Biology subjects

Freedman, B.

Publications and source records attributed to Freedman, B..

9 recordsLinked to original sources

bioRxiv2026

Tendon-derived injectable bio-instructive gel augments tenogenic differentiation of iMSC-SCX cells and extracellular matrix remodeling

In the US, 33 million musculoskeletal injuries have been reported per year, with 50% involving tendons and ligaments in both athletic and aging populations. Tendon repair often results in the formation of biomechanically inferior scar tissue rather than functional regeneration. Local cell therapy is garnering significant interest in tendon repair because it provides a targeted means to repopulate defects with potent therapeutic cells. Here, we developed a porcine tendon-derived thermoresponsive extracellular matrix hydrogel (TG) as an injectable, bio-instructive carrier for scleraxis-overexpressing induced mesenchymal stem cell derived tenocytes (iTenocytes), with the goal of improving cell retention and overall transplantation success. TG was compositionally distinct from purified collagen (PC, from rat tail type 1 collagen) and exhibited favorable material properties for a minimally invasive percutaneous strategy, including thermoresponsive gelation, shear-thinning injectability, retention at the injection site, and controlled biodegradation. In vitro, TG supported three-dimensional cell residence, promoted cell interconnectivity and redistribution at the matrix interface, and increased collagen type I release from iTenocytes compared to those embedded in PC. Transcriptomic and proteomic analyses further showed that TG enhanced programs associated with tenogenic maturation, extracellular matrix assembly, focal adhesion, mechano-transduction, and remodeling. In a rat Achilles tendon partial defect model, both optical imaging and histological analyses demonstrated retention of iTenocytes at the injection site. Additionally, confocal imaging demonstrated retention of iTenocytes within the defect site for up to 10 days. Together, these findings identify TG as a biofunctional injectable carrier that supports the tenogenic characteristics of iTenocytes and enhances their local persistence after transplantation.

cell biology↗

bioRxiv2026

Microenvironment optimization enables kidney organoid longevity with epithelial-endothelial joint basement membrane formation

Kidney organoids degrade in long-term culture and lack joint basement membranes between epithelial and endothelial cells characteristic of renal tissue. Here we show that these limitations can be overcome in static cultures simply by optimizing the microenvironment. Supplementing standard media with tubular-enhancing factors (TEFs) dramatically improves organoid yield and longevity, while vascular-enhancing factors (VEFs) and replating increases endothelial cell yield and invasiveness. A transcriptomic and imaging atlas demonstrates maintenance of nephron structures for six months with increased metabolism, signaling, differentiation, and aging-related pathways. In addition to adherent cultures, these media also enable organoid differentiation and vascularization in suspension cultures and hydrogels. Remarkably, addition of TEFs and VEFs to organoids in suspension induces self-assembly of joint basement membranes between endothelial cells and podocytes or tubules, a major feature of renal tissue. Microenvironment optimization thus enables longitudinal stabilization and higher-order vascularization of kidney organoids, offering a diverse resource for long-term studies and tissue engineering applications.

bioengineering↗

bioRxiv2023

Enterobacter Sp. SM3 Exhibits Run-and-Tumble Motility

The recent discovery of the peritrichous, swarm-competent bacterium Enterobacter sp. SM3 has offered a new opportunity to elucidate the role of swarming motility in the gut microbiome. Here we present first findings of the run-and-tumble behavior of SM3 in both a control solution of motility buffer and upon bulk exposure to the chemoattractants serine and aspartate, drawing a comparison with the well-studied behavior of E. coli. It was found that SM3 runs with an average speed of approximately 30 {micro}m/s for an average duration of 0.77 s. Tumble events occur for an average duration of 0.11 s with a 69{whitebullet} average tumble angle. Both values are similar to that of E. coli. When exposed to serine, SM3 suppresses the frequency of tumble events, which in turn increases the average run duration. In addition, the average tumble angle was found to decrease in response to serine. However, when exposed to up to a millimolar concentration of aspartate, SM3 does not demonstrate a notable change in run-and-tumble parameters. These results suggest that run-and-tumble is the characteristic swimming behavior of SM3 in its planktonic state. These findings serve as a benchmark in a quest to determine the connection among swimming, swarming, and the complex dynamics of the gut microbiome. IMPORTANCEBacteria form the largest domain of living creatures on this planet. Our interactions with bacteria influence us in many ways, not the least of which being in regards to human health. A recently identified species of gut bacteria, Enterobacter sp. SM3, has been shown to reduce intestinal inflammation, suggesting that swarming could play a physiologically beneficial role. In this report, we study the motility of individual SM3 bacteria This study is an essential step towards an overarching goal to understand the influence of bacterial motility on human health.

