Novel conditional mouse models for targeted kidney research : Emphasis on the analysis of the biological function of nephrin

Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.

Genetic studies on recurrent miscarriage

Recurrent miscarriage (RM) is defined as three consecutive pregnancy failures and is estimated to affect ~1% of couples trying to conceive. The cause of RM remains unknown in approximately 50% of cases. In this study, it was hypothesized that some of the underlying factors yet to be discovered are genetic. The aim was to search for mutations in genes AMN, EPCR, TM, and p53 known to cause miscarriage in mouse models and thereby find new genetic causes for unexplained miscarriages in humans. In addition, the mitochondrial genome was studied because mitochondria are involved in processes important in early development. Furthermore, sex chromosome characteristics suggested to underlie miscarriage were also studied. A total of 40 couples and 8 women with unexplained RM were collected for this study and screened for mutations in the candidate genes. Six interesting exonic or potential splice site disrupting variations were detected. However, their phenotypic effects cannot be determined without further investigations. Additionally, an association between the C11992A polymorphism of the p53 gene and RM was detected. The results indicate that women carrying the C/A or A/A genotype have a two-fold higher risk for RM than women with a C/C genotype. This strengthens the results of previous studies reporting that p53 sequence variations may cause miscarriage. The role of variation C11992A in embryonic development is, however, difficult to predict without further studies When screening the mitochondrial genome a heteroplasmic mtDNA variation was found in an unexpected high number of women, as heteroplasmic variations are reported to be rare. One novel variation and 18 previously reported polymorphisms were detected in the mitochondrial genome. Although the detected variations are likely to be neutral polymorphisms, a role in the aetiology of miscarriage cannot be excluded as some mtDNA variations may be pathogenic only when a threshold is reached. Recent publications have reported skewed X chromosome inactivation and Y chromosome microdeletions to be associated with RM. Therefore, these sex chromosome abnormalities in the context of RM were investigated. No associations between skewed X chromosome inactivation or Y chromosome microdeletions and RM in the Finnish patients were detected. Data on ancestral birthplaces of the patients were collected to study any possible geographic clustering, which would indicate a common predisposing factor. The results showed clustering of the birthplaces in eastern Finland in a subset of patients. This suggests a possibility of an enriched susceptibility gene which may contribute to RM.

The meleagrid herpesvirus 1 genome is partially resistant to transposition

The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed. © American Association of Avian Pathologists.

Novel TBK1 truncating mutation in a familial amyotrophic lateral sclerosis patient of Chinese origin

Missense and frameshift mutations in TRAF family member-associated NF-kappa-B activator (TANK)-binding kinase 1 (TBK1) have been reported in European sporadic and familial amyotrophic lateral sclerosis (ALS) cohorts. To assess the role of TBK1 in ALS patient cohorts of wider ancestry, we have analyzed whole-exome sequence data from an Australian cohort of familial ALS (FALS) patients and controls. We identified a novel TBK1 deletion (c.1197delC) in a FALS patient of Chinese origin. This frameshift mutation (p.L399fs) likely results in a truncated protein that lacks functional domains required for adapter protein binding, as well as protein activation and structural integrity. No novel or reported TBK1 mutations were identified in FALS patients of European ancestry. This is the first report of a TBK1 mutation in an ALS patient of Asian origin and indicates that sequence variations in TBK1 are a rare cause of FALS in Australia. © 2015 Elsevier Inc.

Identification and Characterization of a Novel Deoxyhypusine Synthase in Leishmania donovani

Deoxyhypusine synthase, an NAD(+)-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (N-epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.

Genetics of Type III Bartter Syndrome in Spain, Proposed Diagnostic Algorithm

9 p.

