Knowledge, attitudes, beliefs and barriers of healthcare professionals and adults ≥ 65 years about vaccine-preventable diseases in Spain: the ADult Vaccination drIverS and barriErs (ADVISE) study.

Since 2018, Spanish National Immunization Guidelines include vaccination recommendations for adults ≥65 years. To determine whether health-care professionals and the ≥65 years target group value the need for these recommendations, a cross-sectional study was conducted to capture and describe their knowledge, attitudes, beliefs and behaviors about vaccination. Online surveys were administered to representative groups of general practitioners (GPs), primary care nurses and adults ≥65 years from six major cities (and surrounding rural areas) in Spain. Main topics were attitudes and awareness of vaccines, perceptions about vaccination in adults ≥65 years, and impact of the COVID-19 pandemic on vaccination uptake. A total of 286 health-care professionals (185 GPs, 101 nurses) and 400 adults aged ≥65 years participated in the survey. GP and nurse groups agreed strongly about the importance of influenza and pneumococcal vaccination in the target population. Longer patient visit times were identified as a key factor toward promoting vaccination. The ≥65 years sample group, especially those ≥75+ years and/or with chronic diseases, was reasonably positive about the effectiveness and benefits of vaccines. Lower vaccination rates for the pneumococcal than influenza vaccine (29% vs. 80%) in the ≥65 years sample group suggest that efforts are needed to improve pneumococcal vaccine uptake. Aligning with other published works, GPs have a key role in promoting vaccination in the target population. The COVID-19 pandemic appears to have heightened awareness about the importance of vaccination among health-care professionals and adults ≥65 years.

Lysenin Toxin Membrane Insertion Is pH-Dependent but Independent of Neighboring Lysenins.

Pore-forming toxins form a family of proteins that act as virulence factors of pathogenic bacteria, but similar proteins are found in all kingdoms of life, including the vertebrate immune system. They are secreted as soluble monomers that oligomerize on target membranes in the so-called prepore state; after activation, they insert into the membrane and adopt the pore state. Lysenin is a pore-forming toxin from the earthworm Eisenida foetida, of which both the soluble and membrane-inserted structures are solved. However, the activation and membrane-insertion mechanisms have remained elusive. Here, we used high-speed atomic force microscopy to directly visualize the membrane-insertion mechanism. Changing the environmental pH from pH 7.5 to below pH 6.0 favored membrane insertion. We detected a short α-helix in the soluble structure that comprised three glutamic acids (Glu92, Glu94, and Glu97) that we hypothesized may represent a pH-sensor (as in similar toxins, e.g., Listeriolysin). Mutant lysenin still can form pores, but mutating these glutamic acids to glutamines rendered the toxin pH-insensitive. On the other hand, toxins in the pore state did not favor insertion of neighboring prepores; indeed, pore insertion breaks the hexagonal ordered domains of prepores and separates from neighboring molecules in the membrane. pH-dependent activation of toxins may represent a common feature of pore-forming toxins. High-speed atomic force microscopy with single-molecule resolution at high temporal resolution and the possibility of exchanging buffers during the experiments presents itself as a unique tool for the study of toxin-state conversion.

Glasslike Membrane Protein Diffusion in a Crowded Membrane.

Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion.