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.info:eu-repo/semantics/publishedVersio

Neurodegenerative Potential of the Aqueous Leaf Extract of Ocimum gratissimum: A Histological and Biochemical Study

Ocimum gratissimum is an herbaceous perennial shrub which is widely distributed in many regions. It is consumed in food as seasoning locally in Nigeria. In the present study, the effect of the acute administration of the aqueous leaf extract of Ocimum gratissimum (AeOG) on prefrontal cortical neurons was checked to assess its neurotoxicity potential. Thirty adult male Wistar rats weighing between 190-210 g were divided into 5 groups (n=6). Group A (control) received 1 ml of normal saline (p.o), groups B-E received 100, 200, 300 and 400 mg/kg AeOG (p.o) respectively. Treatment lasted for fourteen days. Twenty-four hours after treatment, animals were sacrificed and their brains were removed. The prefrontal cortices neuronal morphology was studied using haematoxylin and eosin (H&E) stain; while activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) were assayed in the cerebral homogenate. AeOG administration at doses 300 and 400 mg/kg cause neuronal fragmentation and central chromatolysis with significant (P<0.05) increases in the activities of cerebral ACP and ALP. Our findings show that the acute use of AeOG caused neuronal fragmentation and central chromatolysis which are response to axonal injuries and may leads to onset of neurodegenerative diseases and affect cognitive and executive functions of the prefrontal cortex.Keywords: Ocimum gratissimum, Acid Phosphatase (ACP), Alkaline Phosphatase (ALP), Neurogenerative diseases, Ra

ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions

AU It is:now Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly established that many viruses that threaten public health : establish condensates via phase transitions to complete their lifecycles, and knowledge on such processes may offer new strategies for antiviral therapy. In the case of influenza A virus (IAV), liquid condensates known as viral inclusions, concentrate the 8 distinct viral ribonucleoproteins (vRNPs) that form IAV genome and are viewed as sites dedicated to the assembly of the 8-partite genomic complex. Despite not being delimited by host membranes, IAV liquid inclusions accumulate host membranes inside as a result of vRNP binding to the recycling endocytic marker Rab11a, a driver of the biogenesis of these structures. We lack molecular understanding on how Rab11a-recycling endosomes condensate specifically near the endoplasmic reticulum (ER) exit sites upon IAV infection. We show here that liquid viral inclusions interact with the ER to fuse, divide, and slide. We uncover that, contrary to previous indications, the reported reduction in recycling endocytic activity is a regulated process rather than a competition for cellular resources involving a novel role for the host factor ATG9A. In infection, ATG9A mediates the removal of Rab11a-recycling endosomes carrying vRNPs from microtubules. We observe that the recycling endocytic usage of microtubules is rescued when ATG9A is depleted, which prevents condensation of Rab11a endosomes near the ER. The failure to produce viral inclusions accumulates vRNPs in the cytosol andAU reduces: Pleasecheckandconfirmthattheeditst genome assembly and the release of infectious virions. We propose that the ER supports the dynamics of liquid IAV inclusions, with ATG9A facilitating their formation. This work advances our understanding on how epidemic and pandemic influenza genomes are formed. It also reveals the plasticity of recycling pathway endosomes to undergo condensation in response to infection, disclosing new roles for ATG9A beyond its classical involvement in autophagy.info:eu-repo/semantics/publishedVersio

Defining basic rules for hardening influenza A virus liquid condensates

In biological systems, liquid and solid-like biomolecular condensates may contain the same molecules but their behaviour, including movement, elasticity and viscosity, is different on account of distinct physicochemical properties. As such, it is known that phase transitions affect the function of biological condensates and that material properties can be tuned by several factors including temperature, concentration and valency. It is, however, unclear if some factors are more efficient than others at regulating their behaviour. Viral infections are good systems to address this question as they form condensates de novo as part of their replication programmes. Here, we used influenza A virus liquid cytosolic condensates, A.K.A viral inclusions, to provide a proof of concept that liquid condensate hardening via changes in the valency of its components is more efficient than altering their concentration or the temperature of the cell. Liquid IAV inclusions may be hardened by targeting vRNP interactions via the known NP oligomerizing molecule, nucleozin, both in vitro and in vivo without affecting host proteome abundance nor solubility. This study is a starting point for understanding how to pharmacologically modulate the material properties of IAV inclusions and may offer opportunities for alternative antiviral strategies.info:eu-repo/semantics/publishedVersio

Influenza A virus Inclusions are Liquid Condensates that can be Pharmacologically Hardened

Influenza A virus (IAV) is relevant to human and veterinary health as it causes seasonal epidemic and sporadic pandemics with excess mortality (Hampson and Mackenzie, 2006; Martin-Benito and Ortin, 2013; Vale-Costa et al., 2016a; Silvia Vale-Costa and Amorim, 2016). A way to reduce the risks of IAV disease is the yearly vaccination programmes, which has a reported 40-60% efficiency, pointing to the need for a more efficacious solution. This is only possible by gaining thorough understanding of IAV biology at the fundamental level, which could then inform the development of new antiviral therapeutics

Liquid Biomolecular Condensates and Viral Lifecycles: Review and Perspectives

Viruses are highly dependent on the host they infect. Their dependence triggers processes of virus–host co-adaptation, enabling viruses to explore host resources whilst escaping immunity. Scientists have tackled viral–host interplay at differing levels of complexity—in individual hosts, organs, tissues and cells—and seminal studies advanced our understanding about viral lifecycles, intra- or inter-species transmission, and means to control infections. Recently, it emerged as important to address the physical properties of the materials in biological systems; membrane-bound organelles are only one of many ways to separate molecules from the cellular milieu. By achieving a type of compartmentalization lacking membranes known as biomolecular condensates, biological systems developed alternative mechanisms of controlling reactions. The identification that many biological condensates display liquid properties led to the proposal that liquid–liquid phase separation (LLPS) drives their formation. The concept of LLPS is a paradigm shift in cellular structure and organization. There is an unprecedented momentum to revisit long-standing questions in virology and to explore novel antiviral strategies. In the first part of this review, we focus on the state-of-the-art about biomolecular condensates. In the second part, we capture what is known about RNA virus-phase biology and discuss future perspectives of this emerging field in virology.ISSN:1999-491

Influenza A virus ribonucleoproteins form liquid organelles at endoplasmic reticulum exit sites

Influenza A virus has an eight-partite RNA genome that during viral assembly forms a complex containing one copy of each RNA. Genome assembly is a selective process driven by RNA-RNA interactions and is hypothesized to lead to discrete punctate structures scattered through the cytosol. Here, we show that contrary to the accepted view, formation of these structures precedes RNA-RNA interactions among distinct viral ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We demonstrate that these viral inclusions display characteristics of liquid organelles, segregating from the cytosol without a delimitating membrane, dynamically exchanging material and adapting fast to environmental changes. We provide evidence that viral inclusions develop close to endoplasmic reticulum (ER) exit sites, depend on continuous ER-Golgi vesicular cycling and do not promote escape to interferon response. We propose that viral inclusions segregate vRNPs from the cytosol and facilitate selected RNA-RNA interactions in a liquid environment.info:eu-repo/semantics/publishedVersio