Biology subjects

Cox, D.

Publications and source records attributed to Cox, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Streptococcus pyogenes transforms the salivary proteome during controlled human infection

Streptococcus pyogenes (Group A Streptococcus, GAS) is among the leading causes of infectious mortality and primarily manifests as acute pharyngitis. The development of an effective vaccine continues to be hindered by an incomplete understanding of the host immune response, particularly in the oropharynx. Here, we provided detailed insights into the dynamic salivary proteome during acute illness in a human clinical trial of controlled infection of 20 healthy adults (CHIVAS-M75 trial). Deep profiling of salivary proteins via mass spectrometry revealed a striking increase in pro-inflammatory plasma proteins such as alpha1-antitrypsin and elements of the complement system during peak infection as a clear indication of the acute phase response in the oral environment. Many of these proteins, such as fibrinogen, are well-defined targets of key streptococcal virulence factors, with others, such as vitronectin, suggested to play a role in the host-pathogen relationship. Several salivary proteins with known antimicrobial properties, such as mucin-7 and cystatin, decreased universally across the infected cohort. For the first time, we have described the local and immediate protein response to acute pharyngitis in the oral cavity, highlighting a general host inflammatory signature and an abundance of GAS virulence targets infiltrating to the site of infection.

molecular biology↗

bioRxiv2026

Neoadjuvant chemotherapy induces transient, regimen-specific immune stromal reprogramming in pancreatic ductal adenocarcinoma

Background: Neoadjuvant chemotherapy (NAC) is increasingly incorporated into the management of pancreatic ductal adenocarcinoma (PDAC), yet how treatment regimen and timing reshape the tumour-immune microenvironment remains poorly defined. Methods: Targeted immune transcriptomic profiling was performed using NanoString on spatially annotated tumour, stromal, and immune-enriched regions from resected PDAC specimens from 23 patients, including NAC-treated and treatment-naive cohorts. Key findings were validated and spatially localised using Xenium in situ transcriptomics. Results: NAC induced extensive transcriptional remodelling of the tumour microenvironment in a regimen- and time-dependent manner. Tumours resected shortly after FOLFIRINOX exhibited coordinated upregulation and strong spatial coupling of the CXCL12-CXCR4 axis. These correlations were attenuated or reversed at longer post-treatment intervals and following gemcitabine-based therapy. Disrupted stromal coordination of the CCL2-CCR2 axis further highlighted context-dependent effects of chemotherapy on chemokine signalling. Across regions, NT5E (CD73) expression inversely correlated with CD8A, consistent with spatially restricted T cell exclusion. Conclusions: NAC dynamically reprograms the immunestromal landscape of PDAC in a regimen- and timing-dependent manner, revealing a transient post-chemotherapy window of immune remodelling. The clinical significance of this phenomenon requires further investigation to determine whether temporally optimised integration of immunomodulatory and stroma-targeted therapies with NAC can improve patient outcomes.

cancer biology↗

bioRxiv2026

A genetically encoded RhoG FRET biosensor reveals spatially compartmentalized RhoG-Rac1 signaling during cell protrusion

RhoG is a member of the Rho-family of small GTPases, and is closely related to the canonical Rac1 GTPase, implicated in membrane trafficking, dorsal ruffling, macropinocytosis, and cell protrusion, but its activity has been difficult to visualize directly in living cells with high spatial and temporal resolution. Here, we developed and validated a genetically encoded, single-chain Forster resonance energy transfer (FRET) biosensor for RhoG based on a C-terminal full-length RhoG and an intramolecular RhoG-binding domain derived from ELMO1. The biosensor showed a robust dynamic range when comparing constitutively active and inactive RhoG mutants, responded appropriately to regulation by RhoGDI, GAPs, and GEFs, and detected growth factor-stimulated RhoG activation in live cells. Imaging in mouse embryonic fibroblasts revealed dynamic RhoG activation at leading-edge protrusions, dorsal ruffles, and forming pinocytic and macropinocytic structures. To define the signaling relationship between RhoG and its closely related family member Rac1, we combined the RhoG biosensor with a near-infrared Rac1 FRET biosensor for simultaneous live-cell imaging. Morphodynamic mapping showed that both RhoG and Rac1 activities were positively coupled to edge protrusion, with strongest correlations near the leading-edge, but their direct coupling varied with distance from the edge, indicating partial spatial decoupling within protrusive regions. Inhibition of Src-family kinases altered RhoG dynamics, strongly suppressed Rac1 coupling to protrusion, and inverted the normal positive correlation between RhoG and Rac1 activities. Signaling microdomain analysis further showed that Src inhibition selectively prolonged Rac1 microdomain lifetimes without significantly affecting RhoG domains. Together, these results establish a new biosensor for direct visualization of RhoG activity and reveal that RhoG and Rac1 are coordinated but spatially and temporally distinct components of protrusion-associated signaling networks, with Src-family kinases playing a central role in maintaining their normal coupling.

