Development of <i>Onchocerca volvulus</i> in humanized NSG mice and detection of parasite biomarkers in urine and serum

by John B. Patton, Sasisekhar Bennuru, Mark L. Eberhard, Jessica A. Hess, April Torigian, Sara Lustigman, Thomas B. Nutman, David Abraham

Background

The study of Onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. Small animal models that support the development of adult parasites have not been identified.

Methodology/Principal findings

We hypothesized that highly immunodeficient NSG mice would support the survival and maturation of O. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. NSG mice were humanized with: (1) umbilical cord derived CD34+ stem cells, (2) fetal derived liver, thymus and CD34+ stem cells or (3) primary human skeletal muscle cells. NSG and humanized NSG mice were infected with 100 O. volvulus infective larvae (L3) for 4 to 12 weeks. When necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. In each of the different humanized mouse models, worms matured from L3 to advanced fourth stage larvae, with both male and female organ development. In addition, worms increased in length by up to 4-fold. Serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 O. volvulus-derived proteins found specifically in either the urine or the serum of the humanized O. volvulus-infected NSG mice.

Conclusions/Significance

The newly identified mouse models for onchocerciasis will enable the development of O. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with O. volvulus.

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Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility

by Ramanagouda Ramanagoudr-Bhojappa, Blake Carrington, Mukundhan Ramaswami, Kevin Bishop, Gabrielle M. Robbins, MaryPat Jones, Ursula Harper, Stephen C. Frederickson, Danielle C. Kimble, Raman Sood, Settara C. Chandrasekharappa

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.

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Boundaries mediate long-distance interactions between enhancers and promoters in the <i>Drosophila Bithorax</i> complex

by Nikolay Postika, Mario Metzler, Markus Affolter, Martin Müller, Paul Schedl, Pavel Georgiev, Olga Kyrchanova

Drosophila bithorax complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously. Paradoxically, seven of the BX-C regulatory domains are separated from their gene target by at least one boundary, and must “jump over” the intervening boundaries. To understand the jumping mechanism, the Mcp boundary was replaced with Fab-7 and Fab-8. Mcp is located between the iab-4 and iab-5 domains, and defines the border between the set of regulatory domains controlling abd-A and Abd-B. When Mcp is replaced by Fab-7 or Fab-8, they direct the iab-4 domain (which regulates abd-A) to inappropriately activate Abd-B in abdominal segment A4. For the Fab-8 replacement, ectopic induction was only observed when it was inserted in the same orientation as the endogenous Fab-8 boundary. A similar orientation dependence for bypass activity was observed when Fab-7 was replaced by Fab-8. Thus, boundaries perform two opposite functions in the context of BX-C–they block crosstalk between neighboring regulatory domains, but at the same time actively facilitate long distance communication between the regulatory domains and their respective target genes.

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Correction: Brain-Computer Interface-Based Communication in the Completely Locked-In State

by Ujwal Chaudhary, Bin Xia, Stefano Silvoni, Leonardo G. Cohen, Niels Birbaumer

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Integrative network-centric approach reveals signaling pathways associated with plant resistance and susceptibility to <i>Pseudomonas syringae</i>

by Elizabeth K. Brauer, George V. Popescu, Dharmendra K. Singh, Mauricio Calviño, Kamala Gupta, Bhaskar Gupta, Suma Chakravarthy, Sorina C. Popescu

Plant protein kinases form redundant signaling pathways to perceive microbial pathogens and activate immunity. Bacterial pathogens repress cellular immune responses by secreting effectors, some of which bind and inhibit multiple host kinases. To understand how broadly bacterial effectors may bind protein kinases and the function of these kinase interactors, we first tested kinase–effector (K-E) interactions using the Pseudomonas syringae pv. tomato–tomato pathosystem. We tested interactions between five individual effectors (HopAI1, AvrPto, HopA1, HopM1, and HopAF1) and 279 tomato kinases in tomato cells. Over half of the tested kinases interacted with at least one effector, and 48% of these kinases interacted with more than three effectors, suggesting a role in the defense. Next, we characterized the role of select multi-effector–interacting kinases and revealed their roles in basal resistance, effector-triggered immunity (ETI), or programmed cell death (PCD). The immune function of several of these kinases was only detectable in the presence of effectors, suggesting that these kinases are critical when particular cell functions are perturbed or that their role is typically masked. To visualize the kinase networks underlying the cellular responses, we derived signal-specific networks. A comparison of the networks revealed a limited overlap between ETI and basal immunity networks. In addition, the basal immune network complexity increased when exposed to some of the effectors. The networks were used to successfully predict the role of a new set of kinases in basal immunity. Our work indicates the complexity of the larger kinase-based defense network and demonstrates how virulence- and avirulence-associated bacterial effectors alter sectors of the defense network.

