3DeltaSC15 strain can be explained by the inability of the strain to lower the water surface tension and to make aerial hyphae hydrophobic.

IncL/M-type plasmids R446b and R471a were originally isolated from Morganella morganii (formerly Proteus morganii) as the first members of the IncL/M group of multidrug resistance (MDR) plasmids ([1][1], [2][2]). IncL/M plasmids are now commonly identified among environmental and clinical isolates

Familial subvalvular aortic stenosis (SAS) is one of the most common congenital heart defects in dogs and is an inherited defect of Newfoundlands, golden retrievers and human children. Although SAS is known to be inherited, specific genes involved in Newfoundlands with SAS have not been defined. We hypothesized that SAS in Newfoundlands is inherited in an autosomal dominant pattern and caused by a single genetic variant. We studied 93 prospectively recruited Newfoundland dogs, and 180 control dogs of 30 breeds. By providing cardiac screening evaluations for Newfoundlands we conducted a pedigree evaluation, genome-wide association study and RNA sequence analysis to identify a proposed pattern of inheritance and genetic loci associated with the development of SAS. We identified a three-nucleotide exonic insertion in phosphatidylinositol-binding clathrin assembly protein (PICALM) that is associated with the development of SAS in Newfoundlands. Pedigree evaluation best supported an autosomal dominant pattern of inheritance and provided evidence that equivocally affected individuals may pass on SAS in their progeny. Immunohistochemistry demonstrated the presence of PICALM in the canine myocardium and area of the subvalvular ridge. Additionally, small molecule inhibition of clathrin-mediated endocytosis resulted in developmental abnormalities within the outflow tract (OFT) of Xenopus laevis embryos. The ability to test for presence of this PICALM insertion may impact dog-breeding decisions and facilitate reduction of SAS disease prevalence in Newfoundland dogs. Understanding the role of PICALM in OFT development may aid in future molecular and genetic investigations into other congenital heart defects of various species. © 2014 The Author(s).

The National Institute of Health and Medical Research (INSERM, France) The Ministry of Research (France) The University Paris Diderot, Sorbonne Paris Cite (France) The National Institute of Blood Transfusion (INTS, France) The Laboratories of Excellence, GR-Ex (France); International audience; Inactivating mutations of KISS-1 receptor (KISS1R) have been recently described as a rare cause of isolated hypogonadotropic hypogonadism transmitted as a recessive trait. Few mutations have been described, and the structure-function relationship of KISS1R remains poorly understood. Here, we have taken advantage of the discovery of a novel mutation of KISS1R to characterize the structure and function of an uncommon protein motif composed of 3 proline-arginine-arginine (PRR) repeats located within the intracellular domain. A heterozygous insertion of 1 PRR repeat in-frame with 3 PRR repeats leading to synthesis of a receptor bearing 4 PRR repeats (PRR-KISS1R) was found in the index case. Functional analysis of PRR-KISS1R showed a decrease of the maximal response to kisspeptin stimulation, associated to a lower cell surface expression without modification of total expression. PRR-KISS1R exerts a dominant negative effect on the synthesis of the wild-type (WT)-KISS1R. This effect was due to the nature of inserted residues but also to the difference of the length of the intracellular domain between PRR-KISS1R and WT-KISS1R. A molecular dynamic analysis showed that the additional PRR constrained this arginine-rich region into a polyproline type II helix. Altogether, this study shows that a heterozygous insertion in KISS1R may lead to hypogonadotropic hypogonadism by a dominant negative effect on the WT receptor. An additional PRR repeat into a proline-arginine-rich motif can dramatically changed the conformation of the intracellular domain of KISS1R and its probable interaction with partner proteins.

Although carbon dioxide (CO 2 ) is known to be essential for Streptococcus pneumoniae growth, it is poorly understood how this respiratory tract pathogen adapts to the large changes in environmental CO 2 levels it encounters during transmission, host colonization, and disease. To identify the molecular mechanisms that facilitate pneumococcal growth under CO 2 -poor conditions, we generated a random S. pneumoniae R6 mariner transposon mutant library representing mutations in 1,538 different genes and exposed it to CO 2 -poor ambient air. With Tn-seq, we found mutations in two genes that were involved in S. pneumoniae adaptation to changes in CO 2 availability. The gene pca , encoding pneumococcal carbonic anhydrase (PCA), was absolutely essential for S. pneumoniae growth under CO 2 -poor conditions. PCA catalyzes the reversible hydration of endogenous CO 2 to bicarbonate (HCO 3 − ) and was previously demonstrated to facilitate HCO 3 − -dependent fatty acid biosynthesis. The gene folC that encodes the dihydrofolate/folylpolyglutamate synthase was required at the initial phase of bacterial growth under CO 2 -poor culture conditions. FolC compensated for the growth-phase-dependent decrease in S. pneumoniae intracellular long-chain ( n > 3) polyglutamyl folate levels, which was most pronounced under CO 2 -poor growth conditions. In conclusion, S. pneumoniae adaptation to changes in CO 2 availability involves the retention of endogenous CO 2 and the preservation of intracellular long-chain polyglutamyl folate pools.