cell biology↗

bioRxiv2026

Lipid-Coated Water-in-Oil Droplets as a Passivation-Free Platform for Cost-Effective Fluorescence Spectroscopy

Fluorescence correlation spectroscopy (FCS) provides valuable information about molecular dynamics, however, experimental setup typically requires labour-intensive passivation to prevent non-specific binding of molecules to sample containers. Furthermore, precious samples can be wasted by having to use relatively high sample volumes in existing sample containers. We overcome these major issues using a simple method of sample encapsulation into water-in-oil droplets, using purified proteins and cell lysates as proof-of-concept. FCS of fluorescently labelled protein samples in the nanomolar (nM) range confirmed that water-in-oil droplets yield more accurate measurements than conventional open-chamber methods. We first optimized the droplet composition to prevent protein coating at the water-oil interface using pegylated-lipids. We then utilized FCS to accurately measure protein concentrations and diffusion speeds in nanolitre volumes. Additionally, we used fluorescence cross-correlation spectroscopy (FCCS) to measure enzymatic cleavage of substrate inside our droplet system, demonstrating the capacity of this platform to measure biological processes at the nanoscale. Overall, conducting FCS in droplets offers a cost-effective, robust, and accessible alternative for measuring molecular dynamics, with promising potential for high-throughput and resource-limited applications. TOC Image + Text O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/734730v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@199c622org.highwire.dtl.DTLVardef@9f195eorg.highwire.dtl.DTLVardef@13a8969org.highwire.dtl.DTLVardef@138692_HPS_FORMAT_FIGEXP M_FIG C_FIG Conventional single-molecule fluorescence requires slow, expensive glass passivation procedures to prevent proteins adsorbing to surfaces. By encapsulating proteins in lipid-coated nanolitre water droplets, the passivation requirement is removed, enabling accurate measurement of protein dynamics in low nanolitre volumes. Water-in-oil droplets thus provide a passivation-free platform for fluorescence correlation spectroscopy.

biochemistry↗

bioRxiv2026

Tie2 signaling in the tumor microenvironment orchestrates breast cancer cell dissemination through TMEM doorways

During breast cancer metastasis, tumor cells migrate toward intratumoral blood vessels and intravasate through stable structures known as TMEM (Tumor Microenvironment of Metastasis) doorways. TMEM doorways, composed of a Mena-expressing tumor cell, a Tie2hi/VEGFhi macrophage, and an endothelial cell, are clinically validated prognostic markers of distant metastasis in breast cancer and represent the exclusive sites of tumor cell intravasation. We previously demonstrated that Tie2 signaling is essential for TMEM doorway function and tumor cell intravasation. In this study, we investigated how Tie2 signaling promotes tumor cell intravasation and metastasis. Because all three TMEM doorway-associated cell types can express Tie2, we sought to determine which of these cells contribute to the Tie2 signaling-dependent vascular opening at TMEM doorways and tumor cell dissemination. We found that endothelial cells associated with TMEM doorways secrete Ang2, which stimulates VEGF-A expression in Tie2hi macrophages. Elevated VEGF-A levels at TMEM doorways increase vascular permeability, facilitating tumor cell entry into the bloodstream. Using tissue staining and line-scan analysis of Tie2 and lineage markers in human and mouse breast cancer models, we observed Tie2 expression in macrophages, tumor cells, and endothelial cells. To assess functional contributions, we selectively disrupted Tie2 in macrophages, endothelial cells, and cancer cells using CRISPR-Cas9 and RNAi approaches and tested in which of these cell-knockouts of Tie2 expression affected transendothelial migration in vitro. Macrophage-specific Tie2 deletion had the greatest impact on tumor cell intravasation. To confirm this finding in vivo, we generated a mouse model with inducible, macrophage-specific Tie2 knockout. Acute, targeted loss of Tie2 specifically in macrophages significantly reduced TMEM doorway associated vascular opening and tumor cell intravasation. Together, these findings establish macrophage Tie2 signaling as a critical driver of TMEM doorway-mediated vascular permeability and metastatic dissemination in breast cancer.

