Biology subjects

Fox, J. M.

Publications and source records attributed to Fox, J. M..

15 recordsLinked to original sources

bioRxiv2026

Enhanced early IgG-mediated complement deposition in the development of chronic chikungunya virus disease

Chikungunya virus (CHIKV) disease typically resolves following acute infection; however, some individuals develop chronic CHIKV disease (CCD) characterized by persistent, debilitating joint pain. Antibodies help clear CHIKV through neutralization and Fc effector functions. Previous studies have associated CCD with a poor neutralizing antibody response; however, the role of Fc effector functions in CCD development remains unclear. Here, purified IgG from post-acute serum of individuals who either resolved CHIKV disease or developed CCD was evaluated for IgG activity and Fc effector functions to identify correlations with disease progression and biomarkers for CCD. Resolution was associated with higher levels of CHIKV-specific IgG and IgG1 and stronger CHIKV neutralization. Regression analysis identified bulk IgG2 as a strong predictor of CHIKV disease progression. Development of CCD was associated with enhanced IgG-mediated complement deposition on infected cells. These findings suggest that localized complement activation at sites of infection may contribute to persistent inflammation underlying CCD.

immunology↗

bioRxiv2026

Bioorthogonal Tuning of Hydrogel Stiffness Promotes Zonal Redifferentiation of Passaged Chondrocytes

Generating bioengineered cartilage that recapitulates the depth-dependent phenotype, structure, and function of native articular cartilage remains a challenge. While cartilage is rich in aggrecan and type II collagen, proper function depends on depth-dependent protein expression. Superficial zone chondrocytes (SZCs) secrete proteoglycan-4 (PRG4) to lubricate the cartilage surface. Deep zone chondrocytes produce type X collagen (COLX) to support compressive loading and load transfer to subchondral bone. We previously demonstrated that passaged full-thickness chondrocytes (FTCs) and zonal chondrocytes can re-express cartilage and zone-specific markers following scaffold-free three-dimensional (3D) culture in redifferentiation media. However, in the absence of an instructive matrix, cells expressed low levels of zone-specific proteins and exhibited limited depth-dependent organization. We hypothesize that synthetic extracellular matrix with zone-specific microenvironmental cues will guide zonal differentiation. To this end, passaged primary bovine chondrocytes were encapsulated in a soft, hyaluronan (HA)-based, cell-adhesive, and protease-degradable hydrogel established via bioorthogonal tetrazine (Tz) ligation with norbornene (Nb). When supplemented with TGF{beta}3, FTCs deposited aggrecan and type II collagen with minimal type I collagen. Application of interfacial tetrazine ligation with trans-cyclooctene (TCO) during cell culture resulted in matrix stiffening, leading to upregulation of COLX expression. Conversely, SZCs cultured in soft hydrogels exhibited the greatest PRG4 expression. Establishment of a trilayered construct with region-specific stiffness via the diffusion-controlled reaction promoted PRG4 and COLX expression in defined zones. Together, these findings demonstrate that tunable HA-based hydrogels can enhance zone-specific chondrocyte phenotypes and promote the formation of zonally organized cartilage.

bioengineering↗

bioRxiv2026

Context-dependent peptide recognition shapes tyrosine kinase substrate specificity beyond consensus motifs

Protein tyrosine kinases (PTKs) regulate cellular biochemistry by phosphorylating tyrosine residues that alter protein function; their substrate preferences define the topology of signaling cascades. Previous studies of PTKs have mapped their average preferences for amino acids surrounding phosphorylation sites, but their sensitivity to sequence variation remains poorly understood. Here, we used microbial biosensors for PTK activity to examine the influence of local sequence context on substrate specificity. Across five well-studied PTKs, we identified amino acid substitutions within consensus substrates that could confer sensitivity to substrate length or enhance selectivity for one PTK over others. Using a secondary decoy screen, we found sequence-diverse substrates with unexpectedly orthogonal PTK compatibilities. Our findings show how context-specific sequence features alter PTK substrate specificity far beyond what might be expected from classical consensus models and establish an experimental framework for defining the limits of substrate overlap between closely related kinases.

