HIV and reservoir

Il maggiore ostacolo all’eradicazione di HIV è la presenza del reservoir virale trascrizionalmente silente ma in grado di replicare e difficilmente accessibile alla terapia. Reservoir di piccole dimensioni correlano con una prognosi migliore e una minor probabilità di rebound virologico in caso di semplificazione di terapia. La quantificazione del reservoir risulta pertanto essenziale per valutare la reale efficacia della terapia. La quantificazione dell’HIV-DNA totale (circolare e integrato) rappresenta una metodica semplice e ampiamente utilizzata per analizzare il reservoir, nonostante ne sovrastimi le reali dimensioni includendo genomi defettivi. Pertanto, inizialmente abbiamo ottimizzato una metodica per la quantificazione delle forme circolari di HIV-DNA. La forma circolare 2-LTR è stata misurata con successo; ciò non è stato possibile per la forma 1-LTR a causa di omologie di sequenza con il DNA genomico. Come parte di uno studio attualmente in corso, sette pazienti sono stati arruolati alla diagnosi (T0) e seguiti con prelievi a 6(T1), 12(T2), 18(T3) e 24(T4) mesi dopo l’inizio della terapia. Dati preliminari ottenuti nel primo anno (T0,T1,T2) mostrano un andamento dell’HIV-DNA totale stabile nel tempo (P>0,1); analogamente, la forma 2-LTR non mostra aumenti significativi tra T0 e T1 (P>0,5). Tuttavia, a T0 è stata osservata una differenza statisticamente significativa tra l’HIV-DNA totale e la forma 2-LTR (P<0,05). La seconda parte dello studio si è focalizzata sul saggio TILDA, un metodo in grado di misurare esclusivamente virus competenti la replicazione. Abbiamo adattato questa metodica alle nostre condizioni sperimentali eseguendola su 3 pazienti HIV-positivi. Infine, considerando il costante aumento dei casi di infezione con sottotipi non-B e che il saggio TILDA è stato sviluppato principalmente per il sottotipo B, il protocollo è stato modificato in modo da rilevare il sottotipo C, il più diffuso a livello mondiale. In particolare, è stato eseguito con successo su 3 pazienti infetti con il sottotipo C.Persistence of transcriptionally silent but replication-competent HIV reservoir in resting CD4+T cells constitutes the major barrier to viral eradication. Small latent reservoirs are associated to a better prognosis and predictive of less likelihood to undergo virological rebound during simplified therapy. Therefore, the amount of HIV-DNA in latently infected cells is critical to evaluate the efficacy of available regimens and avoid the potential “re-seeding” in aviremic patients. Although the quantification of total HIV-DNA (circular and integrated) overestimates reservoir size because it includes not replication-competent genomes, it represents a simple and widely used biomarker reflecting global viral reservoir. Therefore, first we optimized PCR methods for detection of circular forms of HIV-DNA in HIV-positive patients. We successfully measured the 2-LTR form, but not the 1-LTR form due to homologies with genomic DNA sequence. As part of an ongoing project on HIV-DNA, we tested our in house assay on seven PBMCs samples of treatment-naïve patients collected at 0(T0), 6(T1), 12(T2), 18(T3) and 24(T4) months after starting ART. Preliminary data on T0,T1, and T2 time points showed that total HIV-DNA is stable during time (P>0,1); likewise, the amount of 2-LTR forms did not show significant differences between T0 and T1 time points (P>0,5). However, we observed a statistically significant difference between 2-LTR form and total HIV-DNA amounts at T0 (P<0,05). The second part of this study focused on TILDA, a method that detects only transcriptionally-competent proviruses. Hence, we adapted TILDA to our experimental conditions performing the modified assay in three long-term treated patients. Moreover, considering that the frequency of non-B subtype infections is constantly increasing and TILDA has been developed based on subtype B virus sequence, we modified the protocol to detect subtype C virus, the most prevalent subtype worldwide. We successfully perform the new version of TILDA in three patients harboring subtype C viruses

Comparison of the Aptima HIV-1 Quant Dx Assay with the COBAS\uae AmpliPrep/COBAS\uae TaqMan\uae HIV-1 v2.0 Test for HIV-1 Viral Load Quantification in Plasma Samples from HIV-1-Infected Patients.

