Massive NGS data analysis reveals hundreds of potential novel gene fusions in human cell lines

Background: Gene fusions derive from chromosomal rearrangements and the resulting chimeric transcripts are often endowed with oncogenic potential. Furthermore, they serve as diagnostic tools for the clinical classification of cancer subgroups with different prognosis and, in some cases, they can provide specific drug targets. So far, many efforts have been carried out to study gene fusion events occurring in tumor samples. In recent years, the availability of a comprehensive Next Generation Sequencing dataset for all the existing human tumor cell lines has provided the opportunity to further investigate these data in order to identify novel and still uncharacterized gene fusion events. Results: In our work, we have extensively reanalyzed 935 paired-end RNA-seq experiments downloaded from "The Cancer Cell Line Encyclopedia" repository, aiming at addressing novel putative cell-line specific gene fusion events in human malignancies. The bioinformatics analysis has been performed by the execution of four different gene fusion detection algorithms. The results have been further prioritized by running a bayesian classifier which makes an in silico validation. The collection of fusion events supported by all of the predictive softwares results in a robust set of ∼ 1,700 in-silico predicted novel candidates suitable for downstream analyses. Given the huge amount of data and information produced, computational results have been systematized in a database named LiGeA. The database can be browsed through a dynamical and interactive web portal, further integrated with validated data from other well known repositories. Taking advantage of the intuitive query forms, the users can easily access, navigate, filter and select the putative gene fusions for further validations and studies. They can also find suitable experimental models for a given fusion of interest. Conclusions: We believe that the LiGeA resource can represent not only the first compendium of both known and putative novel gene fusion events in the catalog of all of the human malignant cell lines, but it can also become a handy starting point for wet-lab biologists who wish to investigate novel cancer biomarkers and specific drug targets

Association of amniotic uric acid, glucose, lactate and creatinine concentrations and lactate/creatinine ratio with newborn survival in small-sized dogs – preliminary results

In order to define the normal composition of canine amniotic fluid and to detect differences between surviving and non-surviving newborn puppies, the present study determined the uric acid, glucose, lactate and creatinine concentrations and the lactate to creatinine ratio in amniotic fluids collected during elective Caesarean section from small-sized purebred bitches. The possible relationship between newborn survival and the studied parameters, as well as the effects of maternal parity, fetal gender and Apgar score were assessed. The study enrolled 27 small-sized purebred bitches submitted to elective Caesarean section at term. After opening the fetal membranes, amniotic fluid samples were collected aseptically from the amniotic sac of each fetus. The data obtained from 74 amniotic fluid samples collected from 27 bitches showed that amniotic glucose concentration was lower (P < 0.05) in non-surviving than in surviving puppies. Within the normal, surviving puppies, amniotic glucose concentration was higher (P < 0.05) in male than in female newborns, and the lactate/creatinine ratio was significantly higher in multiparous than in primiparous bitches (P < 0.05). These preliminary results demonstrate the relevance of amniotic glucose, but not of uric acid, lactate, creatinine and the lactate to creatinine ratio for detecting puppies at risk of death immediately after birth

Improvement of ALT decay kinetics by all-oral HCV treatment: Role of NS5A inhibitors and differences with IFN-based regimens