cancer biology↗

bioRxiv2025

TMEM Doorway Mediated Metastasis in Pancreatic Ductal Adenocarcinoma by Tie2 Signaling

Pancreatic ductal adenocarcinoma (PDAC) is almost invariably fatal due to early hematogenous dissemination that occurs before the primary tumor is clinically detectable, yet the cellular mechanism of tumor cell intravasation has remained unknown. Using multiphoton intravital imaging in autochthonous and orthotopic PDAC models, we demonstrate that intravasation occurs at Tumor Microenvironment of Metastasis (TMEM) doorways--tri-cellular structures comprising a MENA-expressing tumor cell, a Tie2 macrophage, and an endothelial cell in direct contact. These structures are abundant in human PDAC, enriched for Tie2 macrophages, and markedly reduced after neoadjuvant chemotherapy. Selective pharmacologic inhibition of Tie2 with rebastinib decreases TMEM-associated transient vascular openings, suppresses circulating and hepatic disseminated tumor cells, and--when combined with perioperative FOLFIRINOX after curative-intent resection--improves median survival in murine PDAC. These findings establish TMEM doorways as a common, druggable mechanism of intravasation across epithelial cancers and identify Tie2 macrophages as a therapeutic target to prevent metastatic seeding in PDAC, a disease with no anti-metastatic therapies. TMEM doorway-mediated intravasation in PDAC supports its role as a common gateway for hematogenous metastasis in carcinoma.

cancer biology↗

bioRxiv2025

An AGO2 adaptor expands the functional and evolutionary reach of microRNA targeting

Current models of microRNA (miRNA) silencing posit that RNA-sequence rules are sufficient for canonical targeting of mRNAs by Argonaute 2 (AGO2), the central protein of the miRNA-induced silencing complex (miRISC). Using chimeric eCLIP in CRISPR-edited LIMD1+/+, LIMD1+/-, and LIMD1-/- human small airway epithelial cells (hSAECs), we reveal a transcriptome-wide dependency on LIMD1, an AGO2 adaptor, for effective miRNA targeting and repression. In LIMD1-deficient cells, miRNA loading is uncoupled from productive targeting: despite increased AGO2-miRNA interactions, complexes engage fewer transcripts and sites, reducing occupancy and more than halving both the breadth and depth of targeting. We also observe altered AGO2 positional footprints across targets in LIMD1-deficient cells. LIMD1 dependence is most pronounced at defined RNA contexts: weak (GC-poor) seed pairings, interactions involving evolutionarily young miRNAs or sites that nonetheless form thermodynamically stable duplexes, with these losses particularly enriched in coding sequences of rapidly evolving C2H2-zinc-finger genes. Even within canonical seed repertoires of individual AGO2-miRNAs, LIMD1 is most critical at poorly conserved sites, indicating that LIMD1 broadens miRNA regulation beyond ancient, deeply conserved targets. In culture, LIMD1 deficiency de-represses oncogenic proteins that, in vivo, inversely correlate with LIMD1 levels in normal lung and adenocarcinoma, where LIMD1 is characteristically reduced, and whose dysregulation predicts poor survival. Thus, LIMD1 emerges as a key determinant of miRISC architecture, targeting, and potency, challenging RNA-centric models of miRNA function and exemplifying how adaptor proteins diversify post-transcriptional regulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC="FIGDIR/small/680889v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@e0f4f5org.highwire.dtl.DTLVardef@f23fd9org.highwire.dtl.DTLVardef@3de3f2org.highwire.dtl.DTLVardef@1404af2_HPS_FORMAT_FIGEXP M_FIG LIMD1 defines the scope of miRNA-mediated targeting and repression O_LIAGO2-chimeric eCLIP in CRISPR-edited human small airway epithelial cells (hSAECs) shows LIMD1 is required for productive AGO2-miRNA engagement transcriptome-wide. C_LIO_LILIMD1 deficiency reduces the AGO2-miRNA:targetome. Each AGO2-miRNA binds fewer targets, with lower occupancy per site and per transcript and fewer global silencing events. C_LIO_LILIMD1 dependence is strongest for GC-poor seed-sites, less conserved miRNAs and sites, and thermodynamically stronger duplexes. C_LIO_LIDosage-dependent effects of LIMD1 deficiency are broadly observed. C_LIO_LITarget-mRNA decay and translational repression are reduced in LIMD1-deficient hSAECs, increasing protein output. C_LIO_LIIn vivo, LIMD1 LOH-associated deficiency is prevalent and typically clonal in NSCLC. LIMD1 expression inversely correlates with oncogene levels in normal lung and adenocarcinoma, and target dysregulation predicts poor survival. C_LI C_FIG