Background and aims: HIV\u20101 RNA viral load (VL) in plasma samples of HIV\u20101\u2013positive 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\u20101 RNA detection is, therefore, important for clinical care. This study compared the performance of the recently introduced Aptima HIV\u20101 Quant Dx assay (Hologic, Inc) and the standard COBAS AmpliPrep/COBAS TaqMan HIV\u20101 v2.0 Test (CAP/CTM2) (Roche Molecular System, Inc) for HIV\u20101 RNA quantitation. Methods: Assay performance was assessed using 335 clinical samples, a standard HIV\u20101 low VL panel, and 2 diluted samples from well\u2010characterized patients infected with different HIV\u20101 subtypes tested in 5 replicates over 3 days. All samples were tested on both assays to evaluate inter\u2010assay agreement, both qualitatively and quantitively. Altogether, we evaluated assay sensitivity, linearity, accuracy, precision, repeatability, and reproducibility. Results: Assay agreement for qualitative results in 335 clinical samples was fair (80.6%). Correlation of quantitative assay results (n = 164) was excellent (R2 = 0.97), with 96.3% of the results within the 95% limit of assay agreement ( 120.42 to +0.86 log), and 98.8% within 1 log of each other. Aptima\u2010HIV\u20101 yielded results, on average, 0.22 log higher than CAP/CTM2. Both assays accurately quantitated the HIV\u20101 standard at low VL (R2 65 0.94), with all samples within 0.5 log of the target. Conclusion: Aptima\u2010HIV\u20101 assay demonstrated sensitivity, accuracy, reproducibility, and precision for the detection and quantitation of HIV\u20101 RNA across a wide dynamic range of VLs. Its performance, together with full automation and high throughput, suggests that Aptima\u2010HIV\u20101 could be a suitable assay for reliable monitoring of HIV\u20101 VL in patients undergoing treatment

Constitutive regulation of mitochondrial morphology by Aurora A kinase depends on a predicted cryptic targeting sequence at the N-terminus.

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.MRC CRU

Metallurgical characterization of Reciproc and Reciproc Blue Ni-Ti instruments

Reciproc and Reciproc Blue files are composed of the same NiTi alloy but they undergo different thermal treatment. The Reciproc Blue show a titanium oxide superficial layer as confirmed by Raman spectroscopy and are more flexible than Reciproc M-Wire due to their different thermal treatment. The smaller and denser grains of Reciproc Blue files are responsible for the different transition temperatures of the two instruments

Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency

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

Terrestrial Very-Long-Baseline Atom Interferometry:Workshop Summary

This document presents a summary of the 2023 Terrestrial Very-Long-Baseline Atom Interferometry Workshop hosted by CERN. The workshop brought together experts from around the world to discuss the exciting developments in large-scale atom interferometer (AI) prototypes and their potential for detecting ultralight dark matter and gravitational waves. The primary objective of the workshop was to lay the groundwork for an international TVLBAI proto-collaboration. This collaboration aims to unite researchers from different institutions to strategize and secure funding for terrestrial large-scale AI projects. The ultimate goal is to create a roadmap detailing the design and technology choices for one or more km-scale detectors, which will be operational in the mid-2030s. The key sections of this report present the physics case and technical challenges, together with a comprehensive overview of the discussions at the workshop together with the main conclusions

Analysis of post-fire forest regeneration in a mesalpic environment: compound effect of windthrow and forest fire

