Challenges in Imaging Analyses of Biomolecular Condensates in Cells Infected with Influenza A Virus.

Biomolecular condensates are crucial compartments within cells, relying on their material properties for function. They form and persist through weak, transient interactions, often undetectable by classical biochemical approaches. Hence, microscopy-based techniques have been the most reliable methods to detail the molecular mechanisms controlling their formation, material properties, and alterations, including dissolution or phase transitions due to cellular manipulation and disease, and to search for novel therapeutic strategies targeting biomolecular condensates. However, technical challenges in microscopy-based analysis persist. This paper discusses imaging, data acquisition, and analytical methodologies' advantages, challenges, and limitations in determining biophysical parameters explaining biomolecular condensate formation, dissolution, and phase transitions. In addition, we mention how machine learning is increasingly important for efficient image analysis, teaching programs what a condensate should resemble, aiding in the correlation and interpretation of information from diverse data sources. Influenza A virus forms liquid viral inclusions in the infected cell cytosol that serve as model biomolecular condensates for this study. Our previous work showcased the possibility of hardening these liquid inclusions, potentially leading to novel antiviral strategies. This was established using a framework involving live cell imaging to measure dynamics, internal rearrangement capacity, coalescence, and relaxation time. Additionally, we integrated thermodynamic characteristics by analysing fixed images through Z-projections. The aforementioned paper laid the foundation for this subsequent technical paper, which explores how different modalities in data acquisition and processing impact the robustness of results to detect bona fide phase transitions by measuring thermodynamic traits in fixed cells. Using solely this approach would greatly simplify screening pipelines. For this, we tested how single focal plane images, Z-projections, or volumetric analyses of images stained with antibodies or live tagged proteins altered the quantification of thermodynamic measurements. Customizing methodologies for different biomolecular condensates through advanced bioimaging significantly contributes to biological research and potential therapeutic advancements.

Population homogeneity for the antibody response to COVID-19 BNT162b2/Comirnaty vaccine is only reached after the second dose across all adult age ranges.

While mRNA vaccines are administrated worldwide in an effort to contain the COVID-19 pandemic, the heterogeneity of the humoral immune response they induce at the population scale remains unclear. Here, in a prospective, longitudinal, cohort-study, including 1245 hospital care workers and 146 nursing home residents scheduled for BNT162b2 vaccination, together covering adult ages from 19 to 99 years, we analyse seroconversion to SARS-CoV-2 spike protein and amount of spike-specific IgG, IgM and IgA before vaccination, and 3-5 weeks after each dose. We show that immunogenicity after a single vaccine dose is biased to IgG, heterogeneous and reduced with increasing age. The second vaccine dose normalizes IgG seroconversion in all age strata. These findings indicate two dose mRNA vaccines is required to reach population scale humoral immunity. The results advocate for the interval between the two doses not to be extended, and for serological monitoring of elderly and immunosuppressed vaccinees.

Alternative Biochemistries for Alien Life: Basic Concepts and Requirements for the Design of a Robust Biocontainment System in Genetic Isolation.

The universal genetic code, which is the foundation of cellular organization for almost all organisms, has fostered the exchange of genetic information from very different paths of evolution. The result of this communication network of potentially beneficial traits can be observed as modern biodiversity. Today, the genetic modification techniques of synthetic biology allow for the design of specialized organisms and their employment as tools, creating an artificial biodiversity based on the same universal genetic code. As there is no natural barrier towards the proliferation of genetic information which confers an advantage for a certain species, the naturally evolved genetic pool could be irreversibly altered if modified genetic information is exchanged. We argue that an alien genetic code which is incompatible with nature is likely to assure the inhibition of all mechanisms of genetic information transfer in an open environment. The two conceivable routes to synthetic life are either de novo cellular design or the successive alienation of a complex biological organism through laboratory evolution. Here, we present the strategies that have been utilized to fundamentally alter the genetic code in its decoding rules or its molecular representation and anticipate future avenues in the pursuit of robust biocontainment.