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BioRxiv : the Preprint Server For... Jun 2024Ionizable lipid nanoparticles (LNPs) have been pivotal in combating COVID-19, and numerous preclinical and clinical studies have highlighted their potential in nucleic...
Ionizable lipid nanoparticles (LNPs) have been pivotal in combating COVID-19, and numerous preclinical and clinical studies have highlighted their potential in nucleic acid-based therapies and vaccines. However, the effectiveness of endosomal escape for the nucleic acid cargos encapsulated in LNPs is still low, leading to suboptimal treatment outcomes and side effects. Hence, improving endosomal escape is crucial for enhancing the efficacy of nucleic acid delivery using LNPs. Here, a mechanical oscillation (frequency: 65 Hz) is utilized to prompt the LNP-mediated endosomal escape. The results reveal this mechanical oscillation can induce the combination and fusion between LNPs with opposite surface charges, enhance endosomal escape of mRNA by 14%, and increase the transfection efficiency of mRNA up to 1.67 times in the current study. Additionally, cell viability remains high at 99.3% after treatment with oscillation, which is comparable to that of untreated cells. Furthermore, there is no obvious damage to other membranous organelles. Thus, this work presents a user-friendly and safe approach to enhancing endosomal escape of mRNA and boosting gene expression. As a result, our work can be potentially utilized in both research and clinical fields to facilitate LNP-based delivery by enabling more effective release of LNP-encapsulated cargos from endosomes.
PubMed: 38948864
DOI: 10.1101/2024.06.19.599708 -
BioRxiv : the Preprint Server For... Jun 2024Understanding the phenotypic consequences of naturally occurring genetic changes, as well as their impact on fitness, is fundamental to understanding how organisms adapt...
Understanding the phenotypic consequences of naturally occurring genetic changes, as well as their impact on fitness, is fundamental to understanding how organisms adapt to an environment. This is critical when genetic variants have pleiotropic effects, as determining how each phenotype impacted by a gene contributes to fitness is essential to understand how and why traits have evolved. A striking example of a pleiotropic gene contributing to trait evolution is the gene, coding mutations in which underlie albinism and reductions of sleep in the blind Mexican cavefish, . Here, we characterize the effects of mutations in the gene on larval prey capture. We find that when conspecific surface fish with engineered mutations in the allele are hunting, they use cave-like, wide angle strikes to capture prey. However, unlike cavefish or surface fish in the dark, which rely on lateral line mediated hunting, mutant surface fish use vision when striking at prey from wide angles. Finally, we find that while mutant surface fish do not outcompete pigmented surface siblings in the dark, pigmented fish outcompete albino fish in the light. This raises the possibility that albinism is detrimental to larval feeding in a surface-like lighted environment, but does not have negative consequences for fish in cave-like, dark environments. Together, these results demonstrate that plays a role in larval feeding behavior in . Further, they expand our understanding of the pleiotropic phenotypic consequences of in cavefish evolution.
PubMed: 38948816
DOI: 10.1101/2024.06.17.599419 -
BioRxiv : the Preprint Server For... Jun 2024is an opportunistic fungal pathogen responsible for >150,000 deaths every year with a mortality rate as high as 81%. This high medical burden is due, in part, to an...
UNLABELLED
is an opportunistic fungal pathogen responsible for >150,000 deaths every year with a mortality rate as high as 81%. This high medical burden is due, in part, to an incomplete understanding of its pathogenesis. In a previous study, we identified a cryptococcal atypical pleiotropic drug resistance (PDR) transporter, , that regulated antifungal resistance and host interactions. Here, we follow-up on the role of in cryptococcal virulence. , mice infected with the Δ strain display altered symptomatology and disease progression. Specifically, we observed a significant increase in the innate immune cell populations in the Δ-infected mice when compared to their WT-infected littermates. Furthermore, quantification of pulmonary cytokines/chemokines revealed a robust increase of pro-inflammatory cytokines in mice infected with the Δ mutant strain. Whereas antifungal treatment of Δ-infected animals did not affect survival, treatment with a corticosteroid significantly extended survival, highlighting the importance of a balanced/controlled host immune response. We determined that the hyper-inflammatory immune response occurs, in part, because the loss of the Pdr6 transporter indirectly alters the cryptococcal cell wall architecture and results in the increased exposure of chitin, β-glucan, and other cryptococcal-specific pathogen associated molecular patterns. Taken together, this study provides clinical insights regarding cryptococcal pathogenesis while also providing additional functions of PDR-type ATP-binding cassette (ABC) transporters in pathogenic fungi.
