high-throughput-quantitative-real-time-rt-pcr-assay-for-determining

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.

The truncated hemoglobin from Mycobacterium leprae.

In Listeria monocytogenes the acid tolerance response (ATR) takes place through a programmed molecular response which ensures cell survival under unfavorable conditions. Much evidence links ATR with virulence, but the molecular determinants involved in the reactivity to low pHs and the behavior of acid-exposed bacteria within host cells are still poorly understood. We have investigated the effect of acid adaptation on the fate of L. monocytogenes in human macrophages. Expression of genes encoding determinants for cell invasion and intracellular survival was tested for acid-exposed bacteria, and invasive behavior in the human myelomonocytic cell line THP-1 activated with gamma interferon was assessed. Functional approaches demonstrated that preexposure to an acidic pH enhances the survival of L. monocytogenes in activated human macrophages and that this effect is associated with an altered pattern of expression of genes involved in acid resistance and cell invasion. Significantly decreased transcription of the plcA gene, encoding a phospholipase C involved in vacuolar escape and cell-to-cell spread, was observed in acid-adapted bacteria. This effect was due to a reduction in the quantity of the bicistronic plcA-prfA transcript, concomitant with an increase in the level(s) of the monocistronic prfA mRNA(s). The transcriptional shift from distal to proximal prfA promoters resulted in equal levels of the prfA transcript (and, as a consequence, of the inlA, hly, and actA transcripts) under neutral and acidic conditions. In contrast, the sodC and gad genes, encoding a cytoplasmic superoxide dismutase and the glutamate-based acid resistance system, respectively, were positively regulated at a low pH. Morphological approaches confirmed the increased intracellular survival and growth of acid-adapted L. monocytogenes cells both in vacuoles and in the cytoplasm of interferon gamma-activated THP-1 macrophages. Our data indicate that preexposure to a low pH has a positive impact on subsequent challenge of L. monocytogenes with macrophagic cells.

Effect of acid adaptation on the fate of Listeria monocytogenes in THP-1 human macrophages activated by gamma interferon.

Leprosy is an old, still dreaded infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. During the infectious process, M. leprae is faced with the host macrophagic environment, where the oxidative stress and NO release, combined with low pH, low pO2, and high pCO2, contribute to limit the growth of the bacilli. Comparative genomics has unraveled massive gene decay in M. leprae, linking the strictly parasitic lifestyle with the reductive genome evolution. Compared with Mycobacterium tuberculosis and Mycobacterium bovis, the leprosy bacillus has lost most of the genes involved in the detoxification of reactive oxygen and nitrogen species. The very low reactivity of the unique truncated hemoglobin retained by M. leprae could account for the susceptibility of this exceptionally slow-growing microbe to NO.

Nitric oxide and Mycobacterium leprae pathogenicity.

17beta-Estradiol (E2)-induced rapid functions (from seconds to minutes) can be attributed to a fraction of nuclear estrogen receptor-alpha (ERalpha) localized at the plasma membrane. As a potential mechanism, we postulated that S-palmitoylation of the Cys447 residue may explain the ability of ERalpha to associate to plasma membrane making possible E2-dependent rapid functions [e.g., extracellular regulated kinase (ERK) activation]. Here, we report direct evidence that the mutation of the Cys447 residue to Ala impairs human ERalpha palmitoylation and E2-induced rapid ERK phosphorylation when transfected in ER-devoid HeLa cells. Moreover, the Cys447Ala mutation significantly decreases the E2-induced transactivation of an estrogen responsive element construct probe. Similar effects were obtained treating HeLa cells transfected with wild type ERalpha with the palmitoyl-acyltransferase inhibitor 2-bromo-hexadecanoic acid. Moreover, the deletion of the A-D domains (containing the DNA binding region) of ERalpha had no consequences on [(3)H]palmitate incorporation, whereas no palmitoylation occurred in the ERalpha mutant devoid of the E domain (i.e., ligand binding domain). These results point to the pivotal role of the Cys447 residue in ERalpha palmitoylation and in the modulation of E2-induced non-genomic functions.

S-palmitoylation modulates human estrogen receptor-alpha functions.

Is there an answer? Is it better for a bacterium to be gram-positive or gram-negative?

Is there an answer? Is there a genetic basis for human susceptibility to leprosy?

