No abstract

Highlights Unspecific amplifications were found in 56.4% (495 reactions) of negative samples for SARS-CoV-2. In silico analysis of N2 primers-probe and gel electrophoresis showed dimer formation. Optimization of RT-qPCR conditions reduced the dimerization events. Conditions must be adjusted to avoid extensive test repetition and waste of resources.

ATP-diphosphohydrolases (EC 3.6.1.5), also known as ATPDases, NTPases, NTPDases, EATPases or apyrases, are enzymes that hydrolyze a variety of nucleoside tri- and diphosphates to their respective nucleosides, being their activities dependent on the presence of divalent cations, such as calcium and magnesium. Recently, ATP-diphosphohydrolases were identified on the surface of several parasites, such as Trypanosoma sp, Leishmania sp and Schistosoma sp. In parasites, the activity of ATPdiphosphohydrolases has been associated with the purine recuperation and/or as a protective mechanism against the host organism under conditions that involve ATP or ADP, such as immune responses and platelet activation. These proteins have been suggested as possible targets for the development of new antiparasitic drugs. In this review, we will comprehensively address the main aspects of the location and function of ATP-diphosphohydrolase in parasites. Also, we performed a detailed research in scientific database of recent developments in new natural and synthetic inhibitors of the ATPdiphosphohydrolases in parasites.

Since 2020, humanity has been facing the COVID-19 pandemic, a respiratory disease caused by the SARS-CoV-2. The world’s response to pandemic went through the development of diagnostics, vaccines and medicines. Regarding diagnostics, an enormous challenge was faced due to shortage of materials to collect and process the samples, and to perform reliable mass diagnosis by RT-qPCR. In particular, time-consuming and high cost of nucleic acid extraction procedures have hampered the diagnosis; moreover, several steps in the routine for the preparation of the material makes the extracted sample susceptible to contamination. Here two rapid nucleic acid extraction reagents were compared as extraction procedures for SARS-CoV-2 detection in clinical samples by singleplex and multiplex RT-qPCR analysis, using different transport media, samples with high and low viral load, and different PCR machines. As observed, rapid nucleic acid extraction procedures can be applied for reliable diagnosis using a TaqMan-based assay, over multiple platforms. Ultimately, prompt RNA extraction may reduce costs with reagents and plastics, the chances of contamination, and the overall time to diagnosis by RT-qPCR.

Nesse estudo investigamos a adequabilidade da encapsulação do extrato em acetato de etila do ápice foliar de Baccharis dracunculifolia(BdAA) (Asteraceae), uma planta medicinal brasileira com promissoras aplicações farmacêuticas, em nanopartículas de quitosana (BdAA-Qui). As nanopartículas foram desenvolvidas pelo método de gelificação iônica e caracterizadas em relação ao diâmetro hidrodinâmico médio (DHm), índice de polidispersividade (IP), potencial zeta (PZ), eficiência de encapsulação (EE), com quantificação do Artepillin C, marcador químico do extrato, por CLAE-DAD, morfologia (por microscopia eletrônica de transmissão – MET) e citotoxicidade em linhagem celular de fibroblastos (L929). As BdAA-Qui apresentaram DHm superior às nanopartículas controle (sem o extrato). Esse pode ser considerado o primeiro indício da ocorrência da encapsulação dos constituintes do extrato na matriz polimérica das nanopartículas. Além disso, o IP obtido apresentou valor próximo a de sistemas monodispersos. Essa característica foi comprovada pelas imagens de MET. O valor de PZ positivo (20,4±0,85 mV) é característico da protonação da molécula de quitosana e pode implicar em estabilidade satisfatória das nanopartículas desenvolvidas. Ainda, esse valor foi semelhante àquele obtido para as nanopartículas controle, o que pode sugerir que os constituintes do extrato estejam no interior da nanopartícula e não adsorvidos em sua superfície. Em relação à EE, encontramos um valor elevado (cerca de 85%). Esse dado pode ser devido à interação eletrostática entre a quitosana e o Artepillin C (pka 4.65), o qual encontra-se prioritariamente sob a forma ionizada no pH padronizado para o preparo das nanopartículas (4.7). Por fim, a formulação desenvolvida não alterou a viabilidade das células durante o período de exposição (24 ou 48h). Esse dado preliminar é particularmente importante para garantir a segurança de uso do extrato encapsulado em nanopartículas de quitosana. Pelo exposto, o carreador escolhido mostrou-se adequado para encapsular o extrato BdAA visando futuras aplicações farmacêuticas.

Objectives:The aim of this study was the development and validation of a fast method to quantify artepillin C in green propolis using ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS). Methods: High purity (97.8%) artepillin C was isolated from green propolis using chromatography techniques. Quantification was performed using a C 18 (2.1 x 100 mm; 1.7 μm) column, gradient of water and methanol (with 0.01% formic acid) as mobile phase, at a flow rate of 0.4 mL/min and 45 ºC in temperature. A mass spectrometer operated in selected reaction monitoring mode to monitor the deprotonated molecular ion of artepillin C (m/z 299) > fragment ion (m/z 200.12). Several parameters such as specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, and robustness were determined. Results: The method was linear in the 50 -400 μg/mL range (r 2 = 0.9906), showing LOD = 10.79 μg/mL and LOQ = 32.70 μg/mL with satisfactory intra-day and inter-day precision with relative standard deviation (RSD %) of 1.9% and 3.4%, respectively. The accuracy showed recovery of 93-104%, the method was robust and artepillin C was quantified in green propolis at 6.51%. Conclusions: The proposed method showed advantages in comparison with other methods, such as short analysis time and high selectivity for artepillin C.

The emergence of SARS-CoV-2 variants can affect their detection via RT-qPCR. The Omicron variant has a greater affinity for the upper respiratory system and causes clinical conditions similar to bronchitis, as opposed to the pneumonitis-like conditions caused by other SARS-CoV-2 variants. This characteristic increases the viscosity of clinical samples collected for diagnosis. Coinciding with the arrival of the Omicron variant, we observed a failure in control gene expression in our laboratory. In this report, we have optimized a rapid nucleic acid extraction step to restore gene expression and detect the presence of the SARS-CoV-2 virus. We reevaluated 3000 samples, compared variant types occurring in different time periods, and confirmed that the presence of the Omicron variant was responsible for changes observed in the characteristics of these clinical samples. For samples with large amounts of mucus, such as those containing the Omicron variant, a modification to the nucleic acid extraction step was sufficient to restore the quality of RT-qPCR results.