Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content.

Giant unilamellar vesicles (GUVs) are membrane models used to study membrane properties. Electroformation is one of the methods used to produce GUVs. During electroformation protocol, dry lipid film is formed. The drying of the lipid film induces the cholesterol (Chol) demixing artifact, in which Chol forms anhydrous crystals which do not participate in the formation of vesicles. This leads to a lower Chol concentration in the vesicle bilayers compared to the Chol concentration in the initial lipid solution. To address this problem, we propose a novel electroformation protocol that includes rapid solvent exchange (RSE), plasma cleaning, and spin-coating methods to produce GUVs. We tested the protocol, focusing on vesicles with a high Chol content using different spin-coating durations and vesicle type deposition. Additionally, we compared the novel protocol using completely dry lipid film. The optimal spin-coating duration for vesicles created from the phosphatidylcholine/Chol mixture was 30 s. Multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs) obtained by the extrusion of MLVs through 100 nm membrane pores and LUVs obtained by extrusion of previously obtained LUVs through 50 nm membrane pores, were deposited on an electrode for 1.5/1 Chol/phosphatidylcholine (POPC) lipid mixture, and the results were compared. Electroformation using all three deposited vesicle types resulted in a high GUV yield, but the deposition of LUVs obtained by the extrusion of MLVs through 100 nm membrane pores provided the most reproducible results. Using the deposition of these LUVs, we produced high yield GUVs for six different Chol concentrations (from 0% to 71.4%). Using a protocol that included dry lipid film GUVs resulted in lower yields and induced the Chol demixing artifact, proving that the lipid film should never be subjected to drying when the Chol content is high.

Excess Mortality Stratified by Age and Sex for Croatia and Croatian Counties during the 2020-2021 COVID-19 Pandemic.

Excess mortality is often used to estimate the effect of a certain crisis on the population. It is defined as the number of deaths during a crisis exceeding the expected number based on historical trends. Here, we calculated excess mortality due to the COVID-19 pandemic for Croatia in the 2020-2021 period. The excess was calculated on the national and county level for different age and sex categories. In addition to the absolute number, the excess mortality was also expressed as a ratio of excess deaths to the predicted baseline and excess mortality rate. We showed that using both measures is necessary to avoid incorrect conclusions. The estimated excess mortality on the national level was 14,963, corresponding to an excess percentage of 14.3%. With respect to sex, there was a higher excess mortality rate for men compared to women. An exponential relationship was observed between age and the excess mortality rate.These trends wee representative of most counties as well, with large variations in the magnitude of the effect. However, there were also exceptions to the general rule. The reasons for these deviations were discussed in terms of between-county differences in demographic structure, population density and special events that took place during the pandemic.

Membrane Models and Experiments Suitable for Studies of the Cholesterol Bilayer Domains.

Cholesterol (Chol) is an essential component of animal cell membranes and is most abundant in plasma membranes (PMs) where its concentration typically ranges from 10 to 30 mol%. However, in red blood cells and Schwann cells, PMs Chol content is as high as 50 mol%, and in the PMs of the eye lens fiber cells, it can reach up to 66 mol%. Being amphiphilic, Chol molecules are easily incorporated into the lipid bilayer where they affect the membrane lateral organization and transmembrane physical properties. In the aqueous phase, Chol cannot form free bilayers by itself. However, pure Chol bilayer domains (CBDs) can form in lipid bilayer membranes with the Chol content exceeding 50 mol%. The range of Chol concentrations surpassing 50 mol% is less frequent in biological membranes and is consequently less investigated. Nevertheless, it is significant for the normal functioning of the eye lens and understanding how Chol plaques form in atherosclerosis. The most commonly used membrane models are unilamellar and multilamellar vesicles (MLVs) and supported lipid bilayers (SLBs). CBDs have been observed directly using confocal microscopy, X-ray reflectometry and saturation recovery electron paramagnetic resonance (SR EPR). Indirect evidence of CBDs has also been reported by using atomic force microscopy (AFM) and fluorescence recovery after photobleaching (FRAP) experiments. The overall goal of this review is to demonstrate the advantages and limitations of the various membrane models and experimental techniques suitable for the detection and investigation of the lateral organization, function and physical properties of CBDs.

Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films Formed by Vesicle Fusion.

Giant unilamellar vesicles (GUVs) are artificial membrane models which are of special interest to researchers because of their similarity in size to eukaryotic cells. The most commonly used method for GUVs production is electroformation. However, the traditional electroformation protocol involves a step in which the organic solvent is completely evaporated, leaving behind a dry lipid film. This leads to artifactual demixing of cholesterol (Chol) in the form of anhydrous crystals. These crystals do not participate in the formation of the lipid bilayer, resulting in a decrease of Chol concentration in the bilayer compared to the initial lipid solution. We propose a novel electroformation protocol which addresses this issue by combining the rapid solvent exchange, plasma cleaning and spin-coating techniques to produce GUVs from damp lipid films in a fast and reproducible manner. We have tested the protocol efficiency using 1/1 phosphatidylcholine/Chol and 1/1/1 phosphatidylcholine/sphingomyelin/Chol lipid mixtures and managed to produce a GUV population of an average diameter around 40 µm, with many GUVs being larger than 100 µm. Additionally, compared to protocols that include the dry film step, the sizes and quality of vesicles determined from fluorescence microscopy images were similar or better, confirming the benefits of our protocol in that regard as well.

Applying Explainable Machine Learning Models for Detection of Breast Cancer Lymph Node Metastasis in Patients Eligible for Neoadjuvant Treatment.

BACKGROUND: Due to recent changes in breast cancer treatment strategy, significantly more patients are treated with neoadjuvant systemic therapy (NST). Radiological methods do not precisely determine axillary lymph node status, with up to 30% of patients being misdiagnosed. Hence, supplementary methods for lymph node status assessment are needed. This study aimed to apply and evaluate machine learning models on clinicopathological data, with a focus on patients meeting NST criteria, for lymph node metastasis prediction. METHODS: From the total breast cancer patient data (n = 8381), 719 patients were identified as eligible for NST. Machine learning models were applied for the NST-criteria group and the total study population. Model explainability was obtained by calculating Shapley values. RESULTS: In the NST-criteria group, random forest achieved the highest performance (AUC: 0.793 [0.713, 0.865]), while in the total study population, XGBoost performed the best (AUC: 0.762 [0.726, 0.795]). Shapley values identified tumor size, Ki-67, and patient age as the most important predictors. CONCLUSION: Tree-based models achieve a good performance in assessing lymph node status. Such models can lead to more accurate disease stage prediction and consecutively better treatment selection, especially for NST patients where radiological and clinical findings are often the only way of lymph node assessment.

Effects of Moderate Consumption of Red Wine on Hepcidin Levels in Patients with Type 2 Diabetes Mellitus.

Iron overload is often associated with type 2 diabetes (T2D), indicating that hepcidin, the master regulator of iron homeostasis, might be involved in diabetes pathogenesis. Alcohol consumption may also result in increased body iron stores. However, the moderate consumption of wine with meals might be beneficial in T2D. This effect has been mainly attributed to both the ethanol and the polyphenolic compounds in wine. Therefore, we examined the effects of red wine on hepcidin in T2D patients and non-diabetic controls. The diabetic patients (n = 18) and age- and BMI-matched apparently healthy controls (n = 13) were men, aged 40−65 years, non-smoking, with BMI < 35 kg/m2. Following a 2-week alcohol-free period, both groups consumed 300 mL of red wine for 3 weeks. The blood samples for the iron status analysis were taken at the end of each period. The red wine intake resulted in a decrease in serum hepcidin in both the diabetic subjects (p = 0.045) and controls (p = 0.001). The levels of serum ferritin also decreased after wine in both groups, reaching statistical significance only in the control subjects (p = 0.017). No significant alterations in serum iron, transferrin saturation, or soluble transferrin receptors were found. The suppression of hepcidin, a crucial iron-regulatory hormone and acute-phase protein, in T2D patients and healthy controls, is a novel biological effect of red wine. This may deepen our understanding of the mechanisms of the cardiometabolic effects of wine in T2D.

Optimization of Giant Unilamellar Vesicle Electroformation for Phosphatidylcholine/Sphingomyelin/Cholesterol Ternary Mixtures.