Ferrous oxygenated (Fe(II)O2) hemoglobins (Hb's) and myoglobins (Mb's) have been shown to react very rapidly with NO, yielding NO3(-) and the ferric heme-protein derivative (Fe(III)), by means of the ferric heme-bound peroxynitrite intermediate (Fe(III)OONO), according to the minimum reaction scheme: Fe(II)O2 + NO (k(on))--> Fe(III)OONO (h)--> Fe(III) + NO3(-). For most Hb's and Mb's, the first step (indicated by k(on)) is rate limiting, the overall reaction following a bimolecular behavior. By contrast, the rate of isomerization and dissociation of Fe(III)OONO (indicated by h) is rate limiting in NO scavenging by Fe(II)O2 murine neuroglobin, thus the overall reaction follows a monomolecular behavior. Here, we report the characterization of the NO scavenging reaction by Fe(II)O2 truncated Hb GlbO from Mycobacterium leprae. Values of k(on) (=2.1x10(6) M(-1) s(-1)) and h (=3.4 s(-1)) for NO scavenging by Fe(II)O2 M. leprae GlbO have been determined at pH 7.3 and 20.0 degrees C, the rate of Fe(III)OONO decay (h) is rate limiting. The Fe(III)OONO intermediate has been characterized by optical absorption spectroscopy in the Soret region. These results have been analyzed in parallel with those of monomeric and tetrameric globins as well as of flavoHb and discussed with regard to the three-dimensional structure of mycobacterial truncated Hbs and their proposed role in protection from nitrosative stress.

Nitric oxide scavenging by Mycobacterium leprae GlbO involves the formation of the ferric heme-bound peroxynitrite intermediate.

As a consequence of reductive genome evolution, the obligate intracellular pathogen Mycobacterium leprae has minimized the repertoire of genes implicated in defense against reactive oxygen and nitrogen species. Genes for multiple hemoglobin types coexist in mycobacterial genomes, but M. leprae has retained only glbO, encoding a group-II truncated hemoglobin. Mycobacterium tuberculosis GlbO has been involved in oxygen transfer and respiration during hypoxia, but a role in protection from nitric oxide (NO) has not been documented yet. Here, we report that the in vitro reaction of oxygenated recombinant M. leprae GlbO with NO results in an immediate stoichiometric formation of nitrate, concomitant with heme-protein oxidation. Overexpression of GlbO alleviates the growth inhibition of Escherichia colihmp (flavohemoglobin gene) mutants in the presence of NO-donors, partly complementing the defect in Hmp synthesis. A promoter element upstream of glbO was predicted in silico, and confirmed by using a glbO::lacZ transcriptional fusion in the heterologous Mycobacterium smegmatis system. The glbO::lacZ fusion was expressed through the whole growth cycle of M. smegmatis, and moderately induced by NO. We propose that M. leprae, by retaining the unique truncated hemoglobin GlbO, may have coupled O2 delivery to the terminal oxidase with a defensive mechanism to scavenge NO from respiratory enzymes. These activities would help to sustain the obligate aerobic metabolism required for intracellular survival of leprosy bacilli.

Truncated hemoglobin GlbO from Mycobacterium leprae alleviates nitric oxide toxicity.

A major challenge in developing vaccines for emerging pathogens is their continued evolution and ability to escape human immunity. Therefore, an important goal of vaccine research is to advance vaccine candidates with sufficient breadth to respond to new outbreaks of previously undetected viruses. Ebolavirus (EBOV) vaccines have demonstrated protection against EBOV infection in nonhuman primates (NHP) and show promise in human clinical trials but immune protection occurs only with vaccines whose antigens are matched to the infectious challenge species. A 2007 hemorrhagic fever outbreak in Uganda demonstrated the existence of a new EBOV species, Bundibugyo (BEBOV), that differed from viruses covered by current vaccine candidates by up to 43% in genome sequence. To address the question of whether cross-protective immunity can be generated against this novel species, cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV. This report provides the first demonstration of vaccine-induced protective immunity against challenge with a heterologous EBOV species, and shows that Ebola vaccines capable of eliciting potent cellular immunity may provide the best strategy for eliciting cross-protection against newly emerging heterologous EBOV species.

Demonstration of cross-protective vaccine immunity against an emerging pathogenic Ebolavirus Species.

