A Novel Unsupervised Method to Identify Genes Important in the Anti-viral Response: Application to Interferon/Ribavirin in Hepatitis C Patients

Background: Treating hepatitis C with interferon/ribavirin results in a varied response in terms of decrease in viral titer and ultimate outcome. Marked responders have a sharp decline in viral titer within a few days of treatment initiation, whereas in other patients there is no effect on the virus (poor responders). Previous studies have shown that combination therapy modifies expression of hundreds of genes in vitro and in vivo. However, identifying which, if any, of these genes have a role in viral clearance remains challenging. Aims: The goal of this paper is to link viral levels with gene expression and thereby identify genes that may be responsible for early decrease in viral titer. Methods: Microarrays were performed on RNA isolated from PBMC of patients undergoing interferon/ribavirin therapy. Samples were collected at pre-treatment (day 0), and 1, 2, 7, 14 and 28 days after initiating treatment. A novel method was applied to identify genes that are linked to a decrease in viral titer during interferon/ribavirin treatment. The method uses the relationship between inter-patient gene expression based proximities and inter-patient viral titer based proximities to define the association between microarray gene expression measurements of each gene and viral-titer measurements. Results: We detected 36 unique genes whose expressions provide a clustering of patients that resembles viral titer based clustering of patients. These genes include IRF7, MX1, OASL and OAS2, viperin and many ISG's of unknown function. Conclusion: The genes identified by this method appear to play a major role in the reduction of hepatitis C virus during the early phase of treatment. The method has broad utility and can be used to analyze response to any group of factors influencing biological outcome such as antiviral drugs or anti-cancer agents where microarray data are available. © 2007 Brodsky et al

The genome of the sea urchin Strongylocentrotus purpuratus

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes

Paleo-Immunology: Evidence Consistent with Insertion of a Primordial Herpes Virus-Like Element in the Origins of Acquired Immunity

BACKGROUND:The RAG encoded proteins, RAG-1 and RAG-2 regulate site-specific recombination events in somatic immune B- and T-lymphocytes to generate the acquired immune repertoire. Catalytic activities of the RAG proteins are related to the recombinase functions of a pre-existing mobile DNA element in the DDE recombinase/RNAse H family, sometimes termed the "RAG transposon". METHODOLOGY/PRINCIPAL FINDINGS:Novel to this work is the suggestion that the DDE recombinase responsible for the origins of acquired immunity was encoded by a primordial herpes virus, rather than a "RAG transposon." A subsequent "arms race" between immunity to herpes infection and the immune system obscured primary amino acid similarities between herpes and immune system proteins but preserved regulatory, structural and functional similarities between the respective recombinase proteins. In support of this hypothesis, evidence is reviewed from previous published data that a modern herpes virus protein family with properties of a viral recombinase is co-regulated with both RAG-1 and RAG-2 by closely linked cis-acting co-regulatory sequences. Structural and functional similarity is also reviewed between the putative herpes recombinase and both DDE site of the RAG-1 protein and another DDE/RNAse H family nuclease, the Argonaute protein component of RISC (RNA induced silencing complex). CONCLUSIONS/SIGNIFICANCE:A "co-regulatory" model of the origins of V(D)J recombination and the acquired immune system can account for the observed linked genomic structure of RAG-1 and RAG-2 in non-vertebrate organisms such as the sea urchin that lack an acquired immune system and V(D)J recombination. Initially the regulated expression of a viral recombinase in immune cells may have been positively selected by its ability to stimulate innate immunity to herpes virus infection rather than V(D)J recombination Unlike the "RAG-transposon" hypothesis, the proposed model can be readily tested by comparative functional analysis of herpes virus replication and V(D)J recombination

Preclinical pharmacokinetics and metabolism of a novel prototype DNA-PK inhibitor NU7026

In this study we investigated the in vitro time dependence of radiosensitisation, pharmacokinetics and metabolism of NU7026, a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). At a dose of 10 μM, which is nontoxic to cells per se, a minimum NU7026 exposure of 4 h in combination with 3 Gy radiation is required for a significant radiosensitisation effect in CH1 human ovarian cancer cells. Following intravenous administration to mice at 5 mg kg−1, NU7026 underwent rapid plasma clearance (0.108 l h−1) and this was largely attributed to extensive metabolism. Bioavailability following interperitoneal (i.p.) and p.o. administration at 20 mg kg−1 was 20 and 15%, respectively. Investigation of NU7026 metabolism profiles in plasma and urine indicated that the compound undergoes multiple hydroxylations. A glucuronide conjugate of a bis-hydroxylated metabolite represented the major excretion product in urine. Identification of the major oxidation site as C-2 of the morpholine ring was confirmed by the fact that the plasma clearance of NU7107 (an analogue of NU7026 methylated at C-2 and C-6 of the morpholine ring) was four-fold slower than that of NU7026. The pharmacokinetic simulations performed predict that NU7026 will have to be administered four times per day at 100 mg kg−1 i.p. in order to obtain the drug exposure required for radiosensitisation