openDal punto di vista ecologico i disturbi naturali modificano la struttura e la composizione di un ecosistema. All’interno di un bosco, sono eventi che danno inizio a fenomeni di successione secondaria, vale a dire la sostituzione del soprassuolo esistente con un diverso tipo di formazione forestale. I fenomeni degli incendi boschivi e degli schianti da vento sono tra i principali disturbi naturali di tipo abiotico che interessano i boschi delle Alpi. Nelle Alpi, gli incendi boschivi si concentrano solitamente nel periodo invernale-primaverile per via delle precipitazioni poco abbondanti e l’accumulo di sostanza secca, ma in presenza di stagioni calde e siccitose si possono registrare anche in estate. È questo il caso dell’incendio ad alta severità che si è sviluppato in un popolamento montano di abete rosso nel comune di Frassilongo (TN) nel luglio 2022. Il fuoco ha interessato sia una porzione di pecceta in piedi che un abieteto in cui erano presenti schianti da vento a causa del passaggio della tempesta Vaia nell’ottobre 2018. Il seguente lavoro si è posto l’obiettivo di mettere a confronto le dinamiche in atto nelle due aree per quanto riguarda la rinnovazione forestale post-disturbo in termini di specie, densità e altezza degli individui in funzione di variabili quali parametri topografici (quota, pendenza, esposizione), copertura del suolo e natura della necromassa. Lo studio è stato condotto in 40 aree di saggio distribuite equamente tra le due aree di studio (bosco schiantato e bosco in piedi). I risultati dei test statistici effettuati evidenziano che l’altezza e la densità della rinnovazione sono influenzate dalle caratteristiche del sito in termini di copertura del suolo, in particolare dal tipo e dalla disposizione sul terreno della necromassa.From the ecological point of view, natural disturbances change the structure and composition of an ecosystem. Within a forest, these are events that initiate secondary succession, which is the replacement of the existing stand with a different type of forest formation. Wildland fires and windthrows are among the main natural abiotic disturbances affecting forests in the Alps. Forest fires in the Alps are usually concentrated in the winter-spring period due to low rainfall and accumulation of dry biomass. However, in the presence of hot and dry seasons, a significant number of events are also recorded in summer. This is the case with the high-severity fire that developed in a mountain stand of spruce in the municipality of Frassilongo (TN) in July 2022. The fire affected both a standing part of the spruce forest and an area where slash was present due to storm Vaia in October 2018. The following work aims to compare the dynamics regarding post-disturbance forest regeneration in the two areas, in terms of species, density and height of individuals as a function of variables such as topographic parameters (elevation, slope, exposure), land cover and nature of deadwood. The study was conducted in 40 circular test plots equally divided between the two study areas (windthrown forest and standing forest). The results of the statistical tests show that the height and density of the regeneration are influenced by the characteristics of the site in terms of land cover, mainly by the type of deadwood and its arrangement on the ground

Expression of TDRD9 in a subset of lung carcinomas by CpG island hypomethylation protects from DNA damage

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.This work was supported through funding to C.L. from Cancer Research UK (C3/A10239), Medical Research Council (MR/ M01102X/1) and the Department of Genetics. D.M.G. received support from Cancer Research UK (C3/A18795). R.G. was supported by a PhD studentship from the MRC and additional funding from the Department of Genetics, Cambridge Philosophical Society, Crane’s Charity and DRC. A.M.A. is the recipient of a Yousef Jameel Scholarship from the Cambridge International Trust. A.B. received an Erasmus placement (European Commission Lifelong Learning programme) and M.P.G. was supported by Consolider (CSD2009-00016) and Ministerio de Ciencia e Innovacio´n, Spain, through JdC and Jose Castillejo programmes. J.M. is supported by the German Research Foundation (DFG) (Emmy Noether; MA 5831/1-1) and receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 680042).Peer reviewe

Evaluation of the usage-induced degradation of Genius and Reciproc nickel–titanium reciprocating instruments

The aim of this study was to characterize the main features and the usage-induced degradation of the Genius file after four severely curved root canal instrumentations and to compare their properties to the Reciproc files. Brand new and ex vivo used files were analysed by scanning electron microscopy (SEM) with energy-dispersive X-ray spectrometry (EDS), differential scanning calorimetry (DSC), X-ray diffraction (XRD), optical metallography, and nano-indentation to disclose their morphological, chemical, mechanical, thermal, and phase composition features. Nano-indentation data were statistically analysed using the Student’s t test for normal distribution or the Kolmogorov–Smirnov test for not-normal distributions. SEM analysis showed the presence of micro-cracks near the tip on both files after ex vivo usage test. EDS analysis confirmed that both files are manufactured from an almost equiatomic NiTi alloy. DSC analysis revealed that the transition temperature of the Genius is below 20&nbsp;°C, while that of the Reciproc is above 20&nbsp;°C. XRD analysis of Genius files identified cubic B2 austenite with minor peaks of residual monoclinic B19 martensite, while the contemporaneous presence of martensite, austenite and hexagonal R-phase was observed in the Reciproc files. Significant differences in nanohardness and modulus of elasticity (P &lt;.05) were observed in both Genius and Reciproc files before and after use. The collected results showed that both instruments can be safely used as single-use files