IMPORTANCE
Yeasts of the genus, especially , can cause disease with unacceptably high mortality. This is due to delays in diagnostics, ineffective treatments, and an incomplete understanding of the interactions between this fungus and our immune system. In this study, we expand our knowledge of the biological function of the gene, particularly its effect on modulating the host's immune response. Normally, 's infections are characterized by an anti-inflammatory response that is unable to control the yeast. In the absence of , the response to the infection is a dysregulated pro-inflammatory response that initially controls the fungi but eventually results in death of the host due to too much tissue damage. This is due, in part, to an altered fungal surface. Given the dual role of in modulating antifungal sensitivity and immune responses, this work provides important insights that may lead to new or improved therapeutics.
PubMed: 38948814
DOI: 10.1101/2024.06.17.599354 -
BioRxiv : the Preprint Server For... Jun 2024The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly...
The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly understood. In Arabidopsis, leaf epidermal pavement cells (PC) develop a puzzle-piece shape locally coordinated through apoplastic auxin signaling. Here we show auxin also globally coordinates interdigitation by activating the TIR1/AFB-dependent nuclear signaling pathway. This pathway promotes a transient maximum of auxin at the cotyledon tip, which then moves across the leaf activating local PC polarization, as demonstrated by locally uncaged auxin globally rescuing defects in mutant but not in mutants. Our findings show that hierarchically integrated global and local auxin signaling systems, which respectively depend on TIR1/AFB-dependent gene transcription in the nucleus and TMK-mediated rapid activation of ROP GTPases at the cell surface, control PC interdigitation patterns in Arabidopsis cotyledons, revealing a mechanism for coordinating a local cellular process with the development of whole tissues.
PubMed: 38948792
DOI: 10.1101/2024.06.17.599171 -
BioRxiv : the Preprint Server For... Jun 2024Flow cytometry is a widely used technique for immune cell analysis, offering insights into cell composition and function. Spectral flow cytometry allows for...
Flow cytometry is a widely used technique for immune cell analysis, offering insights into cell composition and function. Spectral flow cytometry allows for high-dimensional analysis of immune cells, overcoming limitations of conventional flow cytometry. However, analyzing data from large antibody panels can be challenging using traditional bi-axial gating strategies. Here, we present a novel analysis pipeline designed to improve analysis of spectral flow cytometry. We employ this method to identify rare T cell populations in aging. We isolated splenocytes from young (2-3 months) and aged (18-19 months) female mice then stained these with a panel of 20 fluorescently labeled antibodies. Spectral flow cytometry was performed, followed by data processing and analysis using Python within a Jupyter Notebook environment to perform batch correction, unsupervised clustering, dimensionality reduction, and differential expression analysis. Our analysis of 3,776,804 T cells from 11 spleens revealed 34 distinct T cell clusters identified by surface marker expression. We observed significant differences between young and aged mice, with certain clusters enriched in one age group over the other. Naïve, effector memory, and central memory CD8 and CD4 T cell subsets exhibited age-associated changes in abundance and marker expression. Additionally, γδ T cell clusters showed differential abundance between age groups. By leveraging high-dimensional analysis methods borrowed from single-cell RNA sequencing analysis, we identified age-related differences in T cell subsets, providing insights into the immune aging process. This approach offers a robust, free, and easily implemented analysis pipeline for spectral flow cytometry data that may facilitate the discovery of novel therapeutic targets for age-related immune dysfunction.
PubMed: 38948780
DOI: 10.1101/2024.06.19.599633 -
BioRxiv : the Preprint Server For... Jun 2024has emerged as a frontrunner among deadly fungal pathogens and is particularly life-threatening for many HIV-infected individuals with compromised immunity. Multiple...
