Biology subjects

Griffin, M.

Publications and source records attributed to Griffin, M..

15 recordsLinked to original sources

bioRxiv2025

Mechanism of NanR transcriptional activation of sialic acid metabolism in Streptococcus pneumoniae.

In Streptococcus pneumoniae, the RpiR transcriptional regulator NanR (SpNanR) senses sialic acid in the environment and upregulates transcription of the nan and siaA operons to increase uptake and metabolism of sialic acid. The molecular basis of this activation is unknown. Here, we demonstrate that SpNanR binds N-acetylmannosamine-6-phosphate, a metabolite of sialic acid catabolism. SpNanR exists in a dimer-tetramer equilibrium, and N-acetylmannosamine-6-phosphate binding strongly stabilizes the tetramer. Crystal structures and site-specific substitutions demonstrate that N-acetylmannosamine-6-phosphate bridges and stabilizes the SpNanR tetramer. SpNanR binds its DNA recognition sequence with nanomolar affinity. Notably, the effector N-acetylmannosamine-6-phosphate does not affect the affinity of SpNanR for DNA. The DNA binding domains are not structurally coupled to the sugar isomerase domains, explaining why N-acetylmannosamine-6-phosphate binding does not affect DNA binding. Structural analysis reveals that sequence specificity arises through distortion of B-DNA and an unusual {pi}-stack formed by two arginine residues in the minor groove, while affinity is driven by backbone contacts. We propose a mechanism by which S. pneumoniae regulates sialic acid metabolism, consistent with our biophysical experiments and in vivo regulatory behavior. These findings define a unique activation mechanism for an RpiR regulator and provide new insights into carbohydrate-responsive gene regulation in pneumococci.

molecular biology↗

bioRxiv2025

Inhibition of p38 MAPK after repetitive mild TBI ameliorates immune signaling and behavioral deficits

BackgroundMild traumatic brain injury (mTBI) can cause long-term functional impairments, and repetitive mTBIs within a window of vulnerability can exacerbate these consequences compared to a single mTBI. However, current interventions for mTBI focus on alleviating symptoms, rather than targeting underlying mechanisms. Following the initial mechanical impact, increasing evidence suggests that the brain undergoes an inflammatory cascade consisting of pro-inflammatory intracellular signaling pathways and production of cytokines, ultimately leading to chronic neuroinflammation and persistent neurological deficits. Prior work in severe traumatic brain injury has shown that the p38 MAPK signaling pathway is a key regulator of microglial activation, proinflammatory cytokines, and synaptic dysfunction, but its role in the context of mTBI remains unclear. As such, this study aimed to determine if inhibition of p38 MAPK would attenuate the inflammatory response and longer-term functional deficits following a weight-drop mouse model of repetitive mTBI. MethodsC57BL/6J male and female mice were injected with a small molecule p38 MAPK inhibitor (SB239063) after each of 5 once-daily weight-drop closed head injuries (CHIs) or sham injuries. Functional outcome was assessed at 4-weeks post injury. Protein and transcriptional alterations associated with the immune response, synaptic function, microglial phenotype, and functional outcomes were assessed at both 4-hours and 4-weeks after the final CHI. ResultsIn females, acute inhibition of p38 MAPK attenuated i) cytokine expression and microglial reactivity at 4-hours post injury and ii) antidepressive-like behavior and synaptic loss at 4-weeks post injury. In males, p38 MAPK inhibition also attenuated microglial reactivity and up-regulation of specific cytokines, although changes in functional outcomes did not reach significance. Interestingly, bulk RNAseq analysis in both sexes showed that acute p38 MAPK inhibition both normalized the effects of injury and upregulated protective genes and pathways associated with recovery and maintenance of brain homeostasis. Together, these findings suggest a role for p38 MAPK in driving the acute and longer-term consequences post repetitive mTBI in a sex-dependent manner, and they suggest therapeutic potential of p38 MAPK inhibition. To our knowledge, this work is the first to investigate the effects of small molecule inhibitor SB239063 as a potential therapeutic treatment administrated following rmTBI.

molecular biology↗

bioRxiv2025

Quantification of collagen and associated features from H&E-stained whole slide pathology images across cancer types using a physics-based deep learning model