biochemistry↗

bioRxiv2026

A Hidden Binding Pocket in the β- ketoacyl-ACP Synthase FabB

Assembly-line enzymes carry out multi-step synthesis of diverse metabolites by using a handful of catalytic motifs in which minor structural differences control substrate specificity and reaction order. Here we examine differences in substrate binding to FabB and FabF, the two {beta}-ketoacyl-ACP synthases (KSs) responsible for fatty acid elongation in Escherichia coli, by exploring a peculiar mutational effect. In FabB, a blocking mutation in the acyl binding pocket yields a shifted, but broad product profile, while in FabF, the same mutation disrupts the binding of acyl chains longer than eight carbons (C8). X-ray crystal structures of the FabB mutant provide an explanation: a second, previously unobserved binding pocket allows medium-to-long acyl chains ([≥] C8) to bind with an alternate conformation. Molecular simulations suggest that this pocket is more stable in FabB than in FabF, where mutations reduce the catalytic competency of longer chains instead of shifting them to the alternate pocket. Our findings indicate that homologous KSs differ not only in their primary binding sites but also in the availability of alternative binding modes that can buffer against mutational effects and enable functional diversification.

biochemistry↗

bioRxiv2025

Time-Resolved Tracking and Secretome Mapping of Intracellular, Cell Surface, and Extracellular Glycoconjugates with a Mini-Tetrazine N-Acetylglucosamine

Glycosylation is instrumental in governing essential biological processes that dictate health outcomes. Herein we describe a mini-tetrazine N-acetylglucosamine (HTz-GlcNAc) that is incorporated into N-linked glycans and through rapid live-cell labeling with trans-cyclooctene (TCO) reagents enables real-time visualization and evaluation of glycoconjugate trafficking in living cells. We utilize cell-permeable and impermeable TCO-fluorophores to perform time-resolved, multicolor live labeling of cell-surface glycoconjugates, as well as intracellular, and internalized glycoconjugates. We observe intracellular glycoconjugate trafficking throughout the secretory pathway of live cells in real-time, as well as cell surface glycan turnover and extracellular vesicle secretion across three days. In the toolkit of probes for metabolic oligosaccharide engineering, HTz-GlcNAc brings the capability of precise interrogation of intracellular and extracellular glycoconjugate trafficking, and selective tagging of intra-and-extracellular vesicles, while providing a handle for the purification and enrichment of glycans for immunoblot and proteomic analyses. TOC image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/682811v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@f65188org.highwire.dtl.DTLVardef@162c58corg.highwire.dtl.DTLVardef@1977b73org.highwire.dtl.DTLVardef@29e9ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Label-free DIA-NN enabled RIME (DIANNeR) reveals glucocorticoid receptor interaction networks in breast, bladder, and blood across normaluntransformed cells, cancer cell lines, and PDXs

Understanding how transcription factors execute tissue-specific programmes requires defining their protein interaction networks in physiologically relevant contexts. However, profiling normal untransformed cells presents fundamental challenges due to intrinsically limited input material. To meet this challenge we integrated data-independent acquisition (DIA), ion mobility separation, and library-free analysis to achieve a 2-fold increase in detection without loss in enrichment. Applied to the glucocorticoid receptor (GR), a ubiquitously expressed nuclear receptor driving pleiotropic responses to standard-of-care anti-inflammatory therapeutics, DIANNeR (DIA-NN enabled RIME) resolves distinct, context-dependent networks across breast, ureter and blood in normal and transformed contexts. Key findings include: detection of a HOXA5-GR interaction in normal epithelium which was undetected in malignant models; an epithelial-restricted SMARCD3-GR interaction with prognostic relevance; and a FOXP3-BCL11B-GR network in primary CD4+ T cell populations undetected by data dependant acquisition (DDA). By defining normal GR tissue interactomes, DIANNeR provides the essential comparator for interpreting network rewiring that drives disease.

cancer biology↗

bioRxiv2024

Requirement for Fc effector function is overcome by binding potency for broadly reactive anti-alphavirus antibodies

