Biology subjects

Chan, C. H.

Publications and source records attributed to Chan, C. H..

15 recordsLinked to original sources

bioRxiv2025

A Bundled Antiparallel Cytochrome Nanowire Structure Suggests Roles in Cell-Cell Electron Transfer and Biofilm Formation

Long-range extracellular electron transfer enables respiring microbes to use minerals, other organisms, or electrodes as electron acceptors by transporting electrons microns away from the cell surface. This process is primarily studied in Geobacter sulfurreducens, which produces at least three different micrometer-long, multi-heme cytochrome nanowires capable of electron transfer. However, the distribution and higher-order structure of these types of cytochrome nanowires remains largely unknown. Here, we employed cryo-electron microscopy to determine the atomic structure of a unique cytochrome nanowire from Desulfuromonas soudanensis WTL, a halophilic, iron- and electrode-reducing bacterium found in deep subsurface brine. These filaments are based on a homolog of the OmcE tetraheme cytochrome, which then assemble into highly ordered bundles of antiparallel filaments. This arrangement likely arises from the association of nanowires extending from adjacent cells. Furthermore, a similar cytochrome bundle structure was observed in Geobacter metallireducens, suggesting that this quaternary structure may be a common feature among nanowires secreted by electroactive microbes. Our findings demonstrate that cytochrome nanowires in diverse taxa can form specialized bundle interfaces, potentially facilitating conductive biofilm formation and representing a novel strategy for microbial electron exchange. More broadly, this work expands understanding of electron transfer mechanisms and demonstrates the production of multi-heme filaments across distinct lineages. These insights could guide future research into cytochrome nanowire secretion and conductive biofilm engineering, with potential applications in electrochemical technologies.

microbiology↗

bioRxiv2025

Primary tumor microbiomes predict distant metastasis of colorectal cancer

Metastasis causes most cancer-related deaths in colorectal carcinoma (CRC), and microbiome markers may have prognostic value. We hypothesized that primary tumor microbiomes predict distant metastases. We analyzed 5-year metastasis-free survival (MFS) in a retrospective cohort of 900 ORIEN CRC tumor microbiomes (RNAseq). ORIEN findings were validated on an independent cohort using 16S rDNA sequencing and pathobiont-specific qPCR. Microbiome alpha diversity was higher in primary tumors than metastases and positively correlated with metastasis risk. Microbiome beta diversity distinguished primary vs. metastasis and predicted 5-year MFS. High primary tumor abundance of B. fragilis and low F. nucleatum were associated with short MFS. Enterobacteriaceae, including E. coli, were enriched in metastases. qPCR identified increased enterotoxigenic B. fragilis and pks+ E. coli detection in CRC metastasizers. Microbial co-occurrence analysis identified a 3-species clique that predicts metastasis (OR 1.9 [1.4-2.6]). Results suggest that primary tumor microbiomes and specific pathobionts are precision markers for metastasis risk.

cancer biology↗

bioRxiv2025

Epigenetic Modulation, Intra-tumoral Microbiome and Immunity in Early Onset Colorectal Cancer

BackgroundThe incidence of colorectal cancer (CRC) in young adults (age of diagnosis < 50 years old) has been rapidly increasing. Although [~]20% of early-onset (EO) CRC cases are due to germline mutations, the etiology of the majority of EOCRC cases remains poorly understood. Non-genetic factors such as environmental exposure and lifestyle changes are likely to have a direct link to the increased incidence of sporadic EOCRC. We hypothesize that such factors may be observable as differences in the EOCRC epigenome, microbiome and immunome. We sought to address this by comparing differences in DNA methylation from the cohort of colorectal cancer patients in The Cancer Genome Atlas (TCGA). Further, we carefully identified intra-tumoral microbes from TCGA and two other datasets and then related the microbes to EOCRC status and deconvolved immune cell abundances. We found that DNA methylation (DNAm) age acceleration by12 years when compared with average-onset CRC (AOCRC) patients. Differentially methylated sites associated with genes are related to CREB signaling in neurons, G protein coupled receptor signaling, phagosome formation and S100 family signaling. These differences were validated in the gene expression from TCGA and a second, larger real-world dataset from the Oncology Research Information Exchange Network (ORIEN). However, no consistent differences were observed in the intra-tumor microbes between EOCRC and AOCRC. Interestingly, the most abundant microbes interacted with the immune systems differently between the EOCRC and AOCRC tumors, characterized by more, larger, positive correlations in EOCRC. These data suggest epigenetic modulation and accelerated aging may play a key role in the development of EOCRC. SIGNIFICANCEWe investigated whether environmentally driven factors contribute to early-onset colorectal cancer (EOCRC). We observed accelerated epigenetic aging in EOCRC and epigenetic changes associated with chronic inflammation. Tumor immune cell abundances correlated more strongly with microbes in EOCRC than average-onset CRC. These data suggest a dysregulation of immune response in EOCRC, driving chronic inflammation and tissue aging.

