Biology subjects

Mayfield, S.

Publications and source records attributed to Mayfield, S..

8 recordsLinked to original sources

bioRxiv2026

A COJEC-chemotherapy resistant model of Th-ALK(F1174L)/MYCN neuroblastoma offers insights into tumour immune evasion and development of the bone marrow metastatic niche

Multi-agent COJEC chemotherapy is the main-stay of induction treatment for patients diagnosed with high-risk neuroblastoma. However, at least 10% of patients will be primary refractory to chemotherapy and only 50% achieve 5-year overall survival. The bone marrow is the most frequent site of metastasis in these patients. Novel approaches are required to improve response rates but the inter- and intra- patient tumour heterogeneity and dynamics of the neuroblastoma immune microenvironment makes anticipation of resistance phenotypes incredibly challenging. We present here a novel immunocompetent C57 Bl/6 model of Th-ALK(F1174L)/MYCN neuroblastoma, in which spontaneous abdominal tumours are driven by expression of mutant Anaplastic Lymphoma Kinase and over-expression of Mycn in the neural crest. We have used this model to generate a personalised dosing schedule inducing COJEC-chemotherapy resistance, in which individual mice receive chemotherapy cycles dependent upon the progression of their neuroblastoma tumours. Using both single cell RNA sequencing and spatial immunophenotyping gave us extraordinary precision in our comprehensive analysis of the tumour intrinsic and microenvironmental factors associated with COJEC resistance. We found that the resistance phenotype was driven by Cdk8 upregulation in adrenergic and mesenchymal tumour cells. Infiltration of immunosuppressive myeloid-derived immune cells and remodeling of the tumour-associated stroma further contributed to COJEC resistance. In the bone marrow we observed expansion of neutrophils and evidence of NETosis associated with micro-metastatic disease. Our results further endorse the development of CDK8-targeting therapeutics for neuroblastoma patients which might boost the anti-tumour immune response. Additional studies will be required to define the roles of neutrophils and neutrophil NETosis in neuroblastoma progression and metastasis. Our C57 Bl/6 model will be pivotal in future preclinical studies of immune-modulating therapeutics.

cancer biology↗

bioRxiv2025

Sustainable production of plastic-degrading enzymes in Chlamydomonas pacifica

The discovery of a new extremophile alga, Chlamydomonas pacifica, provides an opportunity to expand on heterologous protein expression beyond the traditional Chlamydomonas reinhardtii. C. pacifica is a unicellular extremophile capable of surviving at high pH, high temperatures, and high salinity. These various growth conditions allow C. pacifica to outcompete any invading contaminants in open-air environments. Developing this novel species as a platform for recombinant protein production could significantly advance commercial microalgal recombinant protein production. We have previously shown that C. reinhardtii can secrete a plastic-degrading enzyme: a PETase known as PHL7. This PETase is capable of cleaving ester bonds and has been used commercially for the degradation of PET plastics. However, the expression of such an enzyme has yet to be done in open raceway ponds and on a large scale. Here, we describe the culturing of PHL7 transgenic C. pacifica strain in three 80L raceway ponds and the measurements of recombinant enzymatic expression and activity found in the culture media. Our work provides proof of concept that this new organism can produce functional PHL7 enzymes in addition to producing the valuable components that inherently exist in the C. pacifica algae biomass. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/654053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@10065a3org.highwire.dtl.DTLVardef@110c456org.highwire.dtl.DTLVardef@1c9efc6org.highwire.dtl.DTLVardef@9ceea1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Low-Cost Screening of Algae for Extreme Tolerance to pH, Temperature, Salinity, and Light

Bioprospecting algae strains with tolerance to extreme conditions such as pH, temperature, salinity, and light is crucial for advancing biotechnology and environmental applications. However, traditional screening methods often involve significant costs and labor, restricting their accessibility and practical use. In this study, we developed and validated low-cost, high-throughput screening techniques, predominantly employing agar plates and liquid culture assays, to effectively differentiate tolerance levels among various algae strains. The methodologies were optimized using the model microalga Chlamydomonas reinhardtii and its closely related species Chlamydomonas incerta and the recently discovered extremophilic Chlamydomonas pacifica. We systematically evaluated the algae for tolerance to extremes by establishing precise gradients of pH (acidic to alkaline conditions), salinity (0 to 5 M NaCl), temperature (34-42{degrees}C), and light intensity (40 to 2977 E{middle dot}m-{superscript 2}{middle dot}s-{superscript 1}). Our results demonstrated that these cost-effective, agar plate-based methods effectively distinguished algae