Transcriptional analysis of THP-1 cells infected with Leishmania infantum indicates no activation of the inflammasome platform.

Leishmaniasis is caused by intracellular parasites transmitted to vertebrates by sandfly bites. Clinical manifestations include cutaneous, mucosal or visceral involvement depending upon the host immune response and the parasite species. To assure their survival inside macrophages, these parasites developed a plethora of highly successful strategies to manipulate various immune system pathways. Considering that inflammasome activation is critical for the establishment of a protective immune response in many parasite infections, in this study we determined the transcriptome of THP-1 cells after infection with L. infantum, with a particular focus on the inflammasome components. To this end, the human cell line THP-1, previously differentiated into macrophages by PMA treatment, was infected with L. infantum promastigotes. Differentiated THP-1 cells were also stimulated with LPS to be used as a comparative parameter. The gene expression signature was determined 8 hours after by RNA-seq technique. Infected or uninfected THP-1 cells were stimulated with nigericin (NIG) to measure active caspase-1 and TNF-α, IL-6 and IL-1ß levels in culture supernatants after 8, 24 and 48 hours. L. infantum triggered a gene expression pattern more similar to non-infected THP-1 cells and very distinct from LPS-stimulated cells. Some of the most up-regulated genes in L. infantum-infected cells were CDC20, CSF1, RPS6KA1, CD36, DUSP2, DUSP5, DUSP7 and TNFAIP3. Some up-regulated GO terms in infected cells included cell coagulation, regulation of MAPK cascade, response to peptide hormone stimulus, negative regulation of transcription from RNA polymerase II promoter and nerve growth factor receptor signaling pathway. Infection was not able to induce the expression of genes associated with the inflammasome signaling pathway. This finding was confirmed by the absence of caspase-1 activation and IL-1ß production after 8, 24 and 48 hours of infection. Our results indicate that L. infantum was unable to activate the inflammasomes during the initial interaction with THP-1 cells.

A novel molecular mechanism to explain mutations of the HCV protease associated with resistance against covalently bound inhibitors.

NS3 is an important therapeutic target for direct-acting antiviral (DAA) drugs. However, many patients treated with DAAs have unsustained virologic response (UVR) due to the high mutation rate of HCV. The aim of this work was to shed some light on the puzzling molecular mechanisms of the virus's of patients who showed high viral loads even under treatment with DAA. Bioinformatics tools, molecular modelling analyses were employed to identify mutations associated with HCV resistance to boceprevir and possible structural features related to this phenomenon. We identified two mutations of NS3 that may be associated with HCV resistance: D168N and L153I. The substitution D168N was previously reported in the literature as related with drug failure. Additionally, we identified that its molecular resistance mechanism can be explained by the destabilization of receptor-ligand hydrogen bonds. For the L153I mutation, the resistance mechanism is different from previous models reported in the literature. The L153I substitution decreases the S139 deprotonation susceptibility, and consequently, this mutation impairs the covalent binding between the residue S139 from NS3 and the electrophilic trap on boceprevir, which can induce drug failure. These results were supported by the time course analysis of the mutations of the NS3 protease, which showed that boceprevir was designed for enzymes with an L residue at position 153; however, the sequences with I153 are predominant nowadays. The results presented here could be used to infer about resistance in others DAA, mainly protease inhibitors.

Comparative cytogenetics of cichlid fishes through genomic in-situ hybridization (GISH) with emphasis on Oreochromis niloticus.

Cichlidae is the most species-rich freshwater family of Perciformes and has attracted the attention of aquarium hobbyists, aquaculturists, and sport fisherman. Oreochromis niloticus is very important in aquaculture today and is currently used in varied areas of study as an 'experimental model'. Oreochromis niloticus has been characterized using classical and molecular cytogenetic techniques, with special attention paid to heterochromatin structure and the identification of sex chromosomes. In this study, we compare the genome of O. niloticus with that of other cichlids from Africa and South America using genomic in-situ hybridization (GISH). Our results show that at least some elements comprising the pericentromeric heterochromatin of Nile tilapia are species-specific and that the sequence of the majority of the long arm of the largest chromosome pair is conserved within the tilapiine group, which is composed of the genera Tilapia, Oreochromis, and Sarotherodon. It is suggested that the extensive regions of repeated DNA in the largest chromosome pair of O. niloticus resulted from chromosome rearrangement or accumulation caused by recombination suppression during the evolutionary history of the tilapiines.