Biology subjects

Franks, J.

Publications and source records attributed to Franks, J..

8 recordsLinked to original sources

bioRxiv2023

Human Induced Pluripotent Stem Cell based Hepatic-Modeling of Lipid metabolism associated TM6SF2 E167K variant

BACKGROUND AND AIMSTM6SF2 rs58542926 (E167K) is associated with an increase in the prevalence of Metabolic Disfunction-Associated Steatotic Liver Disease (MASLD). Despite all the investigation related to the role of this variant in lipid metabolism, conflicting results in mouse studies underscore the importance of creating a human model for understanding the TM6SF2 mechanism. Therefore, the aim of this study is to generate a reliable human in vitro model that mimic the effects of the TM6SF2 E167K mutation and can be used for future mechanism studies. APPROACH AND RESULTSWe performed gene editing on human-induced pluripotent stem cells (iPSC) derived from a healthy individual to obtain the cells carrying the TM6SF2 E167K mutation. After hepatic differentiation, a decrease in TM6SF2 protein expression was observed in the mutated-induced hepatocyte. An increase in intracellular lipid droplets and a decrease in the efflux of cholesterol and ApoB100 were also observed. Transcriptomics analysis showed up-regulation of genes related to the transport, flux, and oxidation of lipids, fatty acids, and cholesterol in TM6SF2 E167K cells. Additionally, signs of cellular stress were observed in the ER and mitochondria. CONCLUSIONSOur findings indicate that induced hepatocytes generated from iPSC carrying the TM6SF2 E167K recapitulate the effects observed in human hepatocytes from individuals with the TM6SF2 mutation. This study characterizes an in vitro model that can be used as a platform to help in the identification of potential clinical targets and therapies and to understand the mechanism by which the TM6SF2 E167K variant leads to vulnerability to MASLD.

cell biology↗

bioRxiv2023

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in atrophic AMD

Introductory paragraphAge-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.

cell biology↗

bioRxiv2023

In vivo evolution of a Klebsiella pneumoniae capsule defect promotes complement-mediated opsono-phagocytosis and persistence during recurrent infection

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections rarely overwhelm the host but are associated with high mortality. The complement system is a key host defense against bloodstream infection. However, there are varying reports of serum resistance among KPC-Kp isolates. We assessed growth of 59 KPC-Kp clinical isolates in human serum and found increased resistance in 16/59 (27%). We identified five genetically-related bloodstream isolates with varying serum resistance profiles collected from a single patient during an extended hospitalization marked by recurrent KPC-Kp bloodstream infections. We noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ, that emerged during infection was associated with decreased polysaccharide capsule content, and resistance to complement-mediated killing. Surprisingly, disruption of wcaJ increased deposition of complement proteins on the microbial surface compared to the wild-type strain and led to increased complement-mediated opsono-phagocytosis in human whole blood. Disabling opsono-phagocytosis in the airspaces of mice impaired in vivo control of the wcaJ loss-of-function mutant in an acute lung infection model. These findings describe the rise of a capsular mutation that promotes KPC-Kp persistence within the host by enabling co-existence of increased bloodstream fitness and reduced tissue virulence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/542722v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@155736eorg.highwire.dtl.DTLVardef@10d47c0org.highwire.dtl.DTLVardef@e1ab83org.highwire.dtl.DTLVardef@1c33131_HPS_FORMAT_FIGEXP M_FIG Graphical abstract Created with BioRender.com C_FIG

microbiology↗

bioRxiv2023

Genetically engineering endothelial niche in human kidney organoids enables multilineage maturation, vascularization, and de novo cell types

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducible ETS translocation variant 2 (ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+ cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies. Translational StatementDeveloping therapies for patients with kidney diseases relies on a morphologically and physiologically representative in vitro model. Human kidney organoids are an attractive model to recapitulate kidney physiology, however, they are limited by the absence of a vascular network and mature cell populations. In this work, we have generated a genetically inducible endothelial niche that, when combined with an established kidney organoid protocol, induces the maturation of a robust endothelial cell network, induces a more mature podocyte population, and induces the emergence a functional renin population. This advance significantly increases the clinical relevance of human kidney organoids for etiological studies of kidney disease and future regenerative medicine strategies. Graphical AbstractGenetically engineered endothelial niche induces mature cell populations in human kidney organoids O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/542848v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1b129d4org.highwire.dtl.DTLVardef@313ae3org.highwire.dtl.DTLVardef@111c91org.highwire.dtl.DTLVardef@16a8d45_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Development of a nucleoside-modified mRNA vaccine against clade 2.3.4.4b H5 highly pathogenic avian influenza virus

Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. We generated an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. We show that the vaccine is immunogenic in mice and ferrets and prevents morbidity and mortality of ferrets following 2.3.4.4b H5N1 challenge.

microbiology↗

bioRxiv2023

Cryo-FIB workflow for imaging brain tissue via in situ cryo-electron microscopy

Cryo-electron microscopy (cryo-EM) enables the study of protein complexes, cytoskeletal elements, and organelles in three dimensions without the use of chemical fixation. Most cryo-EM studies focus on vitreously frozen individual cells separated from their native tissue contexts. This reliance on imaging of single cells is primarily due to technical challenges associated with preparing fresh tissue sections at a thinness sufficient for visualization via cryo-EM. Highly heterogenous and specialized tissues, such as brain, are especially affected by this limitation as the cellular, subcellular, and synaptic milieus can significantly vary across neuroanatomical locations. To address this limitation, we established new instrumentation and a workflow that consists of: 1) high-pressure freezing of fresh brain tissue; 2) tissue trimming followed by cryo-focused ion beam milling via the H-bar approach to generate ultrathin lamellae; and 3) cryo-EM imaging. Here, we apply this workflow to visualize the fine ultrastructural details of organelles, as well as cytoskeletal and synaptic elements that comprise the cortical neuropil within fresh, unfixed mouse brain tissue. Moreover, we present initial studies that apply principles of the above workflow to the analysis of postmortem human brain tissue. Overall, our work integrates the strengths of cryo-electron microscopy and tissue-based approaches to produce a generalizable workflow capable of visualizing subcellular structures within complex tissue environments.

neuroscience↗

bioRxiv2022

α-Arrestins maintain phospholipid balance and Atg18 distribution to permit efficient autophagy

Cells selectively reorganize their membrane proteome in response to stressors via selective protein trafficking. The -arrestins, a family of conserved protein trafficking adaptors, bind to select membrane proteins and interact with the ubiquitin ligase Rsp5. The -arrestins recruit Rsp5 to its membrane protein substrates, permitting their ubiquitination and endocytosis. To identify new -arrestin functions, we performed a genetic screen to isolate mutants that alter -arrestin-mediated resistance to rapamycin, a drug that inhibits TORC1. Interestingly, loss of many of the ATG genes, which encode the machinery needed for the self-degradative process of autophagy, disrupted -arrestins ability to promote growth on rapamycin. Herein we define a genetic network linking -arrestins to autophagy. We show autophagy impairment in the absence of select -arrestins, with increased autophagosome lifetimes and delayed/reduced delivery of autophagosomes to the vacuole. The -arrestin mutants that impeded autophagy had vacuole morphology defects and increased vacuolar retention of Atg18, a member of the PROPPIN family that is needed to maintain vacuole shape and facilitate lipid transfer to expanding autophagosomes. Atg18 binds phosphatidylinositol 3 phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), and we observed increased PI3P on the vacuole membrane in -arrestin mutants. The levels of Vps34 and Fab1, the kinases responsible for the generation of PI3P and PI(3,5)P2, respectively, were also elevated at vacuole membranes in cells lacking -arrestins. We posit that altered phospholipids in the vacuolar membrane form the basis for the Atg18-Atg2 mislocalization and autophagy defect. These data demonstrate a previously unappreciated link between the -arrestins and autophagy, expanding the functional impact of these trafficking adaptors in responding to nutrient stress. Author SummaryCells survive nutrient starvation by degrading parts of themselves through the process of autophagy. During autophagy, cells make a double membrane, known as an autophagosome (AP), around bits of cytoplasm or organelles. The AP and its engulfed material are delivered to the vacuole, an organelle that helps break down proteins and lipids. These materials can then be used as building blocks to generate the essential components needed for the cell to survive starvation. For cells to undergo efficient autophagy, they need -arrestins, a group of proteins important for deciding where membrane proteins localize. In cells lacking -arrestins, the AP forms slowly, likely due to a problem in growing the AP membrane. This results in less material being delivered to the vacuole via APs when cells do not have -arrestins. This study defines a new role for -arrestins in promoting AP formation and starvation survival.

cell biology↗

bioRxiv2022

WNK kinases sense molecular crowding and rescue cell volume via phase separation

When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

cell biology↗