BackgroundCollagen is the major component of the extracellular matrix (ECM). Collagen structural organization undergoes significant transformation during tumorigenesis. The visualization of collagen in histological tissue sections would aid in the study of tumor growth, encapsulation, and invasion. However, such visualization requires the use of special stains such as Picrosirius Red (PSR) or Massons Trichrome (MT), or more recently, second-harmonic generation imaging (SHG) in unstained tissue sections. However, PSR and MT both suffer from significant inter- (and intra-) lab stain variabilities, and SHG, while considered a ground truth by many, suffers from issues of system complexity/reliability, cost, and speed/throughput. These technical hurdles limit more widespread assessment of collagen in tissue samples. MethodsUsing high-contrast, high-throughput polarization imaging on PSR-stained slides to generate ground truth training polarization images, we developed a deep learning model (iQMAI) to infer the presence of collagen directly from hematoxylin and eosin (H&E)-stained whole-slide images (WSIs) with high specificity. After iQMAI inference across WSIs, individual collagen fibers were extracted, and features describing overall collagen intensity and fiber morphology were computed. iQMAI pixel-and feature-wise outputs were compared to ground truth polarization imaging to assess model performance. The trained iQMAI model was deployed on H&E-stained WSI from the TCGA LUAD, LUSC, LIHC, and PAAD datasets for evaluation. iQMAI-derived collagen features were compared to tissue composition, gene expression, and overall survival. ResultsThe iQMAI model shows significant generalization across multiple indications. iQMAI collagen predictions were similar to polarization imaging measurements of the same sample, with a mean structural similarity index (SSIM) of 0.84 (95% CI 0.69-0.93), a mean patch-wise RMSE of 0.04 (95% CI 0.02-0.08), and a linear correlation (R2=0.93). Comparing features of the collagen fibers extracted from iQMAI vs. polarization images yielded similar linear correlations between computed fiber tortuosity, length, width, and relative angle. The relationship between collagen fiber density and fibroblast density was distinct in non-small cell lung cancer (LUAD and LUSC), hepatocellular carcinoma (LIHC), and pancreatic ductal adenocarcinoma (PAAD). In PAAD, fiber density and fiber width were both negatively associated with the LRRC-15 gene expression signature, and increased fiber width was associated with longer overall survival. ConclusionsiQMAI is a deep learning model that accurately predicts collagen from an H&E-stained WSI, allowing for spatially resolved quantification of collagen morphology and enabling investigation of the interplay between collagen and other TME components. We demonstrate an example of the utility of iQMAI-based collagen assessment in PAAD, where collagen features are associated with immunosuppressive cancer-associated fibroblasts and overall survival. Understanding the relationship between collagen, the tumor microenvironment composition, and disease progression may aid the development of effective immunotherapies in PAAD and other cancer types.

pathology↗

bioRxiv2024

Reprogramming the genome of M13 bacteriophage for all-in-one personalized cancer vaccine

Peptide-based vaccines face limitations in immunogenicity and stability, and challenges in co-delivering antigens and adjuvants effectively. Virus-based nanoparticles, particularly M13 bacteriophage, present a promising solution due to their genetic modifiability, intrinsic adjuvanticity, and efficient antigen presentation capabilities. Here we developed a programmable M13 phage-based personalized cancer vaccine enabling single-step antigen-adjuvant assembly. Specifically, we designed a reprogrammed (RP) phage platform that precisely regulates Toll-like receptor 9 activation by programming its genome sequence and modulates antigen density through genetic engineering. Vaccination studies with RP phages demonstrated that the immune response could be modulated by fine-tuning the adjuvanticity and antigen density, revealing an optimal antigen dose and adjuvanticity for maximum vaccine efficacy. The RP phage induced a remarkable 24-fold increase in neoantigen-specific CD8+ T cells and eradicated established MC-38 tumors when combined with anti-PD-1 therapy. These findings highlight the RP phages potential as a powerful nanovaccine platform for personalized cancer vaccines.

bioengineering↗

bioRxiv2024

Zebrafish Pol-Theta and Human Pol-Theta, Orthologues with Homologous Function

DNA Polymerase Theta (Pol {theta}) is a conserved an A-family polymerase that plays an essential role in repairing double strand breaks, through micro-homology end joining, and bypassing DNA lesions, through translesion synthesis, to protect genome integrity. Despite its essential role in DNA repair, Pol {theta} is inherently error-prone. Recently, key loop regions were identified to play an important role in key functions of Pol {theta}. Here we present a comparative structure-function study of the polymerase domain of zebrafish and human Pol {theta}. We show that these two proteins share a large amount of sequence and structural homology. However, we identify differences in the amino acid composition within the key loop areas shown to drive characteristic Pol {theta} functions. Despite these differences zebrafish Pol {theta} still displays characteristics identify in human Pol {theta}, including DNA template extension in the presence of different divalent metals, microhomology-mediated end joining, and translesion synthesis. These results will support future studies looking to gain insight into Pol {theta} function on the basis of evolutionarily conserved features.

molecular biology↗

bioRxiv2023

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-EM structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 [A] resolution (extending to 2.2 [A] at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 [A] resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the elevator-with-an-operator mechanism of TRAP transporters.

biochemistry↗

bioRxiv2023

Piezo inhibition prevents and rescues scarring by targeting the adipocyte to fibroblast transition

While past studies have suggested that plasticity exists between dermal fibroblasts and adipocytes, it remains unknown whether fat actively contributes to fibrosis in scarring. We show that adipocytes convert to scar-forming fibroblasts in response to Piezo-mediated mechanosensing to drive wound fibrosis. We establish that mechanics alone are sufficient to drive adipocyte-to- fibroblast conversion. By leveraging clonal-lineage-tracing in combination with scRNA-seq, Visium, and CODEX, we define a "mechanically naive" fibroblast-subpopulation that represents a transcriptionally intermediate state between adipocytes and scar-fibroblasts. Finally, we show that Piezo1 or Piezo2-inhibition yields regenerative healing by preventing adipocytes activation to fibroblasts, in both mouse-wounds and a novel human-xenograft-wound model. Importantly, Piezo1-inhibition induced wound regeneration even in pre-existing established scars, a finding that suggests a role for adipocyte-to-fibroblast transition in wound remodeling, the least-understood phase of wound healing. Adipocyte-to-fibroblast transition may thus represent a therapeutic target for minimizing fibrosis via Piezo-inhibition in organs where fat contributes to fibrosis.

cell biology↗

bioRxiv2022

Heritable and sex-specific variation in the development of social behavior in a wild primate

Affiliative social bonds are linked to fitness components in many social mammals. However, despite their importance, little is known about how the tendency to form social bonds develops in young animals, or if the development of social behavior is heritable and thus can evolve. Using four decades of longitudinal observational data from a wild baboon population, we assessed the environmental determinants of an important social developmental milestone in baboons--the age at which a young animal first grooms a conspecific--and we assessed how mother-offspring grooming reciprocity develops during the juvenile period. We found that grooming development differs between the sexes: female infants groom at an earlier age and reach reciprocity in grooming with their mother earlier than males. Using the quantitative genetic animal model, we also found that age at first grooming behavior for both sexes is weakly heritable (h2 = 4.3%). These results show that sex differences in grooming emerge at a young age; that strong, reciprocal social relationships between mothers and daughters begin very early in life; and that age at first grooming is heritable and therefore can be shaped by natural selection.

animal behavior and cognition↗

bioRxiv2022

Allele-specific expression reveals genetic drivers of tissue regeneration in mice

In adult mammals, skin wounds typically heal by scarring rather than through regeneration. In contrast, "super-healer" MRL mice have the unusual ability to regenerate ear punch wounds, yet the molecular basis for this regeneration remains elusive. Here, in hybrid crosses between MRL and non-regenerating mice, we use allele-specific gene expression to identify cis-regulatory variation associated with ear regeneration. Analyzing three major wound cell populations, we identified extensive strain- and tissue- specific cis-regulatory divergence associated with differences in healing outcomes. Genes with cis-regulatory differences specifically in fibroblasts were associated with wound healing phenotypes and pathways, and were enriched near genetic markers associated with ear-healing in a genetic cross. Finally, we demonstrated that one of these genes, Cfh, could be applied ectopically to accelerate wound repair and induce regeneration in typically fibrotic wounds. Overall, our results provide insight into the molecular drivers of regeneration in MRL mice with potential clinical implications.

