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.     

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.     

Determination of lycopene and beta-carotene content in tomato fruits and related products: Comparison of FT-Raman, ATR-IR, and NIR spectroscopy

Tomatoes and various products derived from thermally processed tomatoes are major sources of lycopene, but apart from this micronutrient, other carotenoids such as beta-carotene also are present in the fruit. They occur in tomato fruits and various tomato products in amounts of 2.62-629.00 (lycopene) and 0.23-2.83 mg/100 g (beta-carotene). Standard methods for determining the carotenoid content require the extraction of the analyte as well as other cleanup steps. In this work, FT-Raman, ATR-IR, and NIR spectroscopy are applied in order to establish new, fast, and nondestructive calibration methods for quantification of lycopene and beta-carotene content in tomato fruits and related products. The best prediction quality was achieved using a model based on IR spectroscopy (R2 = 0.98 and 0.97, SECV = 33.20 and 0.16 for lycopene and beta-carotene, respectively). In spite of the fact that Raman spectra of tomato products show characteristic key bands of the investigated carotenoids, this method gives slightly lower reliability (R2 = 0.91 and 0.89, SECV = 74.34 and 0.34 for lycopene and beta-carotene, respectively). NIR spectroscopy, which has been used for quantification purposes in the agricultural sector for several decades, in this study shows the worse prediction quality (R2 = 0.85 and 0.80, SECV = 91.19 and 0.41 for lycopene and beta-carotene, respectively).     

Structural changes of carotenoid astaxanthin in a single algal cell monitored in situ by Raman spectroscopy

The changes of structure of astaxanthin (AXT), a superpotent antioxidant, upon thermal stress were investigated in unicellular microalgae Haematococcus pluvialis by measuring Raman spectra in situ and analyzing obtained results with DFT calculations. Although no visual changes are observed in the Haematococcus cells upon heating, discernible changes in Raman spectra occur from -100 °C systematically up to 150 °C. The exponential increase of the Raman shift of the ν C═C band at ca. 1520 cm(-1) along with the change of the intensity ratio of bands at 1190 and 1160 cm(-1) is observed, that correlates with the changes predicted by calculations for astaxanthin conformers ordered by decreasing energy. It is assumed that AXT molecules, initially in the form of H-aggregates with the trans conformations of the end-rings, interconvert toward more stable gauche forms upon thermal stress of the algae. The applied approach enables one to follow structural changes of the carotenoid upon temperature stress both in a single algal cell and in a multicellular sample in situ. Obtained information might be of use to improve the industrial process of extraction of AXT in its most bioavailable form.     

Feasibility of diffusion-weighted imaging with DWIBS in staging Hodgkin lymphoma in pediatric patients: comparison with PET/CT

Objective

The aim of the study was to evaluate feasibility of diffusion-weighted whole-body imaging with background body signal suppression (DWIBS) method in diagnosing Hodgkin lymphoma in pediatric patients and to compare it with 18F-FDG PET/CT as a gold standard.

Materials and methods

Eleven patients (median age 14) with newly diagnosed Hodgkin lymphoma were examined with 18F-FDG PET/CT and MRI including whole-body DWIBS sequence (b = 0, 800 s/mm²), before the oncologic treatment. About 26 locations of lymphatic tissues were evaluated visually and quantitatively using ADC_mean (DWIBS) and SUV_max (18F-FDG PET/CT), respectively.

Results

All affected lymph node regions (n = 134) diagnosed in 18F-FDG PET/CT were found with DWIBS, presenting decreased diffusion. Significant correlation was found between ADC and SUV values (R² = − 0.37; p = 0.0001). Nevertheless, additional 33 regions were recognized only by DWIBS. They were significantly smaller than regions diagnosed by both methods.

Discussion

Agreement between DWIBS and 18F-FDG PET/CT for detection and staging of malignant lymphoma is high. DWIBS can be used for the evaluation of pediatric Hodgkin lymphoma.

     

Ultrastructural, stereological evaluation of satellite cells derived from musculus soleus in rats kept in hypokinesia conditions

OBJECTIVES: Selection for surgery of patients with abdominal aortic aneurysm (AAA) depends on an assessment of risk from operation compared with risk from aneurysm rupture. A study was performed to assess the levels of co-morbidity and to see whether co-morbidity was different in people with a normal aorta after ultrasonographic examination than in those with an aneurysmal aorta. SETTING AND METHODS: Over a two year period 5392 people (2341 men, 3051 women) aged 65-80 were screened using B-mode linear ultrasound, with maximum measurements taken of transverse, anteroposterior diameters, or both. All subjects were given a questionnaire seeking a history of angina, stroke, claudication, myocardial infarct, respiratory problems, and diabetes. RESULTS: 218 men and women were found to have an AAA of 3 cm or greater. The results of the questionnaire were analysed using logistic regression whereby all the co-morbid conditions were adjusted for each other and for smoking, sex, and age. The only conditions which were significantly associated with AAA in both sexes were myocardial infarction with an odds ratio (OR) of 1.66 (95% confidence interval (CI) 1.06 to 2.60) and claudication with an OR of 1.68 (95% CI 1.17 to 2.42). The association between angina and AAA was of borderline significance (OR = 1.52, 95% CI 1.00 to 2.30). Stroke was significantly associated only in women, with an OR of 3.71 (95% CI 1.42 to 9.69). Rates of diabetes and respiratory disease were not significantly different between people with AAA and normal aortas. CONCLUSIONS: These findings show there is significantly higher co-morbidity in people with ultrasound detected AAA, which might influence outcome from surgery and long term survival.     

In situ flavonoid analysis by FT-Raman spectroscopy: identification, distribution, and quantification of aspalathin in green rooibos (Aspalathus linearis)

FT-Raman spectroscopy was used for the first time for in situ identification of aspalathin and quantification of the dihyrochalcones in dried, green rooibos (Aspalathus linearis). With the support of two-dimensional correlation spectroscopy, characteristic key bands of aspalathin, the main flavonoid and antioxidant occurring in rooibos, were localized and identified in the spectra obtained from various plant samples. Application of Raman mapping revealed the spatial distribution of this valuable dihydrochalcone within the intact dried leaves. Based on the spectral data and reference HPLC values, reliable multivariate calibration models were developed for quantification of aspalathin, nothofagin, and the combined dihydrochalcone contents of dried, green rooibos.