cancer biology↗

bioRxiv2024

AptaGPT: Advancing aptamer design with a generative pre-trained language model

Aptamers, synthetic oligonucleotide ligands, have shown significant promise for therapeutic and diagnostic applications owing to their high specificity and affinity for target molecules. However, the conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for aptamer selection is time-consuming and often yields limited candidates. To address these limitations, we introduce AptaGPT, a novel computational strategy that leverages a Generative Pre-trained Transformer (GPT) model to design and optimize aptamers. By training on SELEX data from early rounds, AptaGPT generated a diverse array of aptamer sequences, which were then computationally screened for binding using molecular docking. The results of this study demonstrated that AptaGPT is an effective tool for generating potential high-affinity aptamer sequences, significantly accelerating the discovery process and expanding the potential for aptamer research. This study showcases the application of generative language models in bioengineering and provides a new avenue for rapid aptamer development.

bioinformatics↗

bioRxiv2023

The tumor microbiome reacts to hypoxia and can influence response to radiation treatment in colorectal cancer

Tumor hypoxia has been shown to predict poor patient outcomes in several cancer types, partially because it reduces radiations ability to kill cells. We investigated whether some of the clinical effects of hypoxia could also be due to its impact on the tumor microbiome. We examined the RNA-seq data from the Oncology Research Information Exchange Network (ORIEN) database of colorectal cancer (CRC) patients treated with radiotherapy. For each tumor, we identified microbial RNAs and related them to the hypoxic gene expression scores calculated from host mRNA. Our analysis showed that the hypoxia expression score predicted poor patient outcomes and identified tumors enriched with certain microbes such as Fusobacterium nucleatum. The presence of other microbes, such as Fusobacterium canifelinum, predicted poor patient outcomes, suggesting a potential interaction between hypoxia, the microbiome, and radiation response. To investigate this concept experimentally, we implanted CT26 CRC cells into both immune-competent BALB/c and immune-deficient athymic nude mice. After growth, where tumors passively acquired microbes from the gastrointestinal tract, we harvested tumors, extracted nucleic acids, and sequenced host and microbial RNAs. We stratified tumors based on their hypoxia score and performed metatranscriptomic analysis of microbial gene expression. In addition to hypoxia-trophic and -phobic microbial populations, analysis of microbial gene expression at the strain level showed expression differences based on the hypoxia score. Hypoxia appears to not only associate with different microbial populations but also elicit an adaptive transcriptional response in intratumoral microbes. SIGNIFICANCETumor hypoxia reduces radiations ability to kill cells. We explored whether some of the clinical effects of hypoxia could also be due to interaction with the tumor microbiome. Hypoxic expression scores associated with certain microbes and elicited an adaptive transcriptional response in others.

immunology↗

bioRxiv2023

APLNR marks a cardiac progenitor derived with human induced pluripotent stem cells

Cardiomyocytes can be readily derived from human induced pluripotent stem cell (hiPSC) lines, yet its efficacy varies across different batches of the same and different hiPSC lines. To unravel the inconsistencies of in vitro cardiac differentiation, we utilized single cell transcriptomics on hiPSCs undergoing cardiac differentiation and identified cardiac and extra-cardiac lineages throughout differentiation. We further identified APLNR as a surface marker for in vitro cardiac progenitors and immunomagnetically isolated them. Differentiation of isolated in vitro APLNR+ cardiac progenitors derived from multiple hiPSC lines resulted in predominantly cardiomyocytes accompanied with cardiac mesenchyme. Transcriptomic analysis of differentiating in vitro APLNR+ cardiac progenitors revealed transient expression of cardiac progenitor markers before further commitment into cardiomyocyte and cardiac mesenchyme. Analysis of in vivo human and mouse embryo single cell transcriptomic datasets have identified APLNR expression in early cardiac progenitors of multiple lineages. This platform enables generation of in vitro cardiac progenitors from multiple hiPSC lines without genetic manipulation, which has potential applications in studying cardiac development, disease modelling and cardiac regeneration.