Avaliação do efeito do inibidor de histona deacetilase Panobinostat (LBH589) no metabolismo energético de células derivadas de câncer de pulmão

Os mecanismos de ação citotóxica do inibidor de histona deacetilase LBH589 foram investigados em associação com o quimioterápico cisplatina (CDDP) em duas linhagens derivadas de câncer de pulmão de não pequenas células (CPNPC). Os resultados foram analisados em relação ao tipo de morte celular associada às alterações em enzimas relacionadas ao metabolismo energético e a via glicolítica. Para realização do trabalho, foram utilizadas as linhagens tumorais A549 (selvagem para o gene de p53) e Calu-1 (nulo para o gene de p53) tratadas com LBH589 em combinação ou não com CDDP. Foram realizadas curvas de tempo e dose-resposta com as drogas isoladamente pelo ensaio de viabilidade celular (MTT) nas duas linhagens para a escolha das melhores condições para o nosso estudo. As condições dos tratamentos isolados com redução da viabilida celular menores que o IC50 de cada fármaco foram selecionados para realização dos tratamentos combinados. As avaliações de apoptose foram realizadas por citometria de fluxo pelo ensaio de Anexina V/PI, e com a marcação de proteínas por Western Blotting. As proteínas relacionadas a via glicolítica foram avaliadas por Western Blotting e a expressão de RNAm por qPCR. Os resultados demonstraram que o LBH589 combinado a CDDP foi capaz de induzir apoptose em 70% das células (Calu-1) e 54,9% (A549) no tempo de 24 horas, e 90% (calu-1) e 62,1% (A549) em 48 horas, independendo, portanto, do status da p53. Os níveis de expressão de enzimas relacionadas com o metabolismos energético também sofreram alterações nos tratamentos estudados. O LBH589 induziu aumento de cerca de 4x dos níveis de RNAm de HK isoformas I e II em ambas as linhagens. Houve também um aumento na expressão proteica das isoformas de HK I e II. Outras enzimas relacionadas a via glicolítica como PFKP, PKM2 e LDHA foram analisadas e apresentaram redução da expressão proteica, principalmente na presença do LBH589. A combinação da CDDP com LBH589 parece ser promissora para o tratamento de CPNPC induzindo apoptose através de alterações no metabolismo energético tumoral.

Label free electrochemiluminescence protocol for sensitive DNA detection with a tris(2,2'-bipyridyl)ruthenium(II) modified electrode based on nucleic acid oxidation

Label free electrochemiluminescence (ECL) DNA detection based on catalytic guanine and adenine bases oxidation using tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)] modified glassy carbon (GC) electrode was demonstrated in this work. The modified GC electrode was prepared by casting carbon nanotubes (CNT)/Nafion/Ru(bpy)(3)(2+) composite film on the electrode surface. ECL signals of doublestranded DNA and their thermally denatured counterparts can be distinctly discriminated using cyclic voltammetry (CV) with a low concentration (3.04 x 10(-8) mol/L for Salmon Testes-DNA). Most importantly, sensitive single-base mismatch detection of p53 gene sequence segment was realized with 3.93 x 10(-10) mol/L employing CV stimulation (ECL signal of C/A mismatched DNA oligonucleotides was 1.5-fold higher than that of fully base-paired DNA oligonucleotides). Label free, high sensitivity and simplicity for single-base mismatch discrimination were the main advantages of the present ECL technique for DNA detection over the traditional DNA sensors.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Ferrochelatase is a conserved downstream target of the blue light-sensing White collar complex in fungi.

Light is a universal signal perceived by organisms, including fungi, in which light regulates common and unique biological processes depending on the species. Previous research has established that conserved proteins, originally called White collar 1 and 2 from the ascomycete Neurospora crassa, regulate UV/blue light sensing. Homologous proteins function in distant relatives of N. crassa, including the basidiomycetes and zygomycetes, which diverged as long as a billion years ago. Here we conducted microarray experiments on the basidiomycete fungus Cryptococcus neoformans to identify light-regulated genes. Surprisingly, only a single gene was induced by light above the commonly used twofold threshold. This gene, HEM15, is predicted to encode a ferrochelatase that catalyses the final step in haem biosynthesis from highly photoreactive porphyrins. The C. neoformans gene complements a Saccharomyces cerevisiae hem15Delta strain and is essential for viability, and the Hem15 protein localizes to mitochondria, three lines of evidence that the gene encodes ferrochelatase. Regulation of HEM15 by light suggests a mechanism by which bwc1/bwc2 mutants are photosensitive and exhibit reduced virulence. We show that ferrochelatase is also light-regulated in a white collar-dependent fashion in N. crassa and the zygomycete Phycomyces blakesleeanus, indicating that ferrochelatase is an ancient target of photoregulation in the fungal kingdom.

