Biology subjects

Moreno, C. L.

Publications and source records attributed to Moreno, C. L..

8 recordsLinked to original sources

bioRxiv2026

TriPort tangential flow filtration: a lab-assembled system for EV isolation across diverse cell-culture sources

Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three- port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to- protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum- free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.

cell biology↗

bioRxiv2026

FBXW11 Activity Regulates Radial Glial Expansion in Human Cerebral Organoids

Human brain development depends on tightly coordinated gene-regulatory programs and the emergence of complex tissue architecture, making large scale functional interrogation difficult using conventional screen models. To overcome this challenge, we used a pooled CRISPR screening approach. Guided by neuro-specific whole-genome screens in Drosophila, we tested 129 poorly characterised human orthologs and found 8 that modify cerebral organoid development. Candidates were validated using individual CRISPR knockouts and mosaic competition assays. Among these candidates we describe FBXW11, a substrate-recognition component of the SCF E3 ubiquitin ligase complex, as a potent negative regulator of cerebral organoid expansion. FBXW11 loss increases radial glial abundance, expands ventricular-like domains, and impairs neuronal maturation. Mechanistically, FBXW11 associates with {beta}-catenin and alters WNT signalling. FBXW11 mutations cause the autosomal-dominant Mendelian syndrome Neurodevelopmental, Jaw, Eye and Digital syndrome (NEDJED), and we found that disease-associated variants mapped preferentially to WD40 substrate-binding repeats and {beta}-catenin contact regions, linking impaired substrate recognition to neurodevelopmental disease. Together, these findings identify FBXW11 as a conserved negative regulator of {beta}-catenin-dependent radial glial expansion and neuronal maturation during human cerebral brain development.

neuroscience↗

bioRxiv2025

Replication of SARS-CoV-2 Omicron lineages is defined by TMPRSS2 use in environments where ACE2 is complexed with solute carriers SLC6A19 and SLC6A20.

The Omicron variant of SARS-CoV-2 emerged in late 2021 and since then Omicron subvariants have continued to evolve and dominate globally. The viral S protein evolved towards highly efficient antibody evasion and replicative capacity in the upper respiratory tract resulting in high transmissibility. At the same time, the mutations acquired in the S protein diminish infection of the lung epithelium and pathogenic potential. The changing entry requirements for Omicron sub-lineages that lead to this shift in tropism remain poorly understood. We resolve the changing replication requirements of SARS-CoV-2 to be related to two distinct pools of ACE2. The first pool relates to ACE2s role in the renin angiotensin system (RAS) and this pool can complex with TMPRSS2 (RAS-ACE2). The second pool relates to ACE2s role as a protein solute carrier chaperone than cannot complex with TMPRSS2 (Chaperone ACE2). Here, we demonstrate that pre-Omicron lineages replicate in a TMPRSS2 dependent manner across both ACE2 pools, whilst Omicron lineages can only spread and replicate using chaperone ACE2. This provides a mechanistic basis for the evolving infectivity requirements of SARS-CoV-2 and furthermore provides approaches to track and monitor ACE2 utilizing coronaviruses. Graphical AbstractMechanistic basis for shift in SARS-CoV-2 tropism with the arrival of Omicron. A. Chaperone ACE2 is defined structurally as a heterodimer of dimers with a solute carrier protein-SLC6A19 or SLC6A20. Here this ACE2 structure can exist uncomplexed from TMPRSS2 and enables TMPRSS2 use by both pre-Omicron and Omicron lineages. B. Renin Angiotensin ACE2 is defined by ACE2 with an exposed collectrin-like domain (CLD), which enables binding of TMPRSS2 or ADAM-17. Here ACE2 can form a complex with TMPRSS2 in a manner that allows pre-Omicron but not Omicron lineages to utilize TMPRSS2 to facilitate infection. Here Omicron lineages are heavily attenuated as they cannot use TMPRSS2 to spread. C. to E. Based on single cell profiles, ACE2 can exist as a chaperone with SLC6A20 in C. the Nasal Cavity or D. primarily as RAS-ACE2 in the lung to respond to acute lung injury. E. The largest pool of ACE2 in our body resides within the small intestine on enterocytes and this further facilitates replication in this tissue by pre-Omicron and Omicron lineages. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/663433v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@172aa47org.highwire.dtl.DTLVardef@109285eorg.highwire.dtl.DTLVardef@394f8corg.highwire.dtl.DTLVardef@958e31_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