cell biology↗

bioRxiv2022

Single amino acid residues control potential-dependent inactivation of an inner membrane bc-cytochrome

During extracellular electron transfer, Geobacter sulfurreducens constitutively expresses the bc-cytochrome CbcL, yet cells containing only this menaquinone oxidase fail to respire above -0.1 V vs. SHE. By identifying mutations within cbcL that permit growth at higher potentials, we provide evidence that this cytochrome is regulated by redox potential. Strains expressing CbcLV205A, CbcLV205G, and CbcLF525Y were capable of growth with high potential electron acceptors including Fe(III) citrate, Mn(IV) oxides, and electrodes poised at +0.1 V vs. SHE. Electrochemical characterization of wild type CbcL revealed oxidative inactivation of electron transfer above -0.1 V, while CbcLV205A, CbcLV205G, and CbcLF525Y remained active. Growth yields of CbcLV205A, CbcLV205G, and CbcLF525Y were only 50% of WT, consistent with CbcL-dependent electron transfer conserving less energy. These data support the hypothesis that CbcL has evolved to rapidly shut off in response to redox potential to divert electrons to higher yield oxidases that coexist in the Geobacter membrane. TOC image and captionO_ST_ABSTunnel diode behaviorC_ST_ABSElectron flux from cells utilizing the menaquinone oxidase CbcL is attenuated by increased redox potential, preventing use of this low-efficiency pathway when driving forces are high enough to conserve energy via other oxidases. Single amino acid substitutions eliminate this switch-off effect and allow function at all potentials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/506072v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@11c9b5corg.highwire.dtl.DTLVardef@11be4c6org.highwire.dtl.DTLVardef@4883bborg.highwire.dtl.DTLVardef@c09223_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Lack of specificity in Geobacter periplasmic electron transfer

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized for menaquinone oxidation depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, separate periplasmic carriers could be needed for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, the role of ppcA and its four paralogs was examined. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower levels than PpcA in the periplasm. Mutants containing only PpcC and PpcE showed defects, but were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c-type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple {Delta}ppcABCDE {Delta}pgcA mutants still grew near wild type rates and displayed normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter, and suggests an as-yet undiscovered periplasmic mechanism supports electron transfer to electrodes. ImportanceMany inner and outer membrane redox proteins used by Geobacter for electron transfer to extracellular acceptors are known to have specific functions. However, how these are connected by periplasmic redox carriers remains poorly understood. Since Geobacter sulfurreducens contains multiple paralogous triheme periplasmic cytochromes, each with their own unique biochemical properties and expression profiles, it has been hypothesized that each cytochrome is involved in different respiratory pathways depending on redox potential or energy conservation needs. Here we show that instead of being specific for single conditions, the many periplasmic cytochromes of Geobacter show evidence of being highly promiscuous. Surprisingly, while any one of 6 triheme cytochromes could support similar growth with soluble or insoluble metals, none of these were required when cells utilized electrodes. These findings could simplify construction of synthetic electron transfer pathways.

microbiology↗

bioRxiv2022

Structure of Geobacter OmcZ filaments suggests extracellular cytochrome polymers evolved independently multiple times