Unprecedented loss of ammonia assimilation capability in a urease-encoding bacterial mutualist.

BACKGROUND: Blochmannia are obligately intracellular bacterial mutualists of ants of the tribe Camponotini. Blochmannia perform key nutritional functions for the host, including synthesis of several essential amino acids. We used Illumina technology to sequence the genome of Blochmannia associated with Camponotus vafer. RESULTS: Although Blochmannia vafer retains many nutritional functions, it is missing glutamine synthetase (glnA), a component of the nitrogen recycling pathway encoded by the previously sequenced B. floridanus and B. pennsylvanicus. With the exception of Ureaplasma, B. vafer is the only sequenced bacterium to date that encodes urease but lacks the ability to assimilate ammonia into glutamine or glutamate. Loss of glnA occurred in a deletion hotspot near the putative replication origin. Overall, compared to the likely gene set of their common ancestor, 31 genes are missing or eroded in B. vafer, compared to 28 in B. floridanus and four in B. pennsylvanicus. Three genes (queA, visC and yggS) show convergent loss or erosion, suggesting relaxed selection for their functions. Eight B. vafer genes contain frameshifts in homopolymeric tracts that may be corrected by transcriptional slippage. Two of these encode DNA replication proteins: dnaX, which we infer is also frameshifted in B. floridanus, and dnaG. CONCLUSIONS: Comparing the B. vafer genome with B. pennsylvanicus and B. floridanus refines the core genes shared within the mutualist group, thereby clarifying functions required across ant host species. This third genome also allows us to track gene loss and erosion in a phylogenetic context to more fully understand processes of genome reduction.

NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.

Outer membrane vesicles (OMVs) are ubiquitously secreted from the outer membrane (OM) of Gram-negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild-type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan (PG) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr, consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin-binding protein 4 (PBP4) during exponential growth. The data support a model in which NlpI negatively regulates PBP4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane (OM-PG) envelope stabilizing cross-links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild-type Gram-negative bacteria can use to modulate vesiculation is by altering PG-OM cross-linking via localized modulation of PG degradation and synthesis.

Modulation of bacterial outer membrane vesicle production by envelope structure and content.

BACKGROUND: Vesiculation is a ubiquitous secretion process of Gram-negative bacteria, where outer membrane vesicles (OMVs) are small spherical particles on the order of 50 to 250 nm composed of outer membrane (OM) and lumenal periplasmic content. Vesicle functions have been elucidated in some detail, showing their importance in virulence factor secretion, bacterial survival, and biofilm formation in pathogenesis. Furthermore, OMVs serve as an envelope stress response, protecting the secreting bacteria from internal protein misfolding stress, as well as external envelope stressors. Despite their important functional roles very little is known about the regulation and mechanism of vesicle production. Based on the envelope architecture and prior characterization of the hypervesiculation phenotypes for mutants lacking the lipoprotein, Lpp, which is involved in the covalent OM-peptidoglycan (PG) crosslinks, it is expected that an inverse relationship exists between OMV production and PG-crosslinked Lpp. RESULTS: In this study, we found that subtle modifications of PG remodeling and crosslinking modulate OMV production, inversely correlating with bound Lpp levels. However, this inverse relationship was not found in strains in which OMV production is driven by an increase in "periplasmic pressure" resulting from the accumulation of protein, PG fragments, or lipopolysaccharide. In addition, the characterization of an nlpA deletion in backgrounds lacking either Lpp- or OmpA-mediated envelope crosslinks demonstrated a novel role for NlpA in envelope architecture. CONCLUSIONS: From this work, we conclude that OMV production can be driven by distinct Lpp concentration-dependent and Lpp concentration-independent pathways.

Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn't show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): an exploratory, randomised, placebo-controlled phase 2 study.

Duchenne muscular dystrophy is caused by dystrophin deficiency and muscle deterioration and preferentially affects boys. Antisense-oligonucleotide-induced exon skipping allows synthesis of partially functional dystrophin. We investigated the efficacy and safety of drisapersen, a 2'-O-methyl-phosphorothioate antisense oligonucleotide, given for 48 weeks.