Aurora A kinase (AURKA) is a major regulator of mitosis and an important driver of cancer progression. The roles of AURKA outside of mitosis, and how these might contribute to cancer progression, are not well understood. Here, we show that a fraction of cytoplasmic AURKA is associated with mitochondria, co-fractionating in cell extracts and interacting with mitochondrial proteins by reciprocal co-immunoprecipitation. We have also found that the dynamics of the mitochondrial network are sensitive to AURKA inhibition, depletion or overexpression. This can account for the different mitochondrial morphologies observed in RPE-1 and U2OS cell lines, which show very different levels of expression of AURKA. We identify the mitochondrial fraction of AURKA as influencing mitochondrial morphology, because an N-terminally truncated version of the kinase that does not localize to mitochondria does not affect the mitochondrial network. We identify a cryptic mitochondrial targeting sequence in the AURKA N-terminus and discuss how alternative conformations of the protein may influence its cytoplasmic fate.

No abstract

A leading pharmacological strategy toward HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

A leading pharmacological strategy towards HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with Latency Reversal Agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs we used fungal secondary metabolites (extrolites) as a source of bio-active molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the P-TEFb inhibitory 7SK snRNP complex to be significantly reduced upon GTX treatment of independent donor CD4+T cells. GTX disrupted 7SK snRNP, releasing active P-TEFb, which then phosphorylated RNA Pol II CTD, inducing HIV transcription. Our data highlight the power of combining a medium throughput bioassay, mycology and orthogonal mass spectrometry to identify novel potentially therapeutic compounds.

Background: Total HIV-DNA load in peripheral blood cell (PBMCs) reflects the global viral reservoir that seems not to be affected by antiretroviral treatment. However, some studies report-ed a different permeability of different drugs in cellular compartments.Objective: To investigate the relation between the amount of total HIV-1 DNA and different treatment strategies.Methods: Total HIV-1 DNA was quantified by real time PCR in PBMCs collected from 161 patients with long-term undetectable HIV-RNA receiving different therapy schedules (3-drug regimens or 2-drug regimen containing Raltegravir as integrase inhibitor).Results: Overall, HIV patients who started therapy with a median pre-ART CD4+ cell count >400 cells/mm3 and HIV viral load of 3 log10 copies/ml, achieved a lower amount of HIV total DNA. No significant correlation was found in DNA size when patients were stratified on the basis of different therapeutic protocols. However, HIV DNA load analysis, when only performed in HIV patients with a median pre-ART CD4+ cell count >200 cells/mm3 and HIV viral load < 3 log10 copies/ml, showed a significative DNA decrease in Raltegravir treated group with respect to the NNRTIs-treated group.Conclusion: The data emphasize that HIV-DNA level represents a predictive factor in long-term sup-pressive therapy patients. In addition, the diminished reservoir, only observed in patients treated with the NRTI-sparing regimen RAL plus PI/r before immunological and virological derangement, sug-gests that latest generation drugs, such as integrase inhibitors, might represent an optimal chance in the management of HIV infection.

Background and aimsHIV‐1 RNA viral load (VL) in plasma samples of HIV‐1–positive patients is used to assess the level of viral replication, the risk of disease progression, and the response and efficacy to antiretroviral treatment. Knowing the performance of different tests for HIV‐1 RNA detection is, therefore, important for clinical care. This study compared the performance of the recently introduced Aptima HIV‐1 Quant Dx assay (Hologic, Inc) and the standard COBAS AmpliPrep/COBAS TaqMan HIV‐1 v2.0 Test (CAP/CTM2) (Roche Molecular System, Inc) for HIV‐1 RNA quantitation.MethodsAssay performance was assessed using 335 clinical samples, a standard HIV‐1 low VL panel, and 2 diluted samples from well‐characterized patients infected with different HIV‐1 subtypes tested in 5 replicates over 3 days. All samples were tested on both assays to evaluate inter‐assay agreement, both qualitatively and quantitively. Altogether, we evaluated assay sensitivity, linearity, accuracy, precision, repeatability, and reproducibility.ResultsAssay agreement for qualitative results in 335 clinical samples was fair (80.6%). Correlation of quantitative assay results (n = 164) was excellent (R 2 = 0.97), with 96.3% of the results within the 95% limit of assay agreement (−0.42 to +0.86 log), and 98.8% within 1 log of each other. Aptima‐HIV‐1 yielded results, on average, 0.22 log higher than CAP/CTM2. Both assays accurately quantitated the HIV‐1 standard at low VL (R 2 ≥ 0.94), with all samples within 0.5 log of the target.ConclusionAptima‐HIV‐1 assay demonstrated sensitivity, accuracy, reproducibility, and precision for the detection and quantitation of HIV‐1 RNA across a wide dynamic range of VLs. Its performance, together with full automation and high throughput, suggests that Aptima‐HIV‐1 could be a suitable assay for reliable monitoring of HIV‐1 VL in patients undergoing treatment.

Aurora A kinase (AURKA) is a major regulator of mitosis and an important driver of cancer progression. The roles of AURKA outside of mitosis, and how these might contribute to cancer progression, are not well understood. Here we show that a fraction of cytoplasmic AURKA is associated with mitochondria, co-fractionating in cell extracts and interacting with mitochondrial proteins by reciprocal co-immunoprecipitation. We have also found that the dynamics of the mitochondrial network are sensitive to AURKA inhibition, depletion or overexpression. This can account for the different mitochondrial morphologies observed in RPE1 and U2OS cell lines, which show very different levels of expression of AURKA. We identify the mitochondrial fraction of AURKA as influencing mitochondrial morphology, since an N-terminally truncated version of the kinase that does not localize to mitochondria does not affect the mitochondrial network. We identify a cryptic mitochondrial targeting sequence in the AURKA N-terminus and discuss how alternative conformations of the protein may influence its cytoplasmic fate.peer-reviewed)

Total cell-associated HIV-1 DNA is a surrogate marker of the HIV-1 reservoir, however, certified systems for its quantification are not available. The Italian HIV DNA Network was launched to validate HIV-1 DNA quantification methods in use at University and Hospital labs. A quality control panel including HIV-1 DNA standards, reconstructed blood samples (RBSs) and DNA from different HIV-1 subtypes was blindly tested by 12 participating labs by quantitative real-time PCR (n = 6), droplet digital PCR (n = 3) or both (n = 3). The median 95% hit rate was 4.6 (3.7–5.5) copies per test and linearity in the tested range was excellent (R2 = 1.000 [1.000–1.000]). The median values obtained across labs were 3,370 (2,287–4,245), 445 (299–498), 59 (40–81) and 7 (6–11) HIV-1 DNA copies, for the 3,584, 448, 56 and 7-copy standards, respectively. With RBSs, measured values were within twofold with respect to the median in two thirds of cases. HIV-1 subtypes were missed (CRF01_AE by 3 labs) or underestimated by > 1 log (subtypes A, C, D, F by one lab; CRF01_AE by one lab; CRF02_AG by one lab). The overall performance was excellent with HIV-1 DNA standards, however detection of different HIV-1 subtypes must be improved.