Artificial vesicles are important tools in membrane research because they enable studying membrane properties in controlled conditions. Giant unilamellar vesicles (GUVs) are specially interesting due to their similarity in size to eukaryotic cells. We focus on optimization of GUV production from phosphatidylcholine/sphingomyelin/cholesterol mixtures using the electroformation method. This mixture has been extensively researched lately due to its relevance for the formation of lipid rafts. We measured the effect of voltage, frequency, lipid film thickness, and cholesterol (Chol) concentration on electroformation successfulness using spin-coating for reproducible lipid film deposition. Special attention is given to the effect of Chol concentrations above the phospholipid bilayer saturation threshold. Such high concentrations are of interest to groups studying the role of Chol in the fiber cell plasma membranes of the eye lens or development of atherosclerosis. Utilizing atomic force and fluorescence microscopy, we found the optimal lipid film thickness to be around 30 nm, and the best frequency-voltage combinations in the range of 2-6 V and 10-100 Hz. Increasing the Chol content, we observed a decrease in GUV yield and size. However, the effect was much less pronounced when the optimal lipid film thickness was used. The results underline the need for simultaneous optimization of both electrical parameters and thickness in order to produce high-quality GUVs for experimental research.

Giant Unilamellar Vesicle Electroformation: What to Use, What to Avoid, and How to Quantify the Results.

Since its inception more than thirty years ago, electroformation has become the most commonly used method for growing giant unilamellar vesicles (GUVs). Although the method seems quite straightforward at first, researchers must consider the interplay of a large number of parameters, different lipid compositions, and internal solutions in order to avoid artifactual results or reproducibility problems. These issues motivated us to write a short review of the most recent methodological developments and possible pitfalls. Additionally, since traditional manual analysis can lead to biased results, we have included a discussion on methods for automatic analysis of GUVs. Finally, we discuss possible improvements in the preparation of GUVs containing high cholesterol contents in order to avoid the formation of artifactual cholesterol crystals. We intend this review to be a reference for those trying to decide what parameters to use as well as an overview providing insight into problems not yet addressed or solved.

Effect of Electrical Parameters and Cholesterol Concentration on Giant Unilamellar Vesicles Electroformation.

Giant unilamellar vesicles (GUVs) are used extensively as models that mimic cell membranes. The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. Thus, a methodological paper pertaining to preparations of model lipid bilayer membranes with high Chol content would significantly help the study of properties of these membranes. Lipid solutions containing 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and Chol were fluorescently labeled with phospholipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC18(3)) and spin-coated to produce thin lipid films. GUVs were formed from these films using the electroformation method and the results were obtained using fluorescent microscopy. Electroformation outcomes were examined for different electrical parameters and different Chol concentrations. A wide range of field frequency-field strength (ff-fs) combinations was explored: 10-10,000 Hz and 0.625-9.375 V/mm peak-to-peak. Optimal values for GUVs preparation were found to be 10-100 Hz and 1.25-6.25 V/mm, with largest vesicles occurring for 10 Hz and 3.75 V/mm. Chol:POPC mixing ratios (expressed as a molar ratio) ranged from 0 to 3.5. We show that increasing the Chol concentration decreases the GUVs size, but this effect can be reduced by choosing the appropriate ff-fs combination.

The mitotic spindle is chiral due to torques within microtubule bundles.

Mitosis relies on forces generated in the spindle, a micro-machine composed of microtubules and associated proteins. Forces are required for the congression of chromosomes to the metaphase plate and their separation in anaphase. However, besides forces, torques may exist in the spindle, yet they have not been investigated. Here we show that the spindle is chiral. Chirality is evident from the finding that microtubule bundles in human spindles follow a left-handed helical path, which cannot be explained by forces but rather by torques. Kinesin-5 (Kif11/Eg5) inactivation abolishes spindle chirality. Our theoretical model predicts that bending and twisting moments may generate curved shapes of bundles. We found that bundles turn by about -2 deg µm-1 around the spindle axis, which we explain by a twisting moment of roughly -10 pNµm. We conclude that torques, in addition to forces, exist in the spindle and determine its chiral architecture.