genomics↗

bioRxiv2022

Electric Field Responsive Nanotransducers for Glioblastoma

Electric field therapies such as Tumor Treating Fields (TTFields) have emerged as a bioelectronic treatment for isocitrate dehydrogenase wild-type and IDH mutant grade 4 astrocytoma Glioblastoma (GBM). TTFields rely on alternating current (AC) electric fields (EF) leading to the disruption of dipole alignment and induced dielectrophoresis during cytokinesis. Although TTFields have a favourable side effect profile, particularly compared to cytotoxic chemotherapy, survival benefits remain limited ([~] 4.9 months) after an extensive treatment regime (20 hours/day for 18 months). The cost of the technology also limits its clinical adoption worldwide. Therefore, the discovery of new technology that can enhance survival benefit and improve the cost per added quality of life year per patient, of these TTFields will be of great benefit to cancer treatment and decrease healthcare costs worldwide. In this work, we report the role of electrically conductive gold (GNPs), dielectric silica oxide (SiO2), and semiconductor zinc oxide (ZnO) nanoparticles (NPs) as transducers for enhancing EF mediated anticancer effects on patient derived GBM cells. Physicochemical properties of these NPs were analyzed using spectroscopic, electron microscopy, and light-scattering techniques. In vitro TTFields studies indicated an enhanced reduction in the metabolic activity of patient-derived Glioma INvasive marginal (GIN 28) and Glioma contrast enhanced core (GCE 28) GBM cells in groups treated with NPs vs. control groups, irrespective of NPs dielectric properties. Our results indicate the inorganic NPs used in this work enhance the intracellular EF effects by virtue of bipolar dielectrophoretic and electrophoretic effects. This work presents preliminary evidence which could help to improve future EF applications for bioelectronic medicine. Furthermore, the merits of spherical morphology, excellent colloidal stability, and low toxicity, make these NPs ideal for future studies for elucidating the detailed mechanism and efficacy upon their delivery in GBM preclinical models.

cancer biology↗

bioRxiv2021

Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes

During the progression of osteoarthritis (OA), degradation of the extracellular matrix alters the biomechanical properties of cartilage, especially the compressive modulus. The mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) is required for chondrocyte mechanotransduction However, how OA-mediated cartilage degradation influences TRPV4 signaling remains unknown. To determine if ATDC5 cells alter TRPV4-mediated calcium signaling and cell phenotype in response to softer substrates, we created PEGDA-RGDS hydrogels with Youngs moduli that simulated healthy (~350 kPa), OA (~175 kPa) and severe OA (~35 kPa) tissue. We found that softer substrates reduced the influx of calcium through TRPV4 when challenging chondrocytes with hypotonic swelling (HTS). Chondrocyte apoptosis also increased on the OA and severe OA gels due to elevated basal [Ca2+]i, which is attenuated with pharmacological agonism of TRPV4. Pharmacological agonism of TRPV4 rescued the expression of aggrecan and TRPV4 in chondrocytes cultured on OA gels and enhanced the type II collagen (col2) expression in cells on the normal and OA gels. These data suggest that the biomechanical properties of degraded cartilage alter TRPV4-mediated mechanotransduction in chondrocytes. Given that TRPV4 reduced apoptosis and improved the chondrogenic capacity of cells, TRPV4 stimulation could provide a potential therapeutic target in patients with early to moderate OA.

cell biology↗

bioRxiv2021

Spatial-transcriptomics reveals unique defining molecular features of fluorescence-sorted 5-aminolevulinic acid+ infiltrative tumor cells associated with glioblastoma recurrence and poor survival