Eine gro&szlig;e Herausforderung bei der Entwicklung von Impfstoffen f&uuml;r aufstrebende Krankheitserreger ist ihre kontinuierliche Weiterentwicklung und die F&auml;higkeit, menschliche Immunit&auml;t zu entkommen. Daher ist ein wichtiges Ziel der Impfstoffforschung Impfstoff Kandidaten mit ausreichender Breite neue Ausbr&uuml;che von zuvor nicht erkannte Viren reagieren voraus. Ebolavirus (EBOV) Impfstoffe demonstrierten gegen eine EBOV Infektion in nicht-menschlichen Primaten (NHP) und Versprechen in menschliche klinische Versuche zeigen aber immun-Schutz tritt nur bei Impfstoffen, deren Antigene auf infekti&ouml;se Herausforderung Arten abgestimmt sind. Ein 2007-h&auml;morrhagisches Fieber-Ausbruch in Uganda bewiesen die Existenz einer neuen EBOV Art, Bundibugyo (BEBOV), die von Viren, die aktuelle Impfstoff Kandidaten Genom-Sequenz von bis zu 43 % fallenden unterschieden. Um die Frage, ob die Kreuz-sch&uuml;tzende Immunit&auml;t gegen diese neuartige Art generiert werden kann zu beheben, wurden Makaken Cynomolgus mit Ausdr&uuml;cken ZEBOV und SEBOV Glykoprotein (GP) vor t&ouml;dlichen Herausforderung mit BEBOV DNA/rAd5-Impfstoffen geimpft. Geimpfte Themen entwickelt, robust, Antigen-spezifische humorale und zellul&auml;re Immunantworten gegen den GP von ZEBOV sowie der zellul&auml;ren Immunit&auml;t gegen BEBOV GP und immunisierten Makaken wurden einheitlich gegen t&ouml;dliche Herausforderung mit BEBOV gesch&uuml;tzt. Dieser Bericht stellt die erste Demonstration der Impfstoff induziert sch&uuml;tzende Immunit&auml;t gegen Challenge mit einer heterologen EBOV-Arten und zeigt, dass Ebola Impfstoffe k&ouml;nnen unangek&uuml;ndigt potente zellul&auml;ren Immunit&auml;t die beste Strategie f&uuml;r Kreuz-Schutz vor neu entstehende heterologe technologieorientierter vorsehen EBOV Arten.

Demonstration der Kreuz-sch&uuml;tzender Impfstoff Immunit&auml;t gegen eine aufkommende pathogenen Ebolavirus-Arten.

Cellular RNA interference (RNAi) provides a natural response against viral infection, but some viruses have evolved mechanisms to antagonize this form of antiviral immunity. To determine whether Ebolavirus (EBOV) counters RNAi by encoding suppressors of RNA silencing (SRSs), we screened all EBOV proteins using an RNAi assay initiated by exogenously delivered small interfering RNAs (siRNAs) against either an EBOV or a reporter gene. In addition to viral protein 35 (VP35), we found that VP30 and VP40 independently act as SRSs. Here, we present the molecular mechanisms of VP30 and VP35. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Taken together, our findings elucidate a new mechanism of RNAi suppression that extends beyond the role of SRSs in double-stranded RNA (dsRNA) binding and IFN antagonism. The presence of three suppressors highlights the relevance of host RNAi-dependent antiviral immunity in EBOV infection and illustrates the importance of RNAi in shaping the evolution of RNA viruses.

Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway.

Filoviruses are enveloped viruses that cause sporadic outbreaks of severe hemorrhagic fever [CDC, MMWR Morb. Mortal. Wkly. Rep. 50:73-77, 2001; Colebunders and Borchert, J. Infect. 40:16-20, 2000; Colebunders et al., J. Infect. Dis. 196(Suppl. 2):S148-S153, 2007; Geisbert and Jahrling, Nat. Med. 10:S110-S121, 2004]. Previous studies revealed that endosomal cysteine proteases are host factors for ebolavirus Zaire (Chandran et al., Science 308:1643-1645, 2005; Schornberg et al., J. Virol. 80:4174-4178, 2006). In this report, we show that infection mediated by glycoproteins from other phylogenetically diverse filoviruses are also dependent on these proteases and provide additional evidence indicating that they cleave GP1 and expose the binding domain for the critical host factor Niemann-Pick C1. Using selective inhibitors and knockout-derived cell lines, we show that the ebolaviruses Zaire and Cote d'Ivoire are strongly dependent on cathepsin B, while the ebolaviruses Sudan and Reston and Marburg virus are not. Taking advantage of previous studies of cathepsin B inhibitor-resistant viruses (Wong et al., J. Virol. 84:163-175, 2010), we found that virus-specific differences in the requirement for cathepsin B are correlated with sequence polymorphisms at residues 47 in GP1 and 584 in GP2. We applied these findings to the analysis of additional ebolavirus isolates and correctly predicted that the newly identified ebolavirus species Bundibugyo, containing D47 and I584, is cathepsin B dependent and that ebolavirus Zaire-1995, the single known isolate of ebolavirus Zaire that lacks D47, is not. We also obtained evidence for virus-specific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family Filoviridae.

Filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences.