The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer

BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions.Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates.The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation

Interchromosomal Duplications on the Bactrocera oleae Y Chromosome Imply a Distinct Evolutionary Origin of the Sex Chromosomes Compared to Drosophila

BACKGROUND: Diptera have an extraordinary variety of sex determination mechanisms, and Drosophila melanogaster is the paradigm for this group. However, the Drosophila sex determination pathway is only partially conserved and the family Tephritidae affords an interesting example. The tephritid Y chromosome is postulated to be necessary to determine male development. Characterization of Y sequences, apart from elucidating the nature of the male determining factor, is also important to understand the evolutionary history of sex chromosomes within the Tephritidae. We studied the Y sequences from the olive fly, Bactrocera oleae. Its Y chromosome is minute and highly heterochromatic, and displays high heteromorphism with the X chromosome. METHODOLOGY/PRINCIPAL FINDINGS: A combined Representational Difference Analysis (RDA) and fluorescence in-situ hybridization (FISH) approach was used to investigate the Y chromosome to derive information on its sequence content. The Y chromosome is strewn with repetitive DNA sequences, the majority of which are also interdispersed in the pericentromeric regions of the autosomes. The Y chromosome appears to have accumulated small and large repetitive interchromosomal duplications. The large interchromosomal duplications harbour an importin-4-like gene fragment. Apart from these importin-4-like sequences, the other Y repetitive sequences are not shared with the X chromosome, suggesting molecular differentiation of these two chromosomes. Moreover, as the identified Y sequences were not detected on the Y chromosomes of closely related tephritids, we can infer divergence in the repetitive nature of their sequence contents. CONCLUSIONS/SIGNIFICANCE: The identification of Y-linked sequences may tell us much about the repetitive nature, the origin and the evolution of Y chromosomes. We hypothesize how these repetitive sequences accumulated and were maintained on the Y chromosome during its evolutionary history. Our data reinforce the idea that the sex chromosomes of the Tephritidae may have distinct evolutionary origins with respect to those of the Drosophilidae and other Dipteran families

Using Shifts in Amino Acid Frequency and Substitution Rate to Identify Latent Structural Characters in Base-Excision Repair Enzymes

Protein evolution includes the birth and death of structural motifs. For example, a zinc finger or a salt bridge may be present in some, but not all, members of a protein family. We propose that such transitions are manifest in sequence phylogenies as concerted shifts in substitution rates of amino acids that are neighbors in a representative structure. First, we identified rate shifts in a quartet from the Fpg/Nei family of base excision repair enzymes using a method developed by Xun Gu and coworkers. We found the shifts to be spatially correlated, more precisely, associated with a flexible loop involved in bacterial Fpg substrate specificity. Consistent with our result, sequences and structures provide convincing evidence that this loop plays a very different role in other family members. Second, then, we developed a method for identifying latent protein structural characters (LSC) given a set of homologous sequences based on Gu's method and proximity in a high-resolution structure. Third, we identified LSC and assigned states of LSC to clades within the Fpg/Nei family of base excision repair enzymes. We describe seven LSC; an accompanying Proteopedia page (http://proteopedia.org/wiki/index.php/Fpg_Nei_Protein_Family) describes these in greater detail and facilitates 3D viewing. The LSC we found provided a surprisingly complete picture of the interaction of the protein with the DNA capturing familiar examples, such as a Zn finger, as well as more subtle interactions. Their preponderance is consistent with an important role as phylogenetic characters. Phylogenetic inference based on LSC provided convincing evidence of independent losses of Zn fingers. Structural motifs may serve as important phylogenetic characters and modeling transitions involving structural motifs may provide a much deeper understanding of protein evolution

Expansion of the Preimmune Antibody Repertoire by Junctional Diversity in Bos taurus

Peer reviewe

Caratterizzazione di alcuni siti della rete accelerometrica nazionale al fine di individuare la risposta sismica locale