Spatiotemporal-heterogeneity of glioblastoma (GBM) originating from the genomic and transcriptional variation in spatially distinct intra-tumor sites, may contribute to subtype switching in GBM prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) has enabled the isolation of infiltrative margin tumor cells (5ALA+ cells) from a background of non-neoplastic cells. We have explored the spatial-transcriptomic (ST) landscape to interrogate molecular signatures unique to infiltrating 5ALA+ cells in comparison to GBM core, rim, and invasive margin non-neoplastic cells. ST analysis reveals that GBM molecular subtype plasticity is not restricted to recurrence, but manifests regionally in a cell-type-specific manner. Whilst GBM core and rim are highly enriched with Classical and Proneural subtypes, the unique enrichment of the Mesenchymal subtype (MES) in 5ALA+ cells supports the hypothesis that MES 5ALA+ cells may drive GBM recurrence. Upregulation of the wound response pathway in 5ALA+ cells signifies the possibility of hijacking the wound healing pathway of neural cells to promote tumor growth. Exon-intron split analysis revealed an upregulation of exonic counts for MES and wound-response genes in 5ALA+ cells, implying that these genes are under active post-transcriptional control. Network analysis suggests that wound response genes, including chemokine CCL2 that recruits regulatory T-cells and monocytic myeloid-derived suppressor cells, are controlled by an IRF8-mediated transcriptional program in 5ALA+ cells. A higher stemness signature both in 5ALA+ cells and non-neoplastic cells of the invasive margin emphasizes the role of this microenvironment in stemness acquisition and defines 5ALA+ cells as a rare sub-population of GBM stem cells. Finally, we establish a link between the unique molecular signatures of 5ALA+ cells and poor survival and GBM recurrence. Characterization of the 5ALA+ infiltrative sub-population offers an opportunity to develop more effective GBM treatments and urges focus away from the GBM proliferative core, upon which failed targeted therapies have been predicated.

cancer biology↗

bioRxiv2020

Divergent molecular signatures of regeneration and fibrosis during wound repair

Regeneration is the "holy grail" of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward either scarring or regenerative fates. We profile scarring versus YAP inhibition-induced wound regeneration at the transcriptional (single-cell RNA-sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell surface barcoding, we integrate these data to reveal fibrotic and regenerative "molecular trajectories" of healing. We show that disrupting YAP mechanical signaling yields regenerative repair orchestrated by fibroblasts with activated Trps1 and Wnt signaling. Our findings serve as a multimodal map of wound regeneration and could have therapeutic implications for pathologic fibroses.

cell biology↗

bioRxiv2020

DNA methylation and survival differences associated with the type of IDH mutation in 1p/19q non-codeleted astrocytomas

Somatic mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 occur at high frequency in several tumour types. Even though these mutations are confined to distinct hotspots, we show that gliomas are the only tumour type with an exceptionally high percentage of IDH1R132H mutations. This high prevalence is important as IDH1R132H is presumed to be relatively poor at producing D-2-hydroxyglutarate (D-2HG) whereas high concentrations of this oncometabolite are required to inhibit TET2 DNA demethylating enzymes. Indeed, patients harbouring IDH1R132H mutated tumours have lower levels of genome-wide DNA-methylation, and an associated increased gene expression, compared to tumours with other IDH1/2 mutations ("non-R132H mutations"). This reduced methylation is seen in multiple tumour types and thus appears independent of site of origin. For 1p/19q non-codeleted glioma patients, we show that this difference is clinically relevant: in samples of the randomised phase III CATNON trial, patients harbouring non-R132H mutated tumours have better outcome (HR 0.41, 95% CI [0.24, 0.71], p=0.0013). Non-R132H mutated tumours also had a significantly lower proportion of tumours assigned to prognostically poor DNA-methylation classes (p<0.001). IDH mutation-type was independent in a multivariable model containing known clinical and molecular prognostic factors. To confirm these observations, we validated the prognostic effect of IDH mutation type on a large independent dataset. The observation that non-R132H mutated 1p/19q non-codeleted gliomas have a more favourable prognosis than their IDH1R132H mutated counterpart is clinically relevant and should be taken into account for patient prognostication. Single sentence summaryAstrocytoma patients with tumours harbouring IDH mutations other than p.R132H have increased DNA methylation levels and longer survival

cancer biology↗

bioRxiv2019

α1A-adrenoceptor inverse agonists and agonists modulate receptor signalling through a conformational selection mechanism

G-Protein Coupled Receptors (GPCRs) transmit signals across the cell membrane via an allosteric network from the ligand-binding site to the G-protein binding site via a series of conserved microswitches. Crystal structures of GPCRs provide snapshots of inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here we analyse the correlation between ligand binding and receptor conformation of the 1A-adrenoceptor, known for stimulating smooth muscle contraction in response to binding noradrenaline. NMR of 13C{varepsilon}H3-methionine labelled 1A-adrenoreceptor mutants, each exhibiting differing signalling capacities, revealed how different classes of ligands modulate receptor conformational equilibria. 13C{varepsilon}H3-methionine residues near the microswitches revealed distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling between the microswitches controls receptor conformation and underlies the mechanism of ligand modulation of GPCR signalling in cells.

biochemistry↗