Background: Intracellular HCV-RNA reduction is a proposed mechanism of action of direct-acting antivirals (DAAs), alternative to hepatocytes elimination by pegylated-interferon plus ribavirin (PR). We modeled ALT and HCV-RNA kinetics in cirrhotic patients treated with currently-used all-DAA combinations to evaluate their mode of action and cytotoxicity compared with telaprevir (TVR)+PR. Study design: Mathematical modeling of ALT and HCV-RNA kinetics was performed in 111 HCV-1 cirrhotic patients, 81 treated with all-DAA regimens and 30 with TVR+PR. Kinetic-models and Cox-analysis were used to assess determinants of ALT-decay and normalization. Results: HCV-RNA kinetics was biphasic, reflecting a mean effectiveness in blocking viral production &gt;99.8%. The first-phase of viral-decline was faster in patients receiving NS5A-inhibitors compared to TVR+PR or sofosbuvir+simeprevir (p&lt;0.001), reflecting higher efficacy in blocking assembly/secretion. The second-phase, noted \u3b4 and attributed to infected-cell loss, was faster in patients receiving TVR+PR or sofosbuvir+simeprevir compared to NS5A-inhibitors (0.27 vs 0.21 d-1, respectively, p = 0.0012). In contrast the rate of ALT-normalization, noted \u3bb, was slower in patients receiving TVR+PR or sofosbuvir+simeprevir compared to NS5A-inhibitors (0.17 vs 0.27 d-1, respectively, p&lt;0.001). There was no significant association between the second-phase of viral-decline and ALT normalization rate and, for a given level of viral reduction, ALT-normalization was more profound in patients receiving DAA, and NS5A in particular, than TVR+PR. Conclusions: Our data support a process of HCV-clearance by all-DAA regimens potentiated by NS5A-inhibitor, and less relying upon hepatocyte death than IFN-containing regimens. This may underline a process of "cell-cure" by DAAs, leading to a fast improvement of liver homeostasis

Procedure for rapid oocyte selection based on quantitative analysis of cumulus cell gene expression

PURPOSE: To develop a procedure for the analysis of gene expression in cumulus cells during the interval between ovum pick up and insemination to select the best oocytes for fertilization. METHODS: Five RNA extraction methods, three reverse transcription procedures followed by Real-time quantitative PCR and one single-step mRNA quantification kit were tested to measure the expression of five genes in cumulus cells. RESULTS: Two RNA extraction kits gave the best combination of efficiency and purity. One reverse transcription procedure gave the best speed and efficiency. The single-step kit required more biological material than would be available from single cumulus oocyte complexes (COCs). CONCLUSIONS: Our test identified a combination of RNA extraction and reverse transcription procedures that enables the level measurement of 5 selected cumulus cell transcripts within 4 h. Using this combination it was possible to obtain a reliable quantification of gene expression in 44 out of 46 individual COCs collected from seven patients

Predicted kinetic profiles obtained by simulations from the viral and ALT kinetic models.

<p>(panel <b>A</b>) Different viral kinetics according to HCV-genotypes, NS5A-inhibitors and interferon administration, (panel <b>B</b>) Different ALT kinetics according to NS5A-inhibitors and interferon administration. ALT, alanine transaminase; IFN, interferon.</p

Population kinetic parameters for the best HCV and ALT kinetic model.

<p>Population kinetic parameters for the best HCV and ALT kinetic model.</p

Decrease of viral load and ALT at different time points under treatment according to NS5A-inhibitors and interferon administration.

<p>These graphs shows the median of predictions of ALT and HCV-RNA in different groups of treatment obtained by simulations from the parameter estimates of the two models. ALT, alanine transaminase; IFN, interferon.</p

Biphasic kinetics of HCV-RNA decay, and ALT drop during all-DAA and TVR+PR treatment and follow-up.

<p>In <i>upper panels</i>, median values with 95% confidence interval of HCV-RNA (black dots) and ALT (grey squares) during all-DAAs (panel <b>A</b>) and TVR+PR (panel <b>B</b>) treatment are reported. End of follow-up is at 12 weeks after treatment discontinuation. Black dotted line represents the lower limit of detection of HCV-RNA (12–15 IU/ml). Grey dotted line represents normality range of ALT values in females (45 IU/ml). Histograms in <i>lower panels</i> represent the percentages of patients with HCV-RNA below the lower limit of detection (panel <b>C</b>) and with normal ALT values (panel <b>D</b>) during all-DAAs (black) and TVR+PR (grey) treatment. Normal ALT values were considered as <55 IU/ml in men, and <45 IU/ml in women. ALT, alanine transaminase; DAA, direct-acting antivirals; EOT, end of treatment; IU, international units; LLOD, lower limit of detection (<12–15 IU/ml, not detected); PR, pegylated interferon and ribavirin; TVR, telaprevir. * p-value <0.05 by Fisher exact test; ** p-value ≤0.001 by Fisher exact test.</p

Cox analysis for factors influencing ALT normalization during treatment.

<p>Cox analysis for factors influencing ALT normalization during treatment.</p