UNLABELLED
has emerged as a frontrunner among deadly fungal pathogens and is particularly life-threatening for many HIV-infected individuals with compromised immunity. Multiple virulence factors contribute to the growth and survival of within the human host, the two most prominent of which are the polysaccharide capsule and melanin. As both of these features are associated with the cell wall, we were interested to explore possible cooperative or competitive interactions between these two virulence factors. Whereas capsule thickness had no effect on the rate at which cells became melanized, build-up of the melanin pigment layer resulted in a concomitant loss of polysaccharide material, leaving melanized cells with significantly thinner capsules than their non-melanized counterparts. When melanin was provided exogenously to cells in a transwell culture system we observed a similar inhibition of capsule growth and maintenance. Our results show that melanin sequesters calcium thereby limiting its availability to form divalent bridges between polysaccharide subunits required for outer capsule assembly. The decreased ability of melanized cells to incorporate exported polysaccharide into the growing capsule correlated with the amount of shed polysaccharide, which could have profound negative impacts on the host immune response.
SIGNIFICANCE STATEMENT
is an opportunistic fungal pathogen that presents a significant health risk for immunocompromised individuals. We report an interaction between the two major cryptococcal virulence factors, the polysaccharide capsule and melanin. Melanin impacted the growth and maintenance of the polysaccharide capsule, resulting in loss of capsular material during melanization. Our results suggest that melanin can act as a sink for calcium, thereby limiting its availability to form ionic bridges between polysaccharide chains on the growing surface of the outer capsule. As polysaccharide is continuously exported to support capsule growth, failure of melanized cells to incorporate this material results in a higher concentration of shed polysaccharide in the extracellular milieu, which is expected to interfere with host immunity.
PubMed: 38948764
DOI: 10.1101/2024.06.20.599928 -
BioRxiv : the Preprint Server For... Jun 2024Cochlear hair cell stereocilia bundles are key organelles required for normal hearing. Often, deafness mutations cause aberrant stereocilia heights or morphology that...
Cochlear hair cell stereocilia bundles are key organelles required for normal hearing. Often, deafness mutations cause aberrant stereocilia heights or morphology that are visually apparent but challenging to quantify. Actin-based structures, stereocilia are easily and most often labeled with phalloidin then imaged with 3D confocal microscopy. Unfortunately, phalloidin non-specifically labels all the actin in the tissue and cells and therefore results in a challenging segmentation task wherein the stereocilia phalloidin signal must be separated from the rest of the tissue. This can require many hours of manual human effort for each 3D confocal image stack. Currently, there are no existing software pipelines that provide an end-to-end automated solution for 3D stereocilia bundle instance segmentation. Here we introduce VASCilia, a Napari plugin designed to automatically generate 3D instance segmentation and analysis of 3D confocal images of cochlear hair cell stereocilia bundles stained with phalloidin. This plugin combines user-friendly manual controls with advanced deep learning-based features to streamline analyses. With VASCilia, users can begin their analysis by loading image stacks. The software automatically preprocesses these samples and displays them in Napari. At this stage, users can select their desired range of z-slices, adjust their orientation, and initiate 3D instance segmentation. After segmentation, users can remove any undesired regions and obtain measurements including volume, centroids, and surface area. VASCilia introduces unique features that measures bundle heights, determines their orientation with respect to planar polarity axis, and quantifies the fluorescence intensity within each bundle. The plugin is also equipped with trained deep learning models that differentiate between inner hair cells and outer hair cells and predicts their tonotopic position within the cochlea spiral. Additionally, the plugin includes a training section that allows other laboratories to fine-tune our model with their own data, provides responsive mechanisms for manual corrections through event-handlers that check user actions, and allows users to share their analyses by uploading a pickle file containing all intermediate results. We believe this software will become a valuable resource for the cochlea research community, which has traditionally lacked specialized deep learning-based tools for obtaining high-throughput image quantitation. Furthermore, we plan to release our code along with a manually annotated dataset that includes approximately 55 3D stacks featuring instance segmentation. This dataset comprises a total of 1,870 instances of hair cells, distributed between 410 inner hair cells and 1,460 outer hair cells, all annotated in 3D. As the first open-source dataset of its kind, we aim to establish a foundational resource for constructing a comprehensive atlas of cochlea hair cell images. Together, this open-source tool will greatly accelerate the analysis of stereocilia bundles and demonstrates the power of deep learning-based algorithms for challenging segmentation tasks in biological imaging research. Ultimately, this initiative will support the development of foundational models adaptable to various species, markers, and imaging scales to advance and accelerate research within the cochlea research community.
PubMed: 38948743
DOI: 10.1101/2024.06.17.599381 -
BioRxiv : the Preprint Server For... Jun 2024Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact...
