TrxT and dhd are dispensable for Drosophila brain development but essential for l(3)mbt brain tumour growth.

Expression of the Drosophila cancer-germline (CG), X-linked, head-to-head gene pair TrxT and dhd is normally germline-specific but becomes upregulated in brain tumours caused by mutation in l(3)mbt. Here, we show that TrxT and dhd play a major synergistic role in the emergence of l(3)mbt tumour-linked transcriptomic signatures and tumour development, which is remarkable, taking into account that these two genes are never expressed together under normal conditions. We also show that TrxT, but not dhd, is crucial for the growth of l(3)mbt allografts, hence suggesting that the initial stages of tumour development and long-term tumour growth may depend on different molecular pathways. In humans, head-to-head inverted gene pairs are abundant among CG genes that map to the X chromosome. Our results identify a first example of an X-linked, head-to-head CG gene pair in Drosophila, underpinning the potential of such CG genes, dispensable for normal development and homoeostasis of somatic tissue, as targets to curtail malignant growth with minimal impact on overall health.

The histone code reader PHD finger protein 7 controls sex-linked disparities in gene expression and malignancy in Drosophila.

The notable male predominance across many human cancer types remains unexplained. Here, we show that Drosophila l(3)mbt brain tumors are more invasive and develop as malignant neoplasms more often in males than in females. By quantitative proteomics, we have identified a signature of proteins that are differentially expressed between male and female tumor samples. Prominent among them is the conserved chromatin reader PHD finger protein 7 (Phf7). We show that Phf7 depletion reduces sex-dependent differences in gene expression and suppresses the enhanced malignant traits of male tumors. Our results identify potential regulators of sex-linked tumor dimorphism and show that these genes may serve as targets to suppress sex-linked malignant traits.

The reference epigenome and regulatory chromatin landscape of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.

Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion.

CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.

Discovery of Cancer Driver Long Noncoding RNAs across 1112 Tumour Genomes: New Candidates and Distinguishing Features.

Long noncoding RNAs (lncRNAs) represent a vast unexplored genetic space that may hold missing drivers of tumourigenesis, but few such "driver lncRNAs" are known. Until now, they have been discovered through changes in expression, leading to problems in distinguishing between causative roles and passenger effects. We here present a different approach for driver lncRNA discovery using mutational patterns in tumour DNA. Our pipeline, ExInAtor, identifies genes with excess load of somatic single nucleotide variants (SNVs) across panels of tumour genomes. Heterogeneity in mutational signatures between cancer types and individuals is accounted for using a simple local trinucleotide background model, which yields high precision and low computational demands. We use ExInAtor to predict drivers from the GENCODE annotation across 1112 entire genomes from 23 cancer types. Using a stratified approach, we identify 15 high-confidence candidates: 9 novel and 6 known cancer-related genes, including MALAT1, NEAT1 and SAMMSON. Both known and novel driver lncRNAs are distinguished by elevated gene length, evolutionary conservation and expression. We have presented a first catalogue of mutated lncRNA genes driving cancer, which will grow and improve with the application of ExInAtor to future tumour genome projects.

ChimPipe: accurate detection of fusion genes and transcription-induced chimeras from RNA-seq data.

BACKGROUND: Chimeric transcripts are commonly defined as transcripts linking two or more different genes in the genome, and can be explained by various biological mechanisms such as genomic rearrangement, read-through or trans-splicing, but also by technical or biological artefacts. Several studies have shown their importance in cancer, cell pluripotency and motility. Many programs have recently been developed to identify chimeras from Illumina RNA-seq data (mostly fusion genes in cancer). However outputs of different programs on the same dataset can be widely inconsistent, and tend to include many false positives. Other issues relate to simulated datasets restricted to fusion genes, real datasets with limited numbers of validated cases, result inconsistencies between simulated and real datasets, and gene rather than junction level assessment. RESULTS: Here we present ChimPipe, a modular and easy-to-use method to reliably identify fusion genes and transcription-induced chimeras from paired-end Illumina RNA-seq data. We have also produced realistic simulated datasets for three different read lengths, and enhanced two gold-standard cancer datasets by associating exact junction points to validated gene fusions. Benchmarking ChimPipe together with four other state-of-the-art tools on this data showed ChimPipe to be the top program at identifying exact junction coordinates for both kinds of datasets, and the one showing the best trade-off between sensitivity and precision. Applied to 106 ENCODE human RNA-seq datasets, ChimPipe identified 137 high confidence chimeras connecting the protein coding sequence of their parent genes. In subsequent experiments, three out of four predicted chimeras, two of which recurrently expressed in a large majority of the samples, could be validated. Cloning and sequencing of the three cases revealed several new chimeric transcript structures, 3 of which with the potential to encode a chimeric protein for which we hypothesized a new role. Applying ChimPipe to human and mouse ENCODE RNA-seq data led to the identification of 131 recurrent chimeras common to both species, and therefore potentially conserved. CONCLUSIONS: ChimPipe combines discordant paired-end reads and split-reads to detect any kind of chimeras, including those originating from polymerase read-through, and shows an excellent trade-off between sensitivity and precision. The chimeras found by ChimPipe can be validated in-vitro with high accuracy.