molecular biology↗

bioRxiv2025

Direct single-molecule visualization of Hsp90-mediated relief of a Hsp70-folding block

Hsp70 and Hsp90 are ubiquitous molecular chaperones that cooperate to promote the correct folding and maturation of client proteins. Despite their central role in proteostasis, the molecular mechanisms by which Hsp90 coordinates with Hsp70 to remodel clients remain poorly understood. In particular, how ATP hydrolysis by Hsp90 is coupled to client engagement and conformational change has been a long-standing question. This gap in understanding is largely due to the challenge of visualizing client conformations during the highly dynamic and heterogeneous interactions with the Hsp70/Hsp90 chaperone machinery. To address this, we used a combination of single-molecule fluorescence resonance energy transfer (smFRET) and total internal reflection fluorescence microscopy to observe individual firefly luciferase client proteins as they are sequentially engaged by Hsp70 and Hsp90. Here, we show that Hsp90 reduces rebinding of Hsp70 to folding intermediates while still allowing engagement to misfolded clients, thereby enabling productive refolding in the presence of typically inhibitory concentrations of Hsp70. Furthermore, Hsp90 uses ATP binding and hydrolysis to actively remodel the conformational landscape of the client, promoting controlled folding through localized compaction across multiple regions. These controlled folding events reduce misfolding and are essential for establishing native interdomain contacts. Using smFRET and kinetic simulations, we further demonstrate that heterogeneous Hsp70 binding generates region-specific folding kinetics and conformational dynamics, which are likely driven by variations in the number of available Hsp70-binding sites. This is then exploited during Hsp70/Hsp90-mediated folding to support localized folding of client subdomains, thereby reducing non-native interactions from distal regions to facilitate proper folding of multi-domain proteins.

biophysics↗

bioRxiv2025

Differential inclusion formation of an aggregation-prone protein reveals differences in the proteostasis capacity of neuronal cell lines

Maintaining proteome integrity is essential for cellular function and survival. Disruptions in proteostasis lead to the aggregation of proteins into inclusions, a process that underlies many neurodegenerative diseases. To quantitatively assess the proteostasis capacity of neuronal cells, we employed an aggregation-prone double mutant form of firefly luciferase (denoted FlucDM) as a reporter protein. We compared two commonly used neuronal cell lines, mouse neuroblastoma cells (Neuro-2a) and a motor neuron-like hybrid line (NSC-34), to evaluate their ability to prevent the aggregation of proteins into intracellular inclusions. We observed a significantly greater propensity of FlucDM to form inclusions in NSC-34 cells compared to Neuro-2a cells. This suggests a reduced capacity of NSC-34 cells for managing aggregation-prone proteins. Proteomic profiling of FlucDM inclusions purified from both cell types revealed cell-type-specific engagement of the proteostasis machinery with aggregation-prone proteins. Comparing the proteomic profiles of key arms of the proteostasis network between these two cell lines revealed that the endoplasmic reticulum (ER) unfolded protein response is differentially expressed. This study establishes a quantitative platform for assessing cellular proteostasis capacity and underscores the importance of cell-type context in proteome maintenance. These insights have implications for understanding the selective vulnerability of neurons in protein misfolding disorders.

