Molecular screening for Plasmodium falciparum resistance markers for artemisinins in Mbita, Kenya

Artemisinins-based combination therapies (ACTs) are being recommended against uncomplicated malaria in endemic areas of Africa. However, in these areas data on their long term usefulness is limited. It has been demonstrated that ACTs resistance may be due to single nucleotide polymorphisms (SNPs) in the chemotherapeutic target, the SERCA-type ATPase protein (PfATPase6). This study analyzed PfATPase6 mutations in asymptomatic infections from samples collected from Mbita, a malaria endemic region in Kenya. Mutations in A623E and S769N residues were screened with gene specific primers followed by sequencing. The study demonstrates that there is no mutation in Mbita, Kenya because neither A623E nor S769N PfATPase6 mutations were detected. Resurgence of infections in this area could be due to re-infections and not drug failure. The study recommends that other sites be assessed for PfATPase 6 mutations to verify the long-term usefulness of ACT and monitor any emergency of resistance.Keywords: ACT (Artemisinins-based Combination Therapy), Molecular, Mutations, PfATPase 6doi: 10.4314/ajcem.v12i3.

Prognostic Significance of CSF-1R Expression in Early Invasive Breast Cancer

Colony-stimulating factor-1 receptor (CSF-1R) signaling promotes an immune suppressive microenvironment enriched in M2 macrophages. Given that CSF-1R inhibitors are under investigation in clinical trials, including in breast cancer, CSF-1R expression and association with immune biomarkers could identify patients who derive greater benefit from combination with immunotherapies. TIMER2.0 and bc-GenExMiner v4.7 were used to assess the correlation of CSF1R mRNA with immune infiltrates and prognosis. Following a prespecified training–validation approach, an optimized immunohistochemistry assay was applied to assess CSF-1R on carcinoma cells and macrophages on breast cancer tissue microarray series representing 2384 patients, coupled to comprehensive clinicopathological, biomarker, and outcome data. Significant positive correlations were observed between CSF1R mRNA and immune infiltrates. High carcinoma CSF-1R correlated with grade 3 tumors >2 cm, hormone receptor negativity, high Ki67, immune checkpoint biomarkers, and macrophages expressing CSF-1R and CD163. High carcinoma CSF-1R was significantly associated with poor survival in univariate and multivariate analyses. Adverse prognostic associations were retained in ER+ cases regardless of the presence of CD8+ T cells. CSF-1R+ macrophages were not prognostic. High carcinoma CSF-1R is associated with aggressive breast cancer biology and poor prognosis, particularly in ER+ cases, and identifies patients in whom biomarker-directed CSF-1R therapies may yield superior therapeutic responses.Medicine, Faculty ofNon UBCPathology and Laboratory Medicine, Department ofReviewedFacult

The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication

Background: HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2α), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. Results: PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2α phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2α phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. Conclusion: In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.</p

The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication

Background: HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2α), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. Results: PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2α phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2α phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. Conclusion: In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.</p