microbiology↗

bioRxiv2022

Superinfection plays an important role in the acquisition of complex Plasmodium falciparum infections among female Anopheles mosquitoes

Studies of human malaria infections with multiple, genetically distinct parasites have illuminated mechanisms of malaria transmission. However, few studies have used the genetic diversity in mosquito infections to understand how transmission is sustained. We identified likely human sources of mosquito infections from a longitudinal cohort in Western Kenya based on genetic similarity between parasites and the timing of infections. We found that several human infections were required to reconstitute each mosquito infection and that multiple parasite clones were likely transmitted from infected humans to mosquitoes in each bite, suggesting that superinfection and co-transmission occur simultaneously and are important mechanisms of transmission. We further investigated this using an individual human and mosquito simulation model and found that co-transmission alone was unlikely to reproduce the high complexity of mosquito infections. We concluded that the superinfection of mosquitoes likely plays an important, but under studied, role in sustaining moderate to high malaria transmission.

systems biology↗

bioRxiv2021

WWOX-Mediated Degradation of AMOTp130 Negatively Affects Egress of Filovirus VP40 VLPs

Ebola (EBOV) and Marburg (MARV) viruses continue to emerge and cause severe hemorrhagic disease in humans. A comprehensive understanding of the filovirus-host interplay will be crucial for identifying and developing antiviral strategies. The filoviral VP40 matrix protein drives virion assembly and egress, in part by recruiting specific WW-domain-containing host interactors via its conserved PPxY Late (L) domain motif to positively regulate virus egress and spread. In contrast to these positive regulators of virus budding, a growing list of WW-domain-containing interactors that negatively regulate virus egress and spread have been identified, including BAG3, YAP/TAZ and WWOX. In addition to host WW-domain regulators of virus budding, host PPxY-containing proteins also contribute to regulating this late stage of filovirus replication. For example, angiomotin (AMOT) is a multi-PPxY-containing host protein that functionally interacts with many of the same WW-domain-containing proteins that regulate virus egress and spread. In this report, we demonstrate that host WWOX, which negatively regulates egress of VP40 VLPs and recombinant VSV-M40 virus, interacts with and suppresses the expression of AMOT. We found that WWOX disrupts AMOTs scaffold-like tubular distribution and reduces AMOT localization at the plasma membrane via lysosomal degradation. In sum, our findings reveal an indirect and novel mechanism by which modular PPxY/WW-domain interactions between AMOT and WWOX regulate PPxY-mediated egress of filovirus VP40 VLPs. A better understanding of this modular network and competitive nature of protein-protein interactions will help to identify new antiviral targets and therapeutic strategies. IMPORTANCEFiloviruses (Ebola [EBOV] and Marburg [MARV]) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we reveal a novel mechanism by which host proteins WWOX and AMOTp130 interact with each other and with the EBOV matrix protein VP40 to regulate VP40-mediated egress of virus like particles (VLPs). Our results highlight the biological impact of competitive interplay of modular virus-host interactions on both the virus lifecycle and the host cell.

microbiology↗

bioRxiv2021

Multivalent designed proteins protect against SARS-CoV-2 variants of concern

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. One-Sentence SummaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

synthetic biology↗

bioRxiv2021

Targeting an anchored phosphatase-deacetylase unit restores renal ciliary homeostasis.