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Genome-wide functional analyses of plant coiled–coil NLR-type pathogen receptors reveal essential roles of their N-terminal domain in oligomerization, networking, and immunity

by Tadeusz Wróblewski, Laurentiu Spiridon, Eliza Cristina Martin, Andrei-Jose Petrescu, Keri Cavanaugh, Maria Jose-Truco, Huaqin Xu, Dariusz Gozdowski, Krzysztof Pawłowski, Richard W. Michelmore, Frank L.W. Takken

The ability to induce a defense response after pathogen attack is a critical feature of the immune system of any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. However, the mechanism by which defense signaling is triggered by these NLRs in plants is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, the complexes are highly heterometric. Also, in contrast to metazoan NLRs, the N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions in plants is proposed to provide resilience to perturbation by pathogens.

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Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair

by Eirini M. Kallimasioti-Pazi, Keerthi Thelakkad Chathoth, Gillian C. Taylor, Alison Meynert, Tracy Ballinger, Martijn J. E. Kelder, Sébastien Lalevée, Ildem Sanli, Robert Feil, Andrew J. Wood

Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.

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O.D.G. Giunte e Commissioni: Giunta delle elezioni e delle immunita’ parlamentari – 19/12/2018

O.D.G. Giunte e Commissioni

Giunta delle elezioni e delle immunita’ parlamentari

Seduta/e: 14 del 19/12/2018

odg-giuntaelezionieimmunita-19122018_.pdf


Tratto da www.senato.it

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Interrogazioni a risposta immediata – Question time

Alle ore 15 ha avuto luogo lo svolgimento di interrogazioni a risposta immediata, trasmesse in diretta televisiva, sui seguenti argomenti: Chiarimenti in ordine alla proposta, rivolta agli avvocati e presente sulla piattaforma Rousseau, di prestare consulenza legale in deroga al principio dell’equo compenso (Conte – LEU); Intendimenti del Governo in materia di esecuzione penale esterna (Bazoli – PD); Elementi in ordine all’accoglimento delle domande di riparazione per ingiusta detenzione e ai possibili effetti sull’esercizio dell’azione disciplinare (Costa – FI); Iniziative volte a promuovere la diffusione dei cosiddetti sportelli di prossimità, anche nel quadro dello sviluppo del processo civile telematico (Molinari – LEGA); Posizione del Governo italiano in relazione al quadro finanziario pluriennale europeo, con particolare riferimento al finanziamento della politica agricola comune (Rossini – MISTO-MIN.LING.); Iniziative volte alla valorizzazione e alla tutela del personale militare e civile della difesa (Galantino – M5S); Iniziative volte a favorire la stabilizzazione del personale precario delle Forze armate, anche alla luce delle disposizioni del decreto-legge n. 87 del 2018 (Lollobrigida – FDI).

Per il Governo sono intervenuti: il Ministro della Giustizia, Alfonso Bonafede; il Ministro per gli Affari Europei, Paolo Savona; la Ministra della Difesa, Elisabetta Trenta.

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Iva e Fatturazione elettronica

Il decreto-legge in materia di “pace fiscale” e semplificazioni (n. 119 del 2018), modificato al Senato, contiene numerose norme volte a semplificare gli adempimenti tributari e, in particolare, gli adempimenti legati alla fatturazione elettronica.
Secondo uno studio pubblicato il 21 settembre 2018 dalla Commissione europea, nel 2016 i paesi dell’UE hanno perso quasi 150 miliardi di euro di entrate provenienti dall’imposta sul valore aggiunto (IVA).
L’Italia si conferma prima in Ue per la più grande evasione dell’Iva in valore nominale, con perdite per le casse dello Stato di 35,9 miliardi di euro. Il nostro Paese, inoltre, è terzo per il maggior divario tra il gettito previsto e quello effettivamente riscosso. La differenza ammonta infatti al 25,9%, dietro solo a Romania (35,88%) e Grecia (29,2%). Dai dati che emergono si registra che rispetto al 2015 c’è stato un lieve miglioramento in termini percentuali, in quanto l’evasione si è ridotta dello 0,23%, ma in valore assoluto c’è stato un aumento di circa 200 milioni di euro. Nel complesso, dal 2012 al 2016, l’Italia ha ridotto l’evasione del 3%.

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Nota sul Copyright: L’utilizzo, la riproduzione, l’estrazione di copia, ovvero la distribuzione delle informazioni testuali e degli elementi multimediali disponibili sul sito della Camera dei deputati è autorizzata esclusivamente nei limiti in cui la stessa avvenga nel rispetto dell’interesse pubblico all’informazione, per finalità non commerciali, garantendo l’integrità degli elementi riprodotti e mediante indicazione della fonte.

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