Susceptibility to re-infection in C57BL/6 mice with recombinant strains of Toxoplasma gondii

AbstractThis work reports results of re-infection of BALB/c and C57BL/6 mice with different recombinant strains of Toxoplasma gondii. Mice were prime-infected with the non-virulent D8 strain and challenged with virulent strains. PCR–RFLP of cS10-A6 genetic marker of T. gondii demonstrated that BALB/c mice were re-infected with the EGS strain, while C57BL/6 mice were re-infected with the EGS and CH3 strains. Levels of IFN-γ and IL-10 after D8 prime-infection were lower in C57BL/6 than in BALB/c mice. Brain inflammation after D8 prime-infection was more intense in C57BL/6 than in BALB/c mice. It was shown that re-infection depends on mice lineage and genotype of the strain used in the challenge

miR-337-3p and Its Targets STAT3 and RAP1A Modulate Taxane Sensitivity in Non-Small Cell Lung Cancers

NSCLC (non-small cell lung cancer) often exhibits resistance to paclitaxel treatment. Identifying the elements regulating paclitaxel response will advance efforts to overcome such resistance in NSCLC therapy. Using in vitro approaches, we demonstrated that over-expression of the microRNA miR-337-3p sensitizes NCI-H1155 cells to paclitaxel, and that miR-337-3p mimic has a general effect on paclitaxel response in NSCLC cell lines, which may provide a novel adjuvant strategy to paclitaxel in the treatment of lung cancer. By combining in vitro and in silico approaches, we identified STAT3 and RAP1A as direct targets that mediate the effect of miR-337-3p on paclitaxel sensitivity. Further investigation showed that miR-337-3p mimic also sensitizes cells to docetaxel, another member of the taxane family, and that STAT3 levels are significantly correlated with taxane resistance in lung cancer cell lines, suggesting that endogenous STAT3 expression is a determinant of intrinsic taxane resistance in lung cancer. The identification of a miR-337-3p as a modulator of cellular response to taxanes, and STAT3 and RAP1A as regulatory targets which mediate that response, defines a novel regulatory pathway modulating paclitaxel sensitivity in lung cancer cells, which may provide novel adjuvant strategies along with paclitaxel in the treatment of lung cancer and may also provide biomarkers for predicting paclitaxel response in NSCLC

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Proposed model of taxane sensitization mediated by miR-337-3p and its targets RAP1A and STAT3.

<p>Proposed model of taxane sensitization mediated by miR-337-3p and its targets RAP1A and STAT3.</p

<i>RAP1A</i> and <i>STAT3</i> are direct targets of miR-337-3p.

<p>(A) miR-337-3p and its predicted interaction with target sites in <i>RAP1A</i> and <i>STAT3</i>. Shown are the structures and sequences of the miRNA:target interactions for miR-337-3p and the 3′UTRs of <i>RAP1A</i> and <i>STAT3</i>, and the predicted free energy of hybridization. The seed sequence is highlighted in red. Bases altered by site-directed mutagenesis are underlined. (B) Luciferase reporter assay. NCI-H1155 cells were co-transfected with the indicated oligos and the luciferase reporter vectors. Luciferase and β-galactosidase activities were measured after 72 h, with luciferase activity normalized to β-galactosidase activity. (C) Expression of <i>RAP1A</i> and <i>STAT3</i> mRNA and protein levels after 72 h of transfection of NCI-H1155 cells with either 50 nM miR-337-3p, 25 nM siRNA directed against each of the genes or negative control oligos. Shown are average qRT-PCR results of three independent transfections and representative Western blot images and quantification of band intensities. (D) <i>RAP1A</i> and <i>STAT3</i> mRNA and protein expression in NCI-H1993 cells. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Over-expression of miR-337-3p sensitizes NCI-H1155 cells to paclitaxel.

<p>(A) Dose-dependent effect of paclitaxel on cell viability in the presence or absence of miR-337-3p mimic. Cell viability was measured using the MTS assay. (*, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant) (B) Cell viability as a function of oligo concentration in the presence or absence of paclitaxel. Cell viability was measured using the ATP concentration assay. (C) Effect of miR-337-3p knockdown with miR-337-3p inhibitor (50 nM) on paclitaxel sensitivity in H1155 cells. Shown is the relative cell viability in the presence of 16 nM paclitaxel normalized to control oligos. (****, p<0.0001) (D) Cell cycle analysis as a function of miR-337-3p overexpression and paclitaxel treatment. The fraction of cells in G₁, S and G₂ phases was estimated using the Watson pragmatic model, with the ratio of G₂ to G₁ fractions for paclitaxel treated cells normalized to that observed for control conditions (R = [G₂/G₁]_paclitaxel/[G₂/G₁]_carrier).</p

siRNA knockdown of regulatory targets of miR-337-3p sensitizes NCI-H1155 cells to paclitaxel.

<p>(A) Effect of knockdown of <i>RAP1A</i> and <i>STAT3</i> expression by siRNA on cell viability in the presence and absence of paclitaxel. Shown in red is cell viability in 16 nM paclitaxel normalized to viability in carrier as a function of increasing concentrations of siRNAs against <i>RAP1A</i> or <i>STAT3</i>. Shown in black is cell viability in the absence of paclitaxel. (B) Effect of combined knockdown of <i>RAP1A</i> and <i>STAT3</i> on paclitaxel sensitivity. Shown is the relative cell viability in the presence 16 nM paclitaxel normalized to control oligos. (C) Knockdown of STAT3 and RAP1A enhances paclitaxel-induced G₂/M arrest as measured by flow cytometry. The ratio (R) of the G₂ to G₁ fractions induced by paclitaxel treatment was determined as above. (D) Cell viability as a function of oligo concentration (miR-337-3p mimic or negative control mimic) in the presence of cucurbitacin. *, p<0.05.</p