Pathophysiological defects in water homeostasis can lead to renal failure. Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder associated with abnormal cytoskeletal dynamics in the kidney collecting ducts and perturbed calcium and cAMP signaling in the ciliary compartment. We show that collecting ducts in mice lacking the A-Kinase anchoring protein AKAP220 exhibit enhanced development of primary cilia. Mechanistic studies reveal that AKAP220-associated protein phosphatase 1 (PP1) mediates this phenotype by promoting changes in the stability of histone deacetylase 6 (HDAC6) with concomitant defects in actin dynamics. This proceeds through a previously unrecognized adaptor function for PP1 as all ciliogenesis and cytoskeletal phenotypes are recapitulated in mIMCD3 knock-in cells expressing a phosphatase-targeting defective AKAP220-{Delta}PP1 mutant. Pharmacological blocking of local HDAC6 activity alters cilia development and reduces cystogenesis in kidney-on-chip and organoid models of ADPKD. These findings identify the AKAP220-PPI-HDAC6 pathway as a key effector in primary cilia development.

cell biology↗

bioRxiv2021

Angiomotin Counteracts the Negative Regulatory Effect of Host WWOX on Viral PPxY-Mediated Egress

Filoviridae family members Ebola (EBOV) and Marburg (MARV) viruses and Arenaviridae family member Lassa virus (LASV) are emerging pathogens that can cause hemorrhagic fever and high rates of mortality in humans. A better understanding of the interplay between these viruses and the host will inform about the biology of these pathogens, and may lead to the identification of new targets for therapeutic development. Notably, expression of the filovirus VP40 and LASV Z matrix proteins alone drives assembly and egress of virus-like particles (VLPs). The conserved PPxY Late (L) domain motifs in the filovirus VP40 and LASV Z proteins play a key role in the budding process by mediating interactions with select host WW-domain containing proteins that then regulate virus egress and spread. To identify the full complement of host WW-domain interactors, we utilized WT and PPxY mutant peptides from EBOV and MARV VP40 and LASV Z proteins to screen an array of GST-WW-domain fusion proteins. We identified WW domain-containing oxidoreductase (WWOX) as a novel PPxY-dependent interactor, and we went on to show that full-length WWOX physically interacts with eVP40, mVP40 and LASV Z to negatively regulate egress of VLPs and of a live VSV/Ebola recombinant virus (M40). Interestingly, WWOX is a versatile host protein that regulates multiple signaling pathways and cellular processes via modular interactions between its WW-domains and PPxY motifs of select interacting partners, including host angiomotin (AMOT). Notably, we demonstrated recently that expression of endogenous AMOT not only positively regulates egress of VLPs, but also promotes egress and spread of live EBOV and MARV. Toward the mechanism of action, we show that the competitive and modular interplay among WWOX-AMOT-VP40/Z regulates VLP and M40 virus egress. Thus, WWOX is the newest member of an emerging group of host WW-domain interactors (e.g. BAG3; YAP/TAZ) that negatively regulate viral egress. These findings further highlight the complex interplay of virus-host PPxY/WW-domain interactions and their potential impact on the biology of both the virus and the host during infection. Author SummaryFiloviruses (Ebola [EBOV] and Marburg [MARV]) and arenavirus (Lassa virus; LASV) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we identified host WW-domain containing protein WWOX as a novel interactor with VP40 and Z, and showed that WWOX inhibited budding of VP40/Z virus-like particles (VLPs) and live virus in a PPxY/WW-domain dependent manner. Our findings are important to the field as they expand the repertoire of host interactors found to regulate PPxY-mediated budding of RNA viruses, and further highlight the competitive interplay and modular virus-host interactions that impact both the virus lifecycle and the host cell.

microbiology↗

bioRxiv2021

Compound FC-10696 Inhibits Egress and Spread of Marburg Virus

Marburg virus (MARV) VP40 protein (mVP40) directs egress and spread of MARV, in part, by recruiting specific host WW-domain containing proteins via its conserved PPxY Late (L) domain motif to facilitate efficient virus-cell separation. We reported previously that small molecule compounds targeting the viral PPxY/host WW-domain interaction inhibited VP40-mediated egress and spread. Here, we report on the antiviral potency of novel compound FC-10696, which emerged from extensive structure activity relationship (SAR) of a previously described series of PPxY inhibitors. We show that FC-10696 inhibits egress of both mVP40 VLPs and egress and spread of authentic MARV from HeLa cells and primary human macrophages. Moreover, FC-10696 treated mice displayed delayed onset of weight loss, clinical signs, and significantly lower viral loads compared to controls, with 14% of animals surviving 21 days following a lethal MARV challenge. Thus, FC-10696 represents a first-in-class, host-oriented inhibitor effectively targeting late stages of the MARV lifecycle.

microbiology↗