UNLABELLED
Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. adheres to surfaces using a polysaccharide adhesin called the holdfast. In , disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several genes involved in flagellar surface sensing. One of these, called , codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting results in a severe motility defect that we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network ( , , and ) contribute to both Δ dependent and Δ dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion.
IMPORTANCE
Biofilms pose a threat in clinical and industrial settings. Surface sensing is an early step in biofilm formation. Studying surface sensing can help develop strategies for combating harmful biofilms. Here, we use the freshwater bacterium to study surface sensing. We characterize a previously unstudied gene, , and find that it localizes to the cell pole in the presence of three proteins that make up a component of the flagellum called the C-ring. Additionally, we find that is required for chemotaxis but dispensable for swimming motility. Lastly, our results show that mutating and other genes required for chemotaxis causes a hyperadhesive phenotype. We propose that surface sensing requires chemotaxis for a robust response to a surface.
PubMed: 38948737
DOI: 10.1101/2024.06.20.599946 -
BioRxiv : the Preprint Server For... Jun 2024Although blood group variation was first described over a century ago, our understanding of the genetic variation affecting antigenic expression on the red blood cell...
UNLABELLED
Although blood group variation was first described over a century ago, our understanding of the genetic variation affecting antigenic expression on the red blood cell surface in many populations is lacking. This deficit limits the ability to accurately type patients, especially as serological testing is not available for all described blood groups, and targeted genotyping panels may lack rare or population-specific variants. Here, we perform serological assays across 24 antigens and whole genome sequencing on 100 Omanis, a population underrepresented in genomic databases. We inferred blood group phenotypes using the most commonly typed genetic variants. The comparison of serological to inferred phenotypes resulted in an average concordance of 96.9%. Among the 22 discordances, we identify seven known variants in four blood groups that, to our knowledge, have not been previously reported in Omanis. Incorporating these variants for phenotype inference, concordance increases to 98.8%. Additionally, we describe five candidate variants in the Lewis, Lutheran, MNS, and P1 blood groups that may affect antigenic expression, although further functional confirmation is required. Notably, we identify several blood group alleles most common in African populations, likely introduced to Oman by gene flow over the last thousand years. These findings highlight the need to evaluate individual populations and their population history when considering variants to include in genotype panels for blood group typing. This research will inform future work in blood banks and transfusion services.
KEY POINTS
Utilizing whole genome sequencing to infer blood types in Omanis demonstrates high sensitivity for most blood groupsPopulation history influences blood group variation, necessitating population-specific genotype panels.
PubMed: 38948735
DOI: 10.1101/2024.06.17.599396 -
BioRxiv : the Preprint Server For... Jun 2024Bridge-like lipid transport proteins (BLTPs) are an evolutionarily conserved family of proteins that localize to membrane contact sites and are thought to mediate the...
Bridge-like lipid transport proteins (BLTPs) are an evolutionarily conserved family of proteins that localize to membrane contact sites and are thought to mediate the bulk transfer of lipids from a donor membrane, typically the endoplasmic reticulum (ER), to an acceptor membrane, such as a that of the cell or an organelle . Despite the fundamental importance of BLTPs for cellular function, the architecture, composition, and lipid transfer mechanisms remain poorly characterized. Here, we present the subunit composition and the cryo-electron microscopy structure of the native LPD-3 BLTP complex isolated from transgenic . LPD-3 folds into an elongated, rod-shaped tunnel whose interior is filled with ordered lipid molecules that are coordinated by a track of ionizable residues that line one side of the tunnel. LPD-3 forms a complex with two previously uncharacterized proteins, here named "Intake" and "Spigot", both of which interact with the N-terminal end of LPD-3 where lipids enter the tunnel. Intake has three transmembrane helices, one of which borders the entrance to the tunnel; Spigot has one transmembrane helix and extends 80 Å along the cytosolic surface of LPD-3. Experiments in multiple model systems indicate that Spigot plays a conserved role in ER-PM contact site formation. Our LPD-3 complex structural data, together with molecular dynamics simulations of the transmembrane region in a lipid bilayer, reveal protein-lipid interactions that suggest a model for how the native LPD-3-complex mediates bulk lipid transport and provide a foundation for mechanistic studies of BLTPs.
PubMed: 38948693
DOI: 10.1101/2024.06.21.600134