cell biology↗

bioRxiv2025

Retrospective metabolomics via dual-dimensional deconvolution using ZT Scan DIA 2.0

We present a scanning data-independent acquisition (DIA) strategy, ZT Scan DIA, combined with dual-dimensional tandem mass spectrometry spectral filtering and deconvolution along both the quadrupole and retention time axes to reconstruct compound-specific MS2 spectra from complex mixtures. This approach is particularly effective for hydrophilic metabolomics data, where spectral similarity-based annotation is widely used, increasing annotation rates by 119-193% compared with conventional data-dependent acquisition (DDA) and window-based DIA methods. In lipidomics, deconvolution improved annotation precision by removing contaminant product ions and enabled separate quantification of co-eluting isomers using MS2 chromatograms, although common diagnostic ions could also be erroneously removed. Nevertheless, optimization of analysis parameters minimized this negative effect. Furthermore, we developed a practical data processing pipeline in which raw ZT Scan DIA-MS2 chromatograms are directly used for isomer separation and MS2-based quantification, covering 1,393 and 3,020 molecules for human plasma and mouse liver tissues, respectively. All data processing steps, including direct import of vendor raw data, are supported in MS-DIAL.

bioengineering↗

bioRxiv2025

Human macrophages secrete both interferon α and interferon β protein during infection with Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) infection activates type I interferons (IFNs) which are crucial mediators of tuberculosis (TB) pathogenesis. Despite assumptions that IFN and IFN{beta} are secreted by macrophages, direct protein quantification in primary human monocyte-derived macrophages is surprisingly lacking. Here, we demonstrate measurable IFN and IFN{beta} secretion by MDMs infected with both virulent (H37Rv) and attenuated (H37Ra) Mtb strains as early as 48 h post-infection, with levels persisting at 120 h. These findings challenge existing assumptions about type I IFN kinetics and highlight the importance of timing in experimental designs and provides a foundation for exploring their role in host-pathogen interactions.

immunology↗

bioRxiv2025

Memorization of novel patterns in working memory in a model based on dendritic bistability

Working memory can hold many types of information and is crucial for cognition. Commonly, models of working memory maintain information such as hues or words by forming memory attractors through structured connectivity. However, real-world information can be novel, making it infeasible to use predefined attractors. In addition, most models--with or without attractors--have focused on maintaining binary categories instead of continuous information in each neuron. In the present study, we investigate how the brain might maintain working memory representations of arbitrary novel patterns with graded values. We propose an unstructured, rate-based network model in which each neuron has multiple dendrites. Each dendrite shows bistable activity, which qualitatively captures the conductance-based dynamics in the corresponding spiking model and emulates fast Hebbian plasticity. This network can flexibly maintain novel graded patterns under various perturbations without fine-tuning of parameters. Through analytical characterization of network dynamics during the encoding and memory periods, we identify different conditions that yield either perfect memories or several types of memory errors. Our analysis reveals a functional separation for network neurons into two groups with distinct behaviors. Overall, this architecture provides robust and analytically tractable storage of novel graded patterns in working memory.

neuroscience↗

bioRxiv2025

Robust maintenance of both stimulus location and amplitude in a working memory model based on dendritic bistability

Working memory is a core feature of cognition that enables items to be maintained and manipulated over short durations of time. Stored information can be binary, such as the presence or absence of an object, or graded, such as the graded intensity or location of a feature. Current computational models of working memory cannot robustly maintain both the graded intensity and spatial location of a stored item. Here, we show how this limitation can be overcome if neurons contain multiple bistable dendritic compartments. First, we illustrate the core mechanism for the storage of graded amplitude information in a simple spiking "autapse" circuit consisting of a single neuron connected to itself. Second, we reduce this model to a rate-based model that permits analytic understanding. Third, we implement this mechanism within a spatially extended architecture in which the spatial location of an item is encoded by the set of active neurons. In contrast to classic spatial working memory models, which only encode the binary presence of an item at a given location, the multi-dendrite-neuron model robustly encodes both the amplitude and location of an item in working memory in a noise-resistant manner and without requiring fine tuning of parameters. We show analytically that the key mechanism permitting the storage of amplitude information is equivalent to that of the simpler autapse circuit. This work provides a solution to the problem of encoding graded information in spatial working memory and demonstrates how dendritic computation can increase the representational capacity and robustness of working memory. Significance StatementAnimals can readily remember both the location of an item and analog features of the item such as its amplitude or intensity. Remarkably, current computational models of working memory require extreme fine tuning of model parameters to perform this task. Here, we show how this limitation can be overcome if neurons have active dendritic processes that enable local dendritic compartments of the neuron to robustly maintain digital "up" ("plateau potential") or "down" states. By activating a variable number of plateau potentials at each location, the models can jointly maintain both the location and amplitude of a stimulus in working memory in a noise-resistant manner. This work demonstrates how local dendritic processes can enhance the computational capabilities of working memory networks.