Le indagini geotecniche finalizzate alla stima della risposta sismica locale si limitano molto spesso ai primi 30 m di profondità, valore che è diventato uno standard per la classificazione delle caratteristiche di un sito. Negli anni ’90 Borcherdt (1994) e Martin e Dobry (1994) suggerirono 30 m come la profondità standard di indagine per la verifica delle strutture. Boore et al. (1993, 1994, 1997) e Boore e Joyner (1997) basarono le regressioni per il calcolo delle leggi predittive del moto del suolo sullo stesso parametro. Nel 1997 negli Stati Uniti il National Earthquake Hazards Reduction Program (NEHRP) nella stesura delle norme tecniche per le costruzioni in zona sismica (FEMA, 1997) utilizza per la prima volta il parametro Vs30 come indice per la classificazione dei suoli, con lo scopo di definirne l’amplificazione. Le norme tecniche per le costruzioni in zona sismica della comunità Europea, EC8 (ENV, 1998) ente da dati provenienti dagli Stati Uniti occidentali e, utilizzando dati provenienti dalla stessa regione, Wald & Mori (2000) segnalano che le VS,30 non sono molto ben correlate con l’entità dell’amplificazione, in quanto esiste una forte dispersione dei dati. La figura 1.1 mostra il rapporto tra le amplificazioni, mediate sull’intervallo di frequenza compreso tra 3-5 Hz. raccomandano lo stesso parametro per suddividere i terreni, anche se le classi differiscono in parte dalla classificazione NEHRP. Infine, anche in Italia, le Norme Tecniche per le Costruzioni (Normative Tecniche per le Costruzioni, Gazzetta Ufficiale del 14/01/2008) adottano la stessa suddivisione dei terreni adottata dall’EC8.L’attendibilità della velocità delle onde di taglio nei primi 30 m (VS,30) come estimatore della risposta sismica di un sito, in termini di frequenza e amplificazione, è tuttavia molto discussa.Innanzitutto il parametro è stato ricavato unicamente da dati provenienti dagli Stati Uniti occidentali e, utilizzando dati provenienti dalla stessa regione, Wald & Mori (2000) segnalano che le Vs30 non sono molto ben correlate con l’entità dell’amplificazione, in quanto esiste una forte dispersione dei dati. La figura 1.1 mostra il rapporto tra le amplificazioni, mediate sull’intervallo di frequenza compreso tra 3-5 Hz. I valori risultano effettivamente molto dispersi, ma questo risultato può essere spiegato col fatto che non tutte le classi di sito hanno frequenza di risonanza compreso in questo intervallo di frequenza. Perciò per alcuni siti la media è stata calcolata nell’intorno della frequenza di risonanza (sulle amplificazioni massime), mentre per altri è stata calcolata sulle armoniche superiori, che hanno ampiezze minori. Lavori eseguiti con dati provenienti da altre regioni sottolineano come le Vs30 non siano buoni estimatori per la predizione di amplificazioni in bacini profondi (Park & Hashash, 2004), per la stima delle amplificazioni in altre regioni (Stewart et al., 2003) o in presenza di inversioni di velocità (Di Giacomo et al., 2005). Uno studio recente, eseguito su dati giapponesi (Zhao et al., 2006) si è evitato l’uso della Vs30 perché strati spessi di terreno rigido posti sopra il substrato roccioso amplificano il moto di lungo periodo, mentre gli strati sottili e soffici tendono ad amplificare il moto di corto periodo: ciò significa che la VS,30 non può rappresentare il periodo predominante del sito, dato che si basa solo sugli strati superficiali. Secondo Mucciarelli e Gallipoli (2006) il confronto tra l’amplificazione sismica al sito e la Vs30 mostra che quest’ultimo parametro non è adeguato per spiegare gli effetti di sito osservati in Italia a causa delle situazioni geologiche particolari che sono diffuse nel nostro paese. La figura 1.2 mostra la distribuzione dell’ampiezza rispetto alla classe di sito, in cui si vede che le classi sono mal discriminate e le mediane delle classi A e B (indicate dalla linea nera) sono uguali. È però necessario notare che questo grafico è stato costruito utilizzando le ampiezze ricavate col metodo dei rapporti spettrali H/V, ma in letteratura (Bard, 1999) è dimostrato che tali rapporti spettrali permettono di stimare la frequenza di risonanza, ma falliscono nella stima del valore di amplificazione. In particolare la Vs30 sottostima gli effetti locali ai siti con inversione di velocità e li sovrastima in siti con bacini profondi. La Vs30 sembra fornire dei buoni risultati solo in siti che abbiano un profilo di velocità monotono, crescente con la profondità e un forte contrasto di impedenza nella prima decina di metri. Questo studio si propone di verificare l’attendibilità della velocità delle onde di taglio valutate nei primi 30 m come estimatore della risposta sismica di un sito. Per questo scopo sono state selezionate 45 stazioni della Rete Accelerometrica Nazionale, di cui si conoscono i profili stratigrafici e i profili di velocità delle onde di taglio e di compressione. Inoltre sono state raccolte le registrazioni strong motion relative ai terremoti registrati da queste stazioni. Gli effetti di sito sono stati valutati in due modi: · Le registrazioni sono state utilizzate per calcolare i rapporti spettrali H/V per ricavare la frequenza fondamentale propria di ciascun sito (f0) e il relativo valore di amplificazione; · I profili di velocità delle onde di taglio sono serviti per ricavare il modello teorico monodimensionale per il calcolo della funzione di trasferimento del sito, eseguito per mezzo del modello proposto da Haskell e Thomson (Haskell, 1953, Thomson 1950), da cui ricavare la f0 e l’amplificazione. I valori ottenuti con i due metodi sono stati poi confrontati per verificare la congruenza dei risultati. I profili di velocità hanno permesso di classificare le stazioni utilizzando la velocità media delle onde di taglio nei primi 30 m (Vs30), secondo la normativa italiana. I risultati ottenuti dalla valutazione della risposta di ciascun sito, espressi in termini di frequenza fondamentale e amplificazione, sono stati correlati con la rispettiva classe di sito per verificare l’attendibilità del parametro delle Vs30 come estimatore degli effetti di sito