Electronic circular dichroism of the Cas9 protein and gRNA : Cas9 ribonucleoprotein complex

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex

Chloroquine-induced accumulation of autophagosomes and lipids in the endothelium

Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome–lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid composition of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1). HMEC-1 cells after CQ exposure were measured using a combined methodology based on label-free Raman and fluorescence imaging. Raman spectroscopy was applied to characterize subtle chemical changes in lipid contents and their distribution in the cells, while the fluorescence staining (LipidTox, LysoTracker and LC3) was used as a reference method. The results showed that CQ was not toxic to endothelial cells and did not result in the endothelial inflammation at concentrations of 1–30 µM. Notwithstanding, it yielded an increased intensity of LipidTox, LysoTracker, and LC3 staining, suggesting changes in the content of neutral lipids, lysosomotropism, and autophagy inhibition, respectively. The CQ-induced endothelial response was associated with lipid accumulation and was characterized by Raman spectroscopy. CQ-induced autophagosome accumulation in the endothelium is featured by a pronounced alteration in the lipid profile, but not in the endothelial inflammation. Raman-based assessment of CQ-induced biochemical changes offers a better understanding of the autophagy mechanism in the endothelial cells

Characteristic of drug induced phospholipidosis in vascular endothelial cells by means of Raman and fluorescence imaging

Fosfolipidoza jest jednym z powikłań stosowania kationowych leków amfifilowych. Ich specyficzna struktura chemiczna powoduje występowanie akumulacji fosfolipidów wewnątrz lizosomów, powodując ich nieprawidłowe funkcjonowanie. Markerem obecności fosfolipidozy są tworzące się wewnątrz komórek ciałka blaszkowate, co jest efektem znanym, jednak mechanizm odpowiedzialny za ich tworzenie wciąż nie jest zgłębiony. Ciałka blaszkowate mogą różnić się w zależności od rodzaju leku lub jego dawki, co utrudnia zrozumienie i przewidywanie pojawienia się tego cytotoksycznego mechanizmu. Prawdopodobnie, fosfolipidoza wpływa na rozwój chorób u osób leczonych kationowymi lekami amfifilowymi. W związku z tym, współcześnie poszukuje się jak najlepszych technik do badania fosfolipidozy, których przykładem jest mikroskopia fluorescencyjna.Komórki śródbłonka stanowią pierwszą barierę pośredniczącą między składnikami krwi a tkankami organizmu. Dlatego prawidłowe funkcjonowanie układu sercowo-naczyniowego jest uzależnione od ich właściwego działania.Niniejsza praca przedstawia wykorzystanie obrazowania ramanowskiego jako techniki alternatywnej do badania fosfolipidozy w komórkach HMEC-1, wywołanej przez: amiodaron, chlorochinę oraz fluoksetynę wraz z mikroskopią fluorescencyjną. Spektroskopia ramanowska pozwala uzyskać informacje spektralne oraz chemiczne i pozwala wskazać diagnostyczne markery fosfolipidozy. Uzyskane wyniki z obrazowania ramanowskiego i fluorescencyjnego komórek śródbłonka naczyniowego w warunkach in vitro pozwolą poszerzyć wiedzę na temat zmian indukowanych w komórkach śródbłonka przez różne stężenia wybranych kationowych leków amfifilowych na poziomie subkomórkowym.Phospholipidosis is a main consequence of using cationic amphiphilic drugs. The specific chemical structure of this group of drugs, influence on accumulation of phospholipids inside of lysosomes, what influences their proper function. The hallmark of phospholipidosis are a newly formed lamellar bodies that are well known but the mechanism which stands for their formation is not fully understood. Lamellar bodies can vary in their chemical composition and size depending on the used drug and dosage, therefore predicting this type of toxicity can be challenging. Raman imaging seems to be a suitable technique to study these subtle changes in cells and can contribute to explanation of the phospholipidosis mechanism. Probably phospholipidosis influences on diseases development, in patients pharmacologically treated with cationic amphiphilic drugs. Nowadays, it is important to find the best technique to examine phospholipidosis like fluorescence microscopy.Endothelial cells create the first barrier which mediates between substances inside the blood and tissues in a human organism. That is why, the proper function of cardiovascular system depends on these cells and their functionality.This work presents Raman imaging as an alternative method to study a phospholipidosis in a HMEC-1 cells caused by amiodarone, chloroquine, and fluoxetine along with fluorescence microscopy. Raman spectroscopy provides spectral and chemical information about markers of phospholipidosis. Raman and fluorescence imaging allow to expand the knowledge about changes induced in HMEC-1 by a varied concentration of selected cationic amphiphilic drugs at subcellular level