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Background: Certain causes of death can be avoided with access to timely prevention and treatment. We quantified trends in avoidable deaths from cardiovascular diseases for European Union (EU) countries from 1995 to 2020 and examined variations by demographics, disease characteristics, and geography., Methods: Retrospective secondary data analysis of avoidable cardiovascular mortality using the WHO Mortality Database. Avoidable causes of death were identified from the OECD and Eurostat list (which uses an age threshold of 75 years). Regression models were used to identify changes in the trends of age-standardized mortality rates and potential years of life lost., Findings: From 1995 to 2020, 11.4 million deaths from cardiovascular diseases in Europe were avoidable, resulting in 213.1 million potential life years lost. Avoidable deaths were highest among males (7.5 million), adults 65-74 years (6.8 million), and with the leading cause of death being ischemic heart disease (6.1 million). From its peak in 1995 until 2020, avoidable mortality from cardiovascular diseases has decreased by 57% across the EU. The difference in avoidable cardiovascular diseases mortality between females and males, and between Eastern and Western Europe has reduced greatly, however gaps continue to persist., Interpretation: Avoidable mortality from cardiovascular diseases has decreased substantially among EU countries, although improvement has not been uniform across diseases, demographic groups or regions. These trends suggest additional policy interventions are needed to ensure that improvements in mortality are continued., Funding: World Health Organization, Regional Office for Europe., Competing Interests: ZT reports a previous summer internship with Novo Nordisk. MM reports consulting fees from Abbott, Cadrenal, Paragonix, Second Heart Assist, Medtronic, Janssen, Natera, and Moderna, and participation on the advisory boards of Mesoblast, Fineheart, Leviticus, and NupulseCV., (© 2024 The Author(s).)

Three-dimensional structure determination of membrane proteins is important to fully understand their biological functions. However, obtaining a high-resolution structure has been a major challenge mainly due to the difficulties in retaining the native folding and function of membrane proteins outside of the cellular membrane environment. These challenges are acute if the protein contains a large soluble domain, as it needs bulk water unlike the transmembrane domains of an integral membrane protein. For structural studies on such proteins either by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, bicelles have been demonstrated to be superior to conventional micelles, yet their temperature restrictions attributed to their thermal instabilities are a major disadvantage. Here, we report an approach to overcome this drawback through searching for an optimum combination of bicellar compositions. We demonstrate that bicelles composed of 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholin (DHepPC), without utilizing additional stabilizing chemicals, are quite stable and are resistant to temperature variations. These temperature-resistant bicelles have a robust bicellar phase and magnetic alignment over a broad range of temperatures, between -15 and 80 °C, retain the native structure of a membrane protein, and increase the sensitivity of solid-state NMR experiments performed at low temperatures. Advantages of two-dimensional separated-local field (SLF) solid-state NMR experiments at a low temperature are demonstrated on magnetically aligned bicelles containing an electron carrier membrane protein, cytochrome b5. Morphological information on different DDPC-based bicellar compositions, varying q ratio/size, and hydration levels obtained from (31)P NMR experiments in this study is also beneficial for a variety of biophysical and spectroscopic techniques, including solution NMR and magic-angle-spinning (MAS) NMR for a wide range of temperatures.

Bicelles are increasingly used as model membranes to suitably mimic the biological cell membrane for biophysical and biochemical studies by a variety of techniques including NMR and X-ray crystallography. Recent NMR studies have successfully utilized bicelles for atomic-resolution structural and dynamic studies of antimicrobial peptides, amyloid peptides, and membrane-bound proteins. Though bicelles composed with several different types of lipids and detergents have been reported, the NMR requirement of magnetic alignment of bicelles limits the temperature range in which they can be used and subsequently their composition. Because of this restriction, low-temperature experiments desirable for heat-sensitive membrane proteins have not been conducted because bicelles could not be aligned. In this study, we characterize the magnetic alignment of bicelles with various compositions for a broad range of temperatures using (31)P static NMR spectroscopy in search of temperature-resistant bicelles. Our systematic investigation identified a temperature range of magnetic alignment for bicelles composed of 4:1 DLPC:DHexPC, 4:1:0.2 DLPC:DHexPC:cholesterol, 4:1:0.13 DLPC:DHexPC:CTAB, 4:1:0.13:0.2 DLPC:DHexPC:CTAB:cholesterol, and 4:1:0.4 DLPC:DHexPC:cholesterol-3-sulfate. The amount of cholesterol-3-sulfate used was based on mole percent and was varied in order to determine the optimal amount. Our results indicate that the presence of 75 wt % or more water is essential to achieve maximum magnetic alignment, while the presence of cholesterol and cholesterol-3-sulfate stabilizes the alignment at extreme temperatures and the positively charged CTAB avoids the mixing of bicelles. We believe that the use of magnetically aligned 4:1:0.4 DLPC:DHexPC:cholesterol-3-sulfate bicelles at as low as -15 °C would pave avenues to study the structure, dynamics, and membrane orientation of heat-sensitive proteins such as cytochrome P450 and could also be useful to investigate protein-protein interactions in a membrane environment.

Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochrome-P4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.