Transcriptional diversity during lineage commitment of human blood progenitors.

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine.

Lamivudine for chronic hepatitis B: a brief review

Until recently, the only generally approved treatment for chronic hepatitis B was alpha-interferon; however, it gives only moderate efficacy in terms of sustained response (biochemical, virological and histological). In fact, only 20 percent to 40 percent of treated patients respond to therapy, with lower percentages (~ 10 percent) among patients infected with precore-mutant strains of HBV (HBeAb HBV-DNA positive). The FDA of the USA approved the use of lamivudine in adult patients affected by chronic hepatitis B in 1998. In this review, we focused on the pharmacokinetic and pharmacodynamic properties and efficacy and tolerability of lamivudine in the treatment of chronic hepatitis B cases that are both HBeAg and anti-HBe-positive.

[Current news about epidemiology of syncytial respiratory virus infection]. / Attualità sulla epidemiologia dell'infezione da virus respiratorio sinciziale.

At present, syncytial respiratory virus is the major agent of respiratory infections in pediatric patients. It can determine an important respiratory distress in particular children, as prematures with an gestational age <32 weeks, children affected by congenital heart diseases or bronchopneumonia dysplasia. In these patients, the prophylaxis with palivizumab is very important to prevent chronic pulmonary diseases.

Oral miltefosine treatment in children with visceral leishmaniasis: a brief review

Visceral leishmaniasis (VL) or kala-azar is an infection disease caused by hemiflagellate protozoan parasites (Leishmania donovani) and transmitted to humans by the phlebotomine sandfly. Leishmaniasis is distributed worldwide and 13 million people are estimated to be infected, with about 1.8 million new cases each year. All antileishmanial drugs are toxic and most have to be used parenterally for prolonged period. The therapy has been further complicated by large number of infected children and declining effectiveness of pentavalent antimonial compounds. Although the lipid formulations of amphotericin B are an important advance in therapy, their high cost precludes their use. Miltefosine, a phosphocholine analogue originally developed as antimalignant drug, has been found to be highly active against Leishmania in vitro and in animal model. Based on these experiences this drug was tried against human visceral leishmaniasis and found to be highly effective in children. The aim of this review is to evidence the pharmacodymamic and pharmacokinetic characteristics and the safety, tolerance and efficacy of this drug for treatment of visceral leishmaniasis in children.

[Wilson's disease in paediatric age: diagnosis and treatment. Recent advances]. / Diagnosi e terapia del morbo di Wilson in età pediatrica. Recenti progressi.

Wilson's disease is an autosomal-recessive disorder caused by mutation in the ATP7B gene, with resultant impairment of biliary excretion of copper. Subsequent copper accumulation, first in the liver but ultimately in the brain and other tissues, produces clinical manifestations that may include hepatic, neurological, psychiatric, ophthalmological, and other derangements. This article discusses the recent progress in diagnosis and treatment of this disease in paediatric age.

Prevalence of HBV genotypes in South American immigrants affected by HBV-related chronic active hepatitis

This study evaluated the prevalence of HBV infection in a population of South American immigrants in Italy and to determine in patients with detectable serum HBV-DNA the HBVgenotypes. Between April 2005 and April 2006 a total of 130 South American immigrants were tested for HBsAg. In HBsAg positive patients the biochemical and virological activity of infection and the possible presence of co-infections (HCV, HDV, HIV) were evaluated. In patients with detectable serum HBV DNA, the HBV genotype was determined by INNOLiPA. Among the 130 subjects tested, 14 (10.7 percent) resulted HBsAg positive. All were men, with a mean age of 22 years (range 19-37) and 12 (85.7 percent) came from Brazil, while 2 (14.3 percent) came from Ecuador. All patients infected by HBV had elevated alanine-aminotransferase serum levels (mean level was 127 IU/L, range 74-312) and serum HBV DNA detectable by PCR-Real Time (mean level 1,037,652 copies/mL, range 19,876-1,377,648). Genotype distribution was as follow: genotype D, 9 (64.2 percent), genotype A, 5 (35.8 percent). All patients infected by genotype D came from Brazil, while among the patients infected by genotype A, three came from Brazil and two from Ecuador. Our study evidences a moderate prevalence of HBV-infection in South American immigrants with the identification of two genotypes, D and A. These genotypes are not the most prevalent in the South America and this is probably the expression of a possible geographical redistribution of HBV genotypes.