While early genetic and low resolution structural observations suggested that extracellular conductive filaments on metal reducing organisms such as Geobacter were composed of Type IV pili, it has now been established that bacterial c-type cytochromes can polymerize to form extracellular filaments capable of long-range electron transport. Atomic structures exist for two such cytochrome filaments, formed from the hexaheme cytochrome OmcS and the tetraheme cytochrome OmcE. Due to the highly conserved heme packing within the central OmcS and OmcE cores, and shared pattern of heme coordination between subunits, it has been suggested that these polymers have a common origin. We have now used cryo-EM to determine the structure of a third extracellular filament, formed from the Geobacter sulfurreducens octaheme cytochrome, OmcZ. In contrast to the linear heme chains in OmcS and OmcE, the packing of hemes, heme:heme angles, and between-subunit heme coordination is quite different in OmcZ. A branched heme arrangement within OmcZ leads to a highly surface exposed heme in every subunit, which may account for the formation of conductive biofilm networks, and explain the higher measured conductivity of OmcZ filaments. This new structural evidence suggests that conductive cytochrome polymers arose independently on more than one occasion from different ancestral multiheme proteins.

biophysics↗

bioRxiv2021

Geobacter sulfurreducens inner membrane cytochrome CbcBA controls electron transfer and growth yield near the energetic limit of respiration.

Geobacter sulfurreducens utilizes extracellular electron acceptors such as Mn(IV), Fe(III), syntrophic partners, and electrodes that vary from +0.4 to -0.3 V vs. Standard Hydrogen Electrode (SHE), representing a potential energy span that should require a highly branched electron transfer chain. Here we describe CbcBA, a bc-type cytochrome essential near the thermodynamic limit of respiration when acetate is the electron donor. Mutants lacking cbcBA ceased Fe(III) reduction at -0.21 V vs. SHE, could not transfer electrons to electrodes between -0.21 and -0.28 V, and could not reduce the final 10% - 35% of Fe(III) minerals. As redox potential decreased during Fe(III) reduction, cbcBA was induced with the aid of the regulator BccR to become one of the most highly expressed genes in G. sulfurreducens. Growth yield (CFU/mM Fe(II)) was 112% of WT in {Delta}cbcBA, and deletion of cbcL (a different bc-cytochrome essential near -0.15 V) in {Delta}cbcBA increased yield to 220%. Together with ImcH, which is required at high redox potentials, CbcBA represents a third cytoplasmic membrane oxidoreductase in G. sulfurreducens. This expanding list shows how these important metal-reducing bacteria may constantly sense redox potential to adjust growth efficiency in changing environments.

microbiology↗

bioRxiv2020

Memory Transfer of Random Time Patterns Across Modalities

Perception is sensitive to statistical regularities in the environment, including temporal characteristics of sensory inputs. Interestingly, temporal patterns implicitly learned within one modality can also be recognised in another modality. However, it is unclear how cross-modal learning transfer affects neural responses to sensory stimuli. Here, we recorded neural activity of human volunteers (N=24, 12 females, 12 males) using electroencephalography (EEG), while participants were exposed to brief sequences of randomly-timed auditory or visual pulses. Some trials consisted of a repetition of the temporal pattern within the sequence, and subjects were tasked with detecting these trials. Unknown to the participants, some trials reappeared throughout the experiment, enabling implicit learning. Replicating previous behavioural findings, we showed that participants benefit from temporal information learned in audition, and that they can apply this information to stimuli presented in vision. Such memory transfer was not observed from vision to audition. However, using an analysis of EEG response learning curves, we showed that learning temporal structures both within and across modalities modulates single-trial EEG response amplitudes in both conditions (audition to vision and vision to audition). Interestingly, the neural correlates of temporal learning within modalities relied on modality-specific brain regions, while learning transfer affected activity in frontal regions, suggesting distinct mechanisms. The cross-modal effect could be linked to frontal beta-band activity. The neural effects of learning transfer were similar both when temporal information learned in audition was transferred to visual stimuli and vice versa. Thus, both modality-specific mechanisms for learning of temporal information, and general mechanisms which mediate learning transfer across modalities, have distinct physiological signatures that are observable in the EEG.