Alphaviruses are emerging public health threats. Broadly reactive anti-alphavirus monoclonal antibodies (mAbs) have been shown to be protective in mouse models of infection. However, the mechanism of Fc-dependent or Fc-independent heterologous protection remains ill-defined in vivo. Here, we used two vaccine-elicited, broadly reactive, anti-alphavirus mAbs, SKT05 and SKT20, to establish correlates of mAb-mediated protection during Venezuelan equine encephalitis virus (VEEV) challenge. SKT20 required Fc effector functions to prevent lethality. In contrast, SKT05-mediated survival was independent of Fc effector functions, which is likely linked to early viral control through potent egress inhibition. However, control of virus replication and spread with SKT05 was Fc-dependent; these findings extended to additional in vivo models with alternative VEEV subtypes and chikungunya virus. During therapeutic delivery of SKT05, Fc effector functions were only required at 3 days post-infection. The necessity of Fc effector functions for SKT20 was related to mAb binding avidity rather than epitope and could be overcome by increasing the dose of SKT20 relative to the functional avidity of SKT05. Collectively, this study identified antibody avidity as a correlate for in vivo efficacy and associated Fc-dependent mechanisms that can be leveraged for therapeutic development of monoclonal antibodies against alphaviruses. One sentence summaryFunctional avidity of broadly reactive anti-alphavirus antibodies dictates requirement for Fc-mediated protection.

microbiology↗

bioRxiv2024

High-efficiency transformation and gene expression in Picosynechococcus sp. PCC 7002

Cyanobacteria are promising microbial platforms for a myriad of biotechnological applications, from sustainable biomaterials to photosynthetic chemical production, but still lack the breadth of genetic tools available for more commonly engineered microbes such as Escherichia coli. This study develops genetic tools to enhance the transformation efficiency and heterologous gene expression in Picosynechococcus sp. PCC 7002, a fast-growing, halotolerant, and naturally competent strain. Integration of fluorescent reporter cassettes across the genome revealed an integration site that yields a fourfold improvement in gene expression relative to previously reported sites. Protocol optimization and engineered DNA methylation in E. coli increased transformation efficiency by over tenfold. This work provides an experimental framework for efficient genome editing and metabolic engineering in the model cyanobacterium PCC 7002.

synthetic biology↗

bioRxiv2024

Activating FcγRs on monocytes are necessary for optimal Mayaro virus clearance

Mayaro virus (MAYV) is an emerging arbovirus. Previous studies have shown antibody Fc effector functions are critical for optimal monoclonal antibody-mediated protection against alphaviruses; however, the requirement of Fc gamma receptors (Fc{gamma}Rs) for protection during natural infection has not been evaluated. Here, we showed mice lacking activating Fc{gamma}Rs (FcR{gamma}-/-) developed prolonged clinical disease with more virus in joint-associated tissues. Viral clearance was associated with anti-MAYV cell surface binding rather than neutralizing antibodies. Lack of Fc-Fc{gamma}R engagement increased the number of monocytes through chronic timepoints. Single cell RNA sequencing showed elevated levels of pro-inflammatory monocytes in joint-associated tissue with increased MAYV RNA present in FcR{gamma}-/- monocytes and macrophages. Transfer of FcR{gamma}-/- monocytes into wild type animals was sufficient to increase virus in joint-associated tissue. Overall, this study suggests that engagement of antibody Fc with activating Fc{gamma}Rs promotes protective responses during MAYV infection and prevents monocytes from being potential targets of infection.

immunology↗

bioRxiv2024

High-density CRISPRi screens reveal adaptive transcriptional gradients in cyanobacteria