Prevalence of HBV-genotypes in immigrants affected by HBV-related chronic active hepatitis

BACKGROUND: The genetic heterogeneity of the HBV genome has been established and eight genotypes can be classified according to the criterion of >8 percent differences in the complete nucleotide sequence of the viral genome. AIMS: To evaluate the prevalence of HBV-infection in a population of immigrants and to determine in patients with detectable serum HBV-DNA the HBV-genotypes. METHODS: Between January 2005 and December 2005 a total of 556 immigrants were tested for HBsAg. In HBsAg positive patients the biochemical and virological activity of infection and the possible presence of co-infections (HCV, HDV, HIV) were evaluated. In patients with detectable serum HBV DNA, the HBV-genotype was determined by INNOLiPA. RESULTS: Among the 556 subjects tested, 60 (10.7 percent) resulted HBsAg positive. All were men, and 42 (70 percent) come from Africa, 10 (16.6 percent) from Asia and 9 (14.4 percent) from East-Europe. 28/60 (46.6 percent) patients presented normal ALT levels (<40 IU/L) and undetectable serum HBV DNA (<100 copies/mL in real-time PCR), while 32 (53.4 percent) patients had ALT levels above laboratory normal values and detectable serum HBV DNA. Genotype distribution was as follow: genotype E, 16 (50 percent), genotype D, 9 (28.1 percent), genotype A, 7 (21.9 percent). CONCLUSION: Our study evidences a moderate prevalence of HBV-infection in immigrants, particularly in sub-Saharan African people, and the potentiality of migratory flow in the introduction of genotype non-D hepatitis B virus, potentially characterized by a different natural history and, possibly, a different response to antiviral treatment.

RACIONAL: A heterogeneidade do genoma do vírus da hepatite B (VHB) foi estabelecida e oito genótipos podem ser classificados de acordo com o critério de diferenças de percentagem maior ou igual a 8 na seqüência completa do nucleotídeo do genoma vira!. OBJETIVOS: Verificar a prevalência da infecção pelo vírus da hepatite B (VHB) em uma população de imigrantes na Itália e determinar os genótipos do VHB em pacientes com níveis séricos detectáveis do VHB-DNA. MÉTODOS: Entre janeiro e dezembro de 2005, o total de 556 imigrantes foram testados para o HbsAg. Se positivos, a atividade bioquímica e viral da infecção e a possível presença de co-infecções (HVC, HVD e HIV) foram examinadas. Nos pacientes positivos para o VHB-DNA, o genótipo do VHB foi determinado pelo método INNOLiPA. RESULTADOS: Entre os 556 pacientes, 60 (10,7 por cento) tinham HbsAg positivo. Todos eram do sexo masculino e 42 (70 por cento), provenientes da Africa, 10 (16,6 por cento) da Asia e 9 (14,4 por cento) do Leste Europeu. 28/60 (46,6 por cento) apresentaram níveis de ALT normais (<40 UI/L) e soro negativo ou indetectável para o VHB-DNA (<100 copies/mL PCR "real-time"), enquanto 32 (53,4 por cento) tinham níveis mais elevados de ALT e soro positivo para VHB-DNA. A distribuição do genótipo foi a seguinte: genótipo E, 16 (50 por cento), genótipo D, 9 (28,1 por cento), genótipo A, 7 (21,1 por cento). CONCLUSÃO: O estudo evidencia a prevalência moderada do HVB em imigrantes, particularmente na população africana, sub-Sahara e o potencial fluxo migratório na introdução da hepatite B, genótipo não-D, potencialmente caracterizada pela história natural e possivelmente levar à diferença no tratamento anti-viral.

Multiple recurrences of vertebral hydatidosis: a case report.

We present a case of an Italian man with a particularly prolonged history of hydatid disease (more than 20 years, with six recurrences) involving both osseus (costal and vertebral) and visceral (pulmonary) sites. The main clinical problems involved in the diagnosis and management of vertebral manifestations of hydatidosis are discussed in the light of the latest clinical research.

Treatment of genotype 1b HCV-related chronic hepatitis: efficacy and toxicity of three different interferon alfa-2b/ribavirin combined regimens in naive patients.

This prospective open-label randomized trial of chronic hepatitis C genotype-1b patients compared compared the efficacy and safety of peg-interferon alfa-2b administered once-weekly versus interferon alfa-2b thrice-weekly or daily, both in combination with ribavirin. Seventy-eight previously untreated patients, with biopsy-documented genotype 1 chronic HCV and persistently elevated ALT levels and detectable HCV RNA, were randomized (26 subjects each) to receive: interferon alfa-2b at 6MIUs.c./three-times-weekly (group A) or interferon alfa-2b, 3MIUs.c./daily (group B) or peg-interferon alfa-2b 1.5mcg/Kg s.c./once-weekly (group C). All regimens included standard weight-based doses of ribavirin (800, 1,000 or 1,200 mg/day) administered for 52-weeks. Patients in the three groups were comparable for age, sex, viral load, ALT value and histological-activity-index (HAI). Therapy was completed by 22, 20 and 23 patients in groups A, B and C, respectively. At the end of treatment, a complete (biochemical and virological) response was observed in 50.0% patients of group A, 57.7% of group B and 65.4% of group C. After an additional 24-weeks of follow-up, a sustained response was observed in 26.9%, 46.1% and 50.0% of patients in groups A, B or C, respectively. Therapy was discontinued by 4, 6 and 2 patients because of adverse events in the above three groups. In naive patients with chronic genotype-lb hepatitis C, a 48 week therapy with peg-interferon or interferon at daily doses combined with ribavirin were both more effective than treatment with thrice-weekly interferon in inducing end of treatment and sustained response. Peg-interferon treatment was better tolerated and provoked significantly fewer therapy discontinuations.