neuroscience↗

bioRxiv2025

Small tau aggregates exhibit disease-specific molecular profiles across tauopathies

Tauopathies are neurodegenerative diseases marked by pathological tau aggregation. While disease-specific folds of insoluble tau filaments have been established, it remains unclear whether the smaller, earlier species also differ across tauopathies. Here, we characterise these small tau aggregates from post-mortem brain of individuals with Alzheimers disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration, Picks disease, and healthy controls. Using two complementary single-molecule assays, we confirm that small tau aggregates vary in abundance, morphology, and post-translational modifications. AD features specific long, fibrillar-shaped aggregates enriched in phospho-epitopes, while PSP aggregates are shorter, round, and selectively phosphorylated at serine-356, a site we identify as correlating with markers of inflammation and apoptosis. Aggregate properties co-vary with cellular stress signatures and align with disease-specific seeding profiles, suggesting distinct pathological mechanisms. These findings suggest that small tau aggregates are not a shared intermediate, but instead encode disease-specific mechanisms, with potential as both biomarkers and therapeutic targets. Key pointsSmall tau aggregates show disease-specific signatures across tauopathies, differing in abundance, morphology, and post-translational modifications. Tau aggregates in AD show enhanced phosphorylation density and structural heterogeneity, including a distinct population of long fibrillar species detectable in the soluble fraction. Alzheimers disease is characterised by specific long, fibrillar-shaped tau aggregates enriched in disease-relevant phospho-epitopes. PSP features round pSer356-positive aggregates that correlate with apoptotic and inflammatory markers, suggesting a distinct mechanism of toxicity. Isoform-specific biosensor assays reveal divergent seeding behaviour: CBD shows strong 4R seeding, while PSP lacks seeding activity. Features of small aggregates co-vary with distinct patterns of gliosis and cell stress, suggesting disease-specific mechanisms of tau-mediated toxicity.

neuroscience↗

bioRxiv2025

Fingerprinting disease-derived protein aggregates reveals unique signature of Motor Neuron Disease

Inappropriate aggregation of TAR DNA-binding protein 43 (TDP-43) is a hallmark of motor neuron disease (MND). Current methods for quantifying heterogeneous aggregate populations in biofluids are limited, precluding their routine use in diagnosis and disease monitoring. Single molecule microscopy methods overcome these limitations to deliver quantitative morphological and compositional fingerprinting of molecular assemblies containing TDP-43. Here, we demonstrate the application of such methods to extracts from donor brain tissues. We show the number and morphology of aggregates derived from frontal cortex and cerebellar samples is sufficient to distinguish MND donors from neurologically normal controls, as well as between different MND cohorts. In addition, we compare proteomic and microscopic compositional profiles, demonstrating how complementary insights delivered by each technique can enhance our understanding of disease mechanisms. These single molecule assays will inform future diagnostic and stratification technologies, improving our ability to deliver patient care in a timely and targeted manner.

molecular biology↗

bioRxiv2024

A CONTRIBUTION TO THE ANATOMY OF TWO RARE CETACEAN SPECIES: THE HOURGLASS DOLPHIN (Lagenorhynchus cruciger) AND THE SPECTACLED PORPOISE (Phocoena dioptrica)

