Repression of HIV-1 reactivation mediated by CRISPR/dCas9-KRAB in lymphoid and myeloid cell models.

BACKGROUND: Despite antiretroviral treatment efficacy, it does not lead to the complete eradication of HIV infection. Consequently, reactivation of the virus from latently infected cell reservoirs is a major challenge toward cure efforts. Two strategies targeting viral latency are currently under investigation: the "shock and kill" and the "block and lock." The "Block and Lock" methodology aims to control HIV-1 latency reactivation, promoting a functional cure. We utilized the CRISPR/dCas9-KRAB platform, which was initially developed to suppress cellular genes transcription, to block drug-induced HIV-1 reactivation in latently infected T cells and myeloid cells. RESULTS: We identified a set of five sgRNAs targeting the HIV-1 proviral genome (LTR1-LTR5), having the lowest nominated off-target activity, and transduced them into the latently infected lymphoid (J-Lat 10.6) and myeloid (U1) cell lines. One of the sgRNAs (LTR5), which binds specifically in the HIV-1 LTR NFκB binding site, was able to promote robust repression of HIV-1 reactivation in latently infected T cells stimulated with Phorbol 12-Myristate 13-Acetate (PMA) and Ingenol B (IngB), both potent protein kinase C (PKC) stimulators. Reactivation with HDAC inhibitors, such as SAHA and Panobinostat, showed the same strong inhibition of reactivation. Additionally, we observed a hundred times reduction of HIV-1 RNA expression levels in the latently infected myeloid cell line, U1 induced with IngB. CONCLUSION: Taken together, our results show that the KRAB fused CRISPR/dCas9 system can robustly prevent the HIV-1 latency reactivation process, mediated by PMA or IngB and SAHA or Panobinostat, both in myeloid and lymphoid HIV-1 latently infected cells. In addition, we demonstrated that KRAB repressor protein is crucial to reactivation resistance phenotype, and we have identified some useful hotspots sequences in HIV-1 LTR for the design sgRNAs.

NGAL como marcador precoce de lesão renal em ratos submetidos à isquemia renal sob anestesia geral balanceada / NGAL as an early marker of renal injury in rats submitted to renal ischemia under general balanced anesthesia

A lesão renal aguda (LRA) caracterizada como redução abrupta da função renal tem incidência variável, dependendo dos critérios utilizados para sua definição. Atualmente, o mais empregado para avaliação de função renal é a dosagem de creatinina plasmática, porém, por sofrer interferência de vários fatores faz com que a mesma não seja o marcador ideal. Um novo biomarcador da função renal, a lipocalina associada à gelatinase dos neutrófilos (NGAL), tem sido estudado como um dos mais precoces e sensíveis marcadores de lesão de rins após isquemia/reperfusão ou lesão nefrotóxica, sendo facilmente identificada no sangue e na urina. A classificação RIFLE para LRA com a validação da NGAL é uma nova perspectiva para o diagnóstico precoce da LRA e instituição de medidas preventivas e protetoras em situações de risco. O objetivo dessa pesquisa foi avaliar a LRA por meio da dosagem plasmática da NGAL em ratos submetidos à isquemia renal sob anestesia geral balanceada e hidratados com Ringer lactato ou com hidroxietilamido e correlacionar a dosagem da NGAL plasmática com a lesão histológica renal. Em trinta ratos Wistar machos (>350g) distribuídos aleatoriamente em três grupos de dez animais, induzidos com isoflurano a 4%, foi realizada intubação orotraqueal e colocados sob ventilação mecânica. Foram cateterizadas a artéria carótida esquerda e a veia jugular direita para monitorização e coletas sanguíneas. ETCO2, PAMI, T foram continuamente monitorizados (Datex, AS3)...

Acute renal injury (ARI), characterized by abrupt renal function decline, has variable incidence, depending on the criteria used for its definition. Presently, the criterion most often used for kidney function evaluation is plasma creatinine dosing; however, because it suffers interference, it is not an ideal marker. A new renal function biomarker, neutrophil gelatinase-associated lipocalin (NGAL) has been studied as one of the earliest and most sensitive kidney injury marker following ischemia/reperfusion or nephrotoxic injury, and it is easily identified in blood and urine. The RIFLE classification for ARI with NGAL validation is a new perspective for early ARI diagnosis and institution of preventive and protective measures in risk situations. This study aimed at evaluating ARI by means of NGAL plasma dosing in rats submitted to renal ischemia under general balanced anesthesia and hydrated with ringer lactate or hydroxyethylamide and at correlating plasma NGAL dosing with histological renal injury. Thirty male Wistar rats (>350g) were used. They were randomly distributed into three groups with ten animals each and induced by 4% isoflurane. Orotracheal intubation was performed and the animals were placed under mechanical ventilation. The left carotid artery and the right jugular vein were catheterized for monitorization and blood collection. ETCO2, PAMI, T were continuously monitorized (Datex, AS3)...