International audience; Gene transfer vectors derived from oncoretroviruses or lentiviruses are the most robust and reliable tools to stably integrate therapeutic transgenes in human cells for clinical applications. Integration of these vectors in the genome may, however, have undesired effects caused by insertional deregulation of gene expression at the transcriptional or post-transcriptional level. The occurrence of severe adverse events in several clinical trials involving the transplantation of stem cells genetically corrected with retroviral vectors showed that insertional mutagenesis is not just a theoretical event, and that retroviral transgenesis is associated with a finite risk of genotoxicity. In addressing these issues, the gene therapy community offered a spectacular example of how scientific knowledge and technology can be put to work to understand the causes of unpredicted side effects, design new vectors, and develop tools and models to predict their safety and efficacy. As an added benefit, these efforts brought new basic knowledge on virus-host interactions and on the biology and dynamics of human somatic stem cells. This review summarizes the current knowledge on the interactions between retroviruses and the human genome and addresses the impact of target site selection on the safety of retroviral vector-mediated gene therapy.

Medulloblastoma, the most common malignant paediatric brain tumour, arises in the cerebellum and disseminates through the cerebrospinal fluid in the leptomeningeal space to coat the brain and spinal cord(1). Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and inmost children at the time of recurrence. Affected children therefore are treated with radiation to the entire developing brain and spinal cord, followed by high-dose chemotherapy, with the ensuing deleterious effects on the developing nervous system(2). The mechanisms of dissemination through the cerebrospinal fluid are poorly studied, and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour(3,4). Here we show that in both mouse and human medulloblastoma, the metastases from an individual are extremely similar to each other but are divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted subclone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could be a major barrier to the development of effective targeted therapies

Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompanied by regional atrophy and pronounced neuron loss in the brain, is caused by mutations in the CLN6 gene. CLN6 is a non-glycosylated endoplasmic reticulum (ER)-resident membrane protein of unknown function. To investigate mechanisms contributing to neurodegeneration in CLN6 disease we examined the nclf mouse, a naturally occurring model of the human CLN6 disease. Prominent autofluorescent and electron-dense lysosomal storage material was found in cerebellar Purkinje cells, thalamus, hippocampus, olfactory bulb and in cortical layer II to V. Another prominent early feature of nclf pathogenesis was the localized astrocytosis that was evident in many brain regions and the more widespread microgliosis. Expression analysis of mutant Cln6 found in nclf mice demonstrated synthesis of a truncated protein with a reduced half-life. Whereas the rapid degradation of the mutant Cln6 protein can be inhibited by proteasomal inhibitors, there was no evidence for ER stress or activation of the unfolded protein response in various brain areas during postnatal development. Age-dependent increases in LC3-II, ubiquitinated proteins, and neuronal p62-positive aggregates were observed, indicating a disruption of the autophagy-lysosome degradation pathway of proteins in brains of nclf mice, most likely due to defective fusion between autophagosomes and lysosomes. These data suggest that proteasomal degradation of mutant Cln6 is sufficient to prevent the accumulation of misfolded Cln6 protein, whereas lysosomal dysfunction impairs constitutive autophagy promoting neurodegeneration.

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however, methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate. © AlphaMed Press.

Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

Commensal and pathogenic Escherichia coli adherence to host and environmental surfaces is mediated by a variety of adhesins. Although extensively studied as a model bacterium, 34% of the genes in the E. coli K-12 genome have no known function. We hypothesized that some of them may correspond to functional adhesins. We characterized E. coli K-12 ycb, ybg, yfc, yad, yra, sfm and yeh operons, which display sequence and organizational homologies to type 1 fimbriae exported by the chaperone/usher pathway. We showed that, although these operons are poorly expressed under laboratory conditions, six of them are nevertheless functional when expressed, and promote adhesion to abiotic and/or epithelial cell surfaces. While the studied fimbriae display different binding specificities, we obtained evidence of synergy/interference with other adhesins such as Ag43 or type 1 fimbriae. We showed that their expression is under the negative control of H-NS and, except for yad, subjected to cAMP receptor protein-mediated activation and carbon catabolite repression. These results therefore demonstrate that ycb, yfc, yad, yra, sfm and yeh operons encode cryptic but functional fimbriae adhesins whose expression following environmental modifications could contribute to E. coli's ability to adhere to and colonize a wide diversity of surfaces in its various ecological niches.