The anatomical description of the hourglass dolphin (Lagenorhynchus cruciger) and the spectacled porpoise (Phocoena dioptrica) remains largely unexplored, due to limited specimen availability and preservation challenges. This study employed digital imaging techniques, conventional histology and computed tomography to provide visualisation of anatomical structures for a detailed analysis. We present a comprehensive analysis of the gross macroscopical and microscopical morphology of two hourglass dolphins and four spectacled porpoises. The hourglass dolphins were characterised by their distinctive black and white pigmentation and a hooked dorsal fin, while the spectacled porpoises were defined by their large dorsal fin, lack of a visible rostrum and unique eye markings. Morphometric measurements and skeletal characteristics aligned with the literature, while internal anatomy (organs and systems) were similar to other odontocetes. Although precise lung measurements were challenging, qualitative assessments indicated relatively large lungs for their body size, supporting the "short dive, big lung" hypothesis and suggesting that these species are not deep divers. The spectacled porpoise dorsal fin was uniquely large with a well-developed blood supply; this is hypothesised to act as a thermoregulatory window, helping to manage body heat. Overall, this study provides new data on the anatomy of the hourglass dolphin and spectacled porpoise, contributing insights that may influence future research on these rare species. The findings highlight the importance of anatomical studies in explaining evolutionary relationships within cetaceans and their ecological roles in the Southern Ocean ecosystems.

evolutionary biology↗

bioRxiv2024

Single-molecule observations of human small heat shock proteins in complex with aggregation-prone client proteins

Small heat shock proteins (sHsps) are molecular chaperones that act to prevent the aberrant aggregation of misfolded proteins. Whilst it is widely suggested that sHsps prevent aggregation by binding to misfolded client proteins, the dynamic and heterogeneous nature of sHsps has hindered attempts to establish the mechanistic details of how sHsp-client protein complexes form. Single-molecule approaches have emerged as a powerful tool to investigate dynamic and heterogeneous interactions such as those that can occur between sHsps and their client proteins. Here, we use total internal reflection fluorescence microscopy to observe and characterise the complexes formed between model aggregation-prone client proteins [firefly luciferase (FLUC), rhodanese, and chloride intracellular channel 1 protein (CLIC)], and the human sHsps B-crystallin (B-c; HSPB1) and Hsp27 (HSPB5). We show that small (monomeric or dimeric) forms of both B-c and Hsp27 bind to misfolded or oligomeric forms of the client proteins at early stages of aggregation, resulting in the formation of soluble sHsp-client complexes. Stoichiometric analysis of these complexes revealed that additional B-c subunits accumulate onto pre-existing sHsp-client complexes to form larger species - this does not occur to the same extent for Hsp27. Instead, Hsp27-client interactions tend to be more transient than those of B-c. Elucidating these mechanisms of sHsp function is crucial to our understanding of how these molecular chaperones act to inhibit protein aggregation and maintain cellular proteostasis.

molecular biology↗

bioRxiv2023

FAK family proteins regulate in vivo breast cancer metastasis via distinct mechanisms.

Breast cancer is the most commonly diagnosed malignancy and the major leading cause of tumor-related deaths in women. It is estimated that the majority of breast tumor-related deaths are a consequence of metastasis, to which no cure exists at present. The FAK family proteins Proline-rich tyrosine kinase (PYK2) and focal adhesion kinase (FAK) are highly expressed in breast cancer, but the exact cellular and signaling mechanisms by which they regulate in vivo tumor cell invasiveness and consequent metastatic dissemination are mostly unknown. Using a PYK2 and FAK knockdown xenograft model we show here, for the first time, that ablation of either PYK2 or FAK decreases primary tumor size and significantly reduces Tumor MicroEnvironment of Metastasis (TMEM) doorway activation, leading to decreased intravasation and reduced spontaneous lung metastasis. Intravital imaging analysis further demonstrates that PYK2, but not FAK, regulates a motility phenotype switch between focal adhesion-mediated fast motility and invadopodia-dependent, ECM-degradation associated slow motility within the primary tumor. Furthermore, we validate our in vivo and intravital imaging results with integrated transcriptomic and proteomic data analysis from xenograft knockdown tumors and reveal new and distinct pathways by which these two homologous kinases regulate breast tumor cell invasiveness and consequent metastatic dissemination. Our findings identify PYK2 and FAK as novel mediators of mammary tumor progression and metastasis and as candidate therapeutic targets for breast cancer metastasis.

cancer biology↗