A chimeric viral platform for directed evolution in mammalian cells

Directed evolution (DE) is a process of mutation and artificial selection to breed biomolecules with new or improved activity1,2. DE platforms are primarily prokaryotic or yeast-based, and stable mutagenic mammalian systems have been challenging to establish and apply3. To this end, we developed PROTein Evolution Using Selection (PROTEUS), a new platform that uses chimeric virus-like vesicles (VLVs) to enable extended mammalian DE campaigns without loss of system integrity. This platform is stable and can generate sufficient diversity for DE in mammalian systems. Using PROTEUS, we altered the doxycycline responsiveness of tetracycline-controlled transactivators, generating a more sensitive TetON-4G tool for gene regulation. PROTEUS is also compatible with intracellular nanobody evolution, and we use it to design a DNA damage-responsive anti-p53 nanobody. Overall, PROTEUS is an efficient and stable platform to direct evolution of biomolecules within mammalian cells.

molecular biology↗

bioRxiv2024

Identification of SEC61B as a novel regulator of calcium flux and platelet hyperreactivity in diabetes mellitus

High platelet reactivity is associated with adverse clinical events and is more frequent in people with diabetes mellitus (DM). To better understand platelet dysfunction in DM, we performed a proteomic analysis in platelets from a matched cohort of 34 people without, and 42 people with type 2 DM. The cohorts were matched by clinical characteristics including age, sex, and coronary artery disease burden. Using high sensitivity unbiased proteomics, we consistently identified over 2,400 intracellular proteins, and detected proteins that are differentially released by platelets from people with diabetes in response to low dose thrombin. Importantly, we identified the endoplasmic reticulum (ER) protein SEC61 translocon subunit beta (SEC61B) was increased in platelets from humans and mice with in vivo hyperglycemia. SEC61B was increased in megakaryocytes in mouse models of diabetes, in association with megakaryocyte ER stress. A rise in cytosolic calcium is a key aspect in platelet activation, and the SEC61 translocon is known to act as a channel for ER calcium leak. We demonstrate that cultured cells overexpressing SEC61B have increased calcium flux and decreased protein synthesis. In accordance, hyperglycemic mouse platelets mobilized more calcium to the cytosol and had lower protein synthesis compared with normoglycemic platelets. Independently, in vitro induction of ER stress increased platelet SEC61B expression and markers of platelet activation. We propose a mechanism whereby ER stress-induced upregulation of platelet SEC61B leads to increased cytosolic calcium, potentially contributing to platelet hyperactivity in people with diabetes. Key PointsO_LIPlatelet SEC61B is increased in hyperglycemia and contributes to increased endoplasmic reticulum (ER) calcium leak C_LIO_LIIncreased ER calcium leak is associated with ER stress and platelet hyperactivity C_LI

cell biology↗

bioRxiv2023

TMPRSS2 activation of Omicron lineage Spike glycoproteins is regulated by TMPRSS2 cleavage of ACE2