BACKGROUND: Inherited apolipoprotein (Apo) A-I deficiency is an orphan disorder characterized by high-density lipoprotein (HDL)-cholesterol deficiency and premature atherosclerosis. Constitutive over-expression of ApoA-I might provide a means to treat this disease. The present study provides a comprehensive evaluation of adeno-associated virus (AAV)-mediated ApoA-I gene delivery to express human (h)ApoA-I and correct the low HDL-cholesterol phenotype associated with ApoA-I deficiency.METHODS: In an effort to maximize AAV-mediated gene expression, we performed head-to-head comparisons of recombinant AAVs with pseudotype capsids 1, 2, 6 and 8 administered by different routes with the use of five different liver-specific promoters in addition to cytomegalovirus as single-stranded or as self-complementary (sc) AAV vectors.RESULTS: Intravenous administration of 1 x 10(13) gc/kg scAAV8, in combination with the liver-specific promoter LP1, in female ApoA-I(-/-) mice resulted in hApoA-I expression levels of 634 +/- 69 mg/l, which persisted for the duration of the study (15 weeks). This treatment resulted in full recovery of HDL-cholesterol levels with correction of HDL particle size and apolipoprotein composition. In addition, we observed increased adrenal cholesterol content and a significant increase in bodyweight in treated mice.CONCLUSIONS: The present study demonstrates that systemic delivery of a scAAV8 vector provides a means for efficient liver expression of hApoA-I, thereby correcting the lipid abnormalities associated with murine ApoA-I deficiency. Importantly, the study demonstrates that AAV-based gene therapy can be used to express therapeutic proteins at a high level for a prolonged period of time and, as such, provides a basis for further development of this strategy to treat hApoA-I deficiency.

International audience; The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa, and identified mutants with attenuated virulence towards Dictyostelium. These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa.

In legume nitrogen-fixing symbioses, rhizobial nod genes are obligatory for initiating infection thread formation and root nodule development. Here we show that the common nod genes, nodD1ABC, whose products synthesize core Nod factor, a chitin-like oligomer, are also required for the establishment of the three-dimensional architecture of the biofilm of Sinorhizobium meliloti. Common nod gene mutants form a biofilm that is a monolayer. Moreover, adding Nod Factor antibody to S. meliloti cells inhibits biofilm formation, while chitinase treatment disrupts pre-formed biofilms. These results attest to the involvement of core Nod factor in rhizobial biofilm establishment. However, luteolin, the plant-derived inducer of S. meliloti's nod genes, is not required for mature biofilm formation, although biofilm establishment is enhanced in the presence of this flavonoid inducer. Because biofilm formation is plant-inducer-independent and because all nodulating rhizobia, both alpha- and beta-proteobacteria have common nod genes, the role of core Nod factor in biofilm formation is likely to be an ancestral and evolutionarily conserved function of these genes.

Aliphatic glucosinolates are compounds which occur in high concentrations in Arabidopsis thaliana and other Brassicaceae species. They are important for the resistance of the plant to pest insects. Previously, the biosynthesis of these compounds was shown to be regulated by transcription factors MYB28 and MYB29. We now show that MYB28 and MYB29 are partially redundant, but in the absence of both, the synthesis of all aliphatic glucosinolates is blocked. Untargeted and targeted biochemical analyses of leaf metabolites showed that differences between single and double knock-out mutants and wild type plants were restricted to glucosinolates. Biosynthesis of long-chain aliphatic glucosinolates was blocked by the myb28 mutation, while short-chain aliphatic glucosinolates were reduced by about 50% in both the myb28 and the myb29 single mutants. Most remarkably, all aliphatic glucosinolates were completely absent in the double mutant. Expression of glucosinolate biosynthetic genes was slightly but significantly reduced by the single myb mutations, while the double mutation resulted in a drastic decrease in expression of these genes. Since the myb28myb29 double mutant is the first Arabidopsis genotype without any aliphatic glucosinolates, we used it to establish the relevance of aliphatic glucosinolate biosynthesis to herbivory by larvae of the lepidopteran insect Mamestra brassicae. Plant damage correlated inversely to the levels of aliphatic glucosinolates observed in those plants: Larval weight gain was 2.6 fold higher on the double myb28myb29 mutant completely lacking aliphatic glucosinolates and 1.8 higher on the single mutants with intermediate levels of aliphatic glucosinolates compared to wild type plants.