Cyanobacteria are the oldest form of photosynthetic life on Earth and contribute to primary production in nearly every habitat, from permafrost to hot springs. Despite longstanding interest in the biochemical basis of environmental adaptation in these microbes, it remains poorly understood and challenging to re-wire. This study uses a high-density, genome-wide CRISPR interference (CRISPRi) screen to examine the influence of gene-specific transcriptional variation on the growth of Synechococcus sp. PCC 7002 under environmental extrema. Surprisingly, many partial knockdowns enhanced fitness under cold monochromatic conditions. Notably, transcriptional repression of a gene for a core subunit of the NDH-1 complex, which is important for photosynthesis and carbon uptake, improved growth rates under both red and blue light but at distinct, color-specific optima. In general, most genes with fitness-improving knockdowns were distinct to each light color, evidencing unique stress responses and alleviation mechanisms. Multi-target transcriptional repression produced nonadditive effects. Findings reveal diverse mechanisms of environmental adaptation in cyanobacteria and provide a new approach for using gradients in sgRNA activity to pinpoint biochemically influential transcriptional changes in cells. SIGNIFICANCE STATEMENTCyanobacteria are the most abundant photosynthetic organisms on Earth, where they endure a striking variety of environmental fluctuations. This study examines the biochemical basis of environmental adaptation in Synechococcus sp. PCC 7002, an important model strain, by modulating the expression of every gene in its genome. Results show that partial, but not complete, reduction in the expression of a subset of influential genes can improve growth under cold monochromatic conditions. Optimal expression levels differ between red and blue light and shift with multi-gene adjustments. Findings show how minor transcriptional adjustments can yield major improvements in growth under environmental extrema and provide a powerful systems-level approach for studying--and fine-tuning--the adaptive capacity of microbes.

microbiology↗

bioRxiv2023

A biophysical rationale for the selective inhibition of PTP1B over TCPTP by nonpolar terpenoids

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including type 2 diabetes, obesity, and cancer. However, a high degree of structural similarity between the catalytic domains of these enzymes has made the development of selective pharmacological inhibitors an enormous challenge. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over TCPTP, two PTPs with high sequence conservation. Here, we use molecular modeling with experimental validation to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations indicate that PTP1B and TCPTP contain a conserved h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically influential WPD loop, which it links to the L-11 loop and 3 and 7 helices--the C-terminal side of the catalytic domain. Terpenoid binding to either of two proximal allosteric sites--an site and a {beta} site--can disrupt the allosteric network. Interestingly, binding to the site forms a stable complex with only PTP1B; in TCPTP, where two charged residues disfavor binding at the site, the terpenoids bind to the {beta} site, which is conserved between the two proteins. Our findings indicate that minor amino acid differences at the poorly conserved site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate, more broadly, how minor differences in the conservation of neighboring--yet functionally similar--allosteric sites can have very different implications for inhibitor selectivity.

bioengineering↗

bioRxiv2022

Identification of CD317-Positive Pro-inflammatory Immune Stromal Cells in Human Mesenchymal Stromal Cell Preparations

Mesenchymal stromal cell (MSC) heterogeneity clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterised by CD317 expression (CD317pos (29.77{+/-}3.00% of the total MSC population), comprising CD317dim (28.10{+/-}4.60%) and CD317bright (1.67{+/-}0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarisation towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

cell biology↗

bioRxiv2020

A single intranasal dose of chimpanzee adenovirus-vectored vaccine confers sterilizing immunity against SARS-CoV-2 infection

The Coronavirus Disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of systemic and mucosal IgA and T cell responses, completely prevents SARS-CoV-2 infection in the upper and lower respiratory tracts, and likely confers sterilizing immunity in most animals. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission, and curtailing pandemic spread.

microbiology↗

bioRxiv2020

Potently neutralizing human antibodies that block SARS-CoV-2 receptor binding and protect animals

The COVID-19 pandemic is a major threat to global health for which there are only limited medical countermeasures, and we lack a thorough understanding of mechanisms of humoral immunity1,2. From a panel of monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein isolated from the B cells of infected subjects, we identified several mAbs that exhibited potent neutralizing activity with IC50 values as low as 0.9 or 15 ng/mL in pseudovirus or wild-type (wt) SARS-CoV-2 neutralization tests, respectively. The most potent mAbs fully block the receptor-binding domain of S (SRBD) from interacting with human ACE2. Competition-binding, structural, and functional studies allowed clustering of the mAbs into defined classes recognizing distinct epitopes within major antigenic sites on the SRBD. Electron microscopy studies revealed that these mAbs recognize distinct conformational states of trimeric S protein. Potent neutralizing mAbs recognizing unique sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two murine models of SARS-CoV-2 infection, passive transfer of either COV2-2916 or COV2-2130 alone or a combination of both mAbs protected mice from severe weight loss and reduced viral burden and inflammation in the lung. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic cocktails.

immunology↗

bioRxiv2019

Minimally disruptive optical control of protein tyrosine phosphatase 1B

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)--an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer--and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

biochemistry↗