neuroscience↗

bioRxiv2020

Domain Binding and Isotype Dictate the Activity of Anti-human OX40 Antibodies

Previous data suggests that anti-OX40 mAb can elicit anti-tumour effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. The contributions of these different effector mechanisms has not previously been systematically evaluated, particularly for mAb directed to human OX40. Therefore, we generated a novel human OX40 knock-in (KI) mouse to evaluate a panel of anti-hOX40 mAb and show that their activities relate directly to two key properties: 1) isotype – with mIgG1 mAb evoking receptor agonism and CD8+ T cell expansion and mIgG2a mAb evoking deletion of Treg and; 2) epitope - with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumour models by engaging these different mechanisms. These findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T cell expansion or Treg depletion might be preferable according to the composition of different tumours.Summary Human trials with anti-OX40 antibodies have been disappointing indicating that optimal reagents have not yet been developed. Here, using a new panel of antibodies, we show that isotype and epitope combine to determine agonistic and therapeutic activity.Competing Interest StatementMSC is a retained consultant for BioInvent International and has performed educational and advisory roles for Baxalta and Boehringer Ingleheim. He has received research funding from Roche, Gilead, Bioinvent International and GSK. BF, IT, LM and MS are employees of Bioinvent International.View Full Text

immunology↗

bioRxiv2020

Stiffness of Nanoparticulate Mineralized Collagen Scaffolds Triggers Osteogenesis via Mechanotransduction and Canonical Wnt Signaling

The ability of the extracellular matrix (ECM) to instruct progenitor cell differentiation has generated excitement for the development of materials-based regenerative solutions. We previously described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material capable of inducing in vivo skull regeneration approaching 60% of the biomechanical properties of native calvarium without exogenous growth factors or ex vivo progenitor cell-priming, suggesting promise as a first-generation material for skull regeneration. Here, we evaluated the contribution of titrating stiffness to osteogenicity by comparing non-crosslinked (NX-MC) and crosslinked (MC) forms of MC-GAG. While both materials were osteogenic, MC demonstrated an increased expression of osteogenic markers and mineralization compared to NX-MC. Both materials were capable of autogenously activating the canonical bone morphogenetic protein receptor (BMPR) signaling pathway with phosphorylation of Smad1/5 (small mothers against decapentaplegic-1/5). However, unlike NX-MC, hMSCs cultured on MC demonstrated significant elevations in the major mechanotransduction mediators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) expression coincident with {beta}-catenin activation in the canonical Wnt signaling pathway. Inhibition of YAP/TAZ activation reduced osteogenic marker expression, mineralization, and {beta}-catenin activation in MC with a much lesser of an effect on NX-MC. YAP/TAZ inhibition also resulted in a reciprocal increase in Smad1/5 phosphorylation as well as BMP2 expression. Our results indicate that increasing MC-GAG stiffness induces osteogenic differentiation via the mechanotransduction mediators YAP/TAZ and the canonical Wnt signaling pathway, whereas the canonical BMPR signaling pathway is activated in a manner independent of mechanical cues.

bioengineering↗

bioRxiv2018

cGAMP signaling controls a transient surface-associated lifestyle

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3,3-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 [A]-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

biochemistry↗

bioRxiv2016

Genome scale mutational analysis of Geobacter sulfurreducens reveals distinct molecular mechanisms for respiration of poised electrodes vs. Fe(III) oxides

Geobacter sulfurreducens generates electricity by coupling intracellular oxidation of organic acids with electron transfer to the cell exterior, while maintaining a conductive connection to electrode surfaces. This unique ability has been attributed to the bacteriums capacity to also respire extracellular terminal electron acceptors that require contact, such as insoluble metal oxides. To expand the molecular understanding of electricity generation mechanisms, we constructed Geobacter sulfurreducens transposon mutant (Tn-Seq) libraries for growth with soluble fumarate or an electrode surface as the electron acceptor. Mutant libraries with over 33,000 unique transposon insertions and an average of 9 transposon insertions per kb allowed identification of 1,214 genomic features essential for growth with fumarate, including over 270 genes with one or more functional homologs that could not be resolved by previous annotation or in silico modeling. Tn-Seq analysis of electrode-grown cells identified mutations in over 50 genes encoding cytochromes, processing systems for proline-rich proteins, sensory systems, extracellular structures, polysaccharides, metabolic enzymes and hypothetical proteins that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of genes identified via Tn-Seq revealed a new putative c-type cytochrome conduit complex (extABCD) essential for growth with electrodes, which was not required for Fe(III)-oxide reduction. In addition, mutants lacking components of a putative methyl-accepting chemotaxis/cyclic dinucleotide sensing network (esnABCD) were defective in electrode growth, but grew normally with Fe(III)-oxides. These results suggest that G. sulfurreducens possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to other environmental electron acceptors.

microbiology↗