The unknown genetic basis for improved phenol production by a recombinant Pseudomonas putida S12 derivative bearing the tpl (tyrosine-phenol lyase) gene was investigated via comparative transcriptomics, nucleotide sequence analysis, and targeted gene disruption. We show upregulation of tyrosine biosynthetic genes and possibly decreased biosynthesis of tryptophan caused by a mutation in the trpE gene as the genetic basis for the enhanced phenol production. In addition, several genes in degradation routes connected to the tyrosine biosynthetic pathway were upregulated. This either may be a side effect that negatively affects phenol production or may point to intracellular accumulation of tyrosine or its intermediates. A number of genes identified by the transcriptome analysis were selected for targeted disruption in P. putida S12TPL3. Physiological and biochemical examination of P. putida S12TPL3 and these mutants led to the conclusion that the metabolic flux toward tyrosine in P. putida S12TPL3 was improved to such an extent that the heterologous tyrosine-phenol lyase enzyme had become the rate-limiting step in phenol biosynthesis. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The expression of the vaccinia virus intermediate I3 gene depends on trans-acting factors which are present in an active state prior to DNA replication. However, activation of transcription requires DNA replication in cis (J. C. Vos and H. G. Stunnenberg, EMBO J., 7:3487-3492, 1988). We have made deletion and linker scanner mutations of the I3 promoter to determine the sequence requirements for transcriptional activity and the dependence of DNA replication. The I3 promoter appears to consist of two elements which are essential and sufficient for accurate transcription initiation both in vivo and in vitro. An upstream and a downstream sequence element were defined ranging from -20 to -9 and +1 to +9, respectively. The upstream element appears to be highly homologous to a sequence in the intermediate I8 promoter. A 3-bp substitution in the upstream I3 promoter element resulted in a change of transcriptional specificity from intermediate to late. Finally, the mutations did not result in an activation of the intermediate promoter prior to DNA replication.

Summary of gene-trap likelihood models The ability of these expression and length-dependent models toexplain trapping probabilities was quantified by the percentreduction in deviance compared to a null model with no covariatesin the datasets after hot-spot removal, analogous to the use of R-squared in linear regression models. The percent reduction indeviance was greater for SA-plasmid and SA-retroviral models(34% and 19%, respectively) than for the poly-A model (6%). Forthe expression-dependent SA vectors, these models explaina substantial proportion of the deviance. The low explanatoryability of the poly-A vector model reflects the relatively lower effectof expression on gene-trap likelihood, and to some extent thesmaller effect of length.Regardless of whether a gene has been inactivated by genetrapping in our experimental data, our models can predict thelikelihood that a gene will be trapped in a single trapping event.These predictions serve as ‘‘trapping scores’’ for each gene. Theraw trapping scores were corrected for the effects of both hotspotsand gene-trap events that could not be mapped to a gene. Thecorrected scores, reported for the 7435 well-defined genes includedin our dataset in Table S2, allow the overall trapping efficiencies ofdifferent vectors to be compared.To validate our model, we compared the expected number oftraps from the SA-retroviral model with the observed number oftraps in gene-trap cell lines produced by Lexicon Genetics [28].This validation set contained 48,809 cell lines from LexiconGenetics that could be annotated to our gene set (data in TableS2). The Spearman’s correlation coefficient for the comparisonwas 0.429 (P,0.005). This level of concordance gives us furtherconfidence that gene-trap likelihood was successfully modeled.Although these models describe a significant proportion of thevariance in trap likelihood between mouse genes, other factorsundoubtedly contribute to trap likelihood, and therefore, trappingscores should be interpreted on a limited scope for individualgenes. For instance, the expression-based vectors used in this studyfail to trap secreted and membrane-bound proteins (data notshown). Other genomic factors that control vector insertion,transcription, splicing, translation and protein localization all likelyplay some role in determining trap likelihood. Further examina-tion of genes where the number of observed traps departs frommodel predictions may help identify other important mechanismsaffecting insertional mutagenesis.Our models have other limitations relating to experimental andmodeling constraints. The restricted number of total genes forwhich high-confidence annotation and ESC expression data wereavailable reduced our dataset size. We also were limited to usingexpression measurements from a single ESC line, and differentglobal expression states potentially exist between the different ESClines used in gene trapping. These constraints may affect theaccuracy of our models, contributing to the overall noise, andthese effects may be stronger at the end of the length andexpression scales, where there are fewer data points.