Continued high-level spread of SARS-CoV-2 has enabled an accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) dependent entry by primary SARS-CoV-2 isolates that occurred upon arrival of Omicron lineages. Mechanistically we show this shift to be a function of two distinct ACE2 pools based on TMPRS22 association with the ACE2 Collectrin-Like Domain (CLD). In engineered cells overexpressing ACE2 and TMPRSS2, ACE2/TMPRSS2 complexes led to either augmentation or attenuation of viral infectivity of pre-Omicron and Omicron lineages, respectively. Mutagenesis of the ACE2-CLD TMPRSS2 cleavage site in ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 activation of viral fusion. Our data supports the evolution of Omicron lineages towards the use of ACE2 unable to form complexes with TMPRSS2 and consistent with ACE2 structure and function as a chaperone for many tissue specific amino acid transport proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/558930v4_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1c86cf0org.highwire.dtl.DTLVardef@16815feorg.highwire.dtl.DTLVardef@7be9e1org.highwire.dtl.DTLVardef@137de1c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO ACE2-TMPRSS2 pool model and evolution of SARS-CoV-2 tropism.A. &-B. ACE2-TMPRSS2 pool model to reconcile the evolving entry requirements of SARS-CoV-2 and changes in viral tropism in vivo. A. Both SARS-CoV-1 and early SARS-CoV-2 (pre-Omicron) lineages have molecular dual tropism, with efficient entry when ACE2 (blue protein) can form complexes with TMPRSS2 (green protein) and in settings where TMPRSS2 is excluded from ACE2 (C4-ACE2 CLD). In both settings ACE2 initially engages SARS-CoV-2 spike and fusion is then triggered through TMPRSS2 cleavage of the Spike S2 domain B. Over time, the dual tropism for two distinct pools of ACE2 (with and without TMPRSS2) has been lost, with consolidation towards ACE2 where TMPRSS2 is no longer in a complex. With the arrival of Omicron lineages, ACE2-TMPRSS2 complexes could no longer enable efficient Spike S2 cleavage and fusogenic activation (TMPRSS2 "off" confirmation"). Rather, only TMPRSS2 uncoupled from ACE2 could facilitate the latter cleavage of S2. C. Over time this has further consolidated over generations of omicron lineages from 2022 lineages (BA.1, BA.2 and BA.5) through to 2023 lineages (XBB.1.5) and now in 2024 JN.1 lineages such as KP.3. Overall, this supports the initial molecular tropism of early SARS-CoV-2 clades to be similar to that observed for SARS-CoV-1, with dual tropism across both ACE2 pools and replication proceeding in tissues where ACE2-TMPRSS2 complexes would be prevalent (e.g. Lung). The evolution away from ACE2-TMPRSS2 complexes towards ACE2 where TMPRSS2 is structurally uncoupled (e.g. ACE2 as a chaperone for solute carriers SLCA619 or SLCA620) is consistent selection of this ACE2 pool in a manner that has sustained transmission fitness within the human population. C_FIG

microbiology↗

bioRxiv2023

P-Selectin promotes SARS-CoV-2 interactions with platelets and the endothelium

COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood1,2. To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes that, when upregulated, alter the outcome of authentic SARS-CoV-2 infection. From this, we identify 34 new genes that can alter the course of infection, including the innate immune receptor P-selectin, which we show is a novel SARS-CoV-2 spike receptor. At the cellular level expression of P-selectin does not confer tropism for SARS-CoV-2, instead it acts to suppress infection. More broadly, P-selectin can also promote binding to SARS-CoV-2 variants, SARS-CoV-1 and MERS, acting as a general spike receptor for highly pathogenic coronaviruses. P-selectin is expressed on platelets and endothelium3, and we confirm SARS-CoV-2 spike interactions with these cells are P-selectin-dependent and can occur under shear flow conditions. In vivo, authentic SARS-CoV-2 uses P-selectin to home to airway capillary beds where the virus interacts with the endothelium and platelets, and blocking this interaction can clear vascular-associated SARS-CoV-2 from the lung. Together we show for the first time that coronaviruses can use the leukocyte recruitment system to control tissue localization, and this fundamental insight may help us understand and control highly pathogenic coronavirus disease progression.

immunology↗

bioRxiv2021

LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here we use whole genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe COVID-19 infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

cell biology↗