Filoviruses sind umh&uuml;llten Viren, sporadische Ausbr&uuml;che der schwere h&auml;morrhagische Fieber [CDC, MMWR Morb. Sterblichen. Wkly. Rep.-50:73-77, 2001; Colebunders und Borchert, J. zu infizieren. 30-20, 2000; Colebunders Et Al., J. zu infizieren. DIS 196(Suppl. 2):S148-S153, 2007; Geisbert und Jahrling, Nat.-Med. 10:S110-S121, 2004]. Fr&uuml;here Studien gezeigt, dass Endosomen Cystein-Proteasen Host Faktoren f&uuml;r Zaire-Ebolavirus (Chandran Et Al., Science 308:1643-1645, 2005; Schornberg Et Al., J. Virol. 80:4174-4178, 2006). In diesem Bericht zeigen wir, dass die Infektion von Glykoproteinen aus anderen phylogenetisch diverse Filoviruses vermittelt auch diese Proteasen abh&auml;ngig sind und zus&auml;tzliche nachweisen, dass sie GP1 Cleave und bindende Dom&auml;ne f&uuml;r kritische Host Factor Niemann-Pick C1 verf&uuml;gbar zu machen. Mit selektiver Hemmer und k.o. abgeleiteten Zelllinien, zeigen wir, dass die Ebolaviruses Zaire und die Elfenbeink&uuml;ste stark abh&auml;ngig von Kathepsin B, w&auml;hrend die Ebolaviruses Sudan sind und Reston und Marburg-Virus nicht. Unter Ausnutzung der fr&uuml;heren Studien Kathepsin B Inhibitor-resistenter Viren (Wong Et Al., J. Virol. 84:163-175, 2010), wir fanden, dass die Virus-spezifische Unterschiede in der Anforderung f&uuml;r Kathepsin B mit Sequenz-Polymorphismen auf R&uuml;ckst&auml;nde 47 in GP1 und 584 in der GP2 korreliert sind. Wir diese Ergebnisse auf die Analyse von zus&auml;tzlichen Ebolavirus Isolaten angewendet und richtig vorausgesagt, dass die neu identifizierten Ebolavirus Art Bundibugyo mit D47 und I584, Kathepsin B abh&auml;ngig ist und dass Ebolavirus Zaire-1995, die einzigen bekannten Isolieren des Ebolavirus Zaire, die D47, fehlt nicht. Wir erhalten auch Beweise f&uuml;r Virus-spezifische Unterschiede in der Rolle des Kathepsin L, einschlie&szlig;lich der Zusammenarbeit mit Kathepsin B. Diese Studien empfehlen dringend, dass die Verwendung von Endosomen Cystein-Proteasen als Host Faktoren f&uuml;r die Einreise eine allgemeine Eigenschaft der Mitglieder der Familie Filoviridae ist.

Filoviruses Endosomen Cystein-Proteasen f&uuml;r die Einreise ben&ouml;tigen aber weisen unterschiedliche Protease &quot;Einstellungen&quot;.

Central Africa is a "hotspot" for emerging infectious diseases (EIDs) of global and local importance, and a current outbreak of ebolavirus is affecting multiple countries simultaneously. Ebolavirus is suspected to have caused recent declines in resident great apes. While ebolavirus vaccines have been proposed as an intervention to protect apes, their effectiveness would be improved if we could diagnostically confirm Ebola virus disease (EVD) as the cause of die-offs, establish ebolavirus geographical distribution, identify immunologically naïve populations, and determine whether apes survive virus exposure.

Article	Year
The truncated hemoglobin from Mycobacterium leprae. Biochemical and biophysical research communications\| PubMed ID: 12074585	2002
Effect of acid adaptation on the fate of Listeria monocytogenes in THP-1 human macrophages activated by gamma interferon. Infection and immunity\| PubMed ID: 12117947	2002
Nitric oxide and Mycobacterium leprae pathogenicity. IUBMB life\| PubMed ID: 12489635	2002
S-palmitoylation modulates human estrogen receptor-alpha functions. Biochemical and biophysical research communications\| PubMed ID: 15033483	2004
Is there an answer? Is it better for a bacterium to be gram-positive or gram-negative? IUBMB life\| PubMed ID: 15370885	2004
Is there an answer? Is there a genetic basis for human susceptibility to leprosy? IUBMB life\| PubMed ID: 16036571	2005
Nitric oxide scavenging by Mycobacterium leprae GlbO involves the formation of the ferric heme-bound peroxynitrite intermediate. Biochemical and biophysical research communications\| PubMed ID: 16307730	2006
Truncated hemoglobin GlbO from Mycobacterium leprae alleviates nitric oxide toxicity. Microbial pathogenesis\| PubMed ID: 16524692	2006
Demonstration of cross-protective vaccine immunity against an emerging pathogenic Ebolavirus Species. PLoS pathogens\| PubMed ID: 20502688	2010
Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway. Journal of virology\| PubMed ID: 21228243	2011
Filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences. Journal of virology\| PubMed ID: 22238307	2012
A new approach for monitoring ebolavirus in wild great apes. PLoS neglected tropical diseases\| PubMed ID: 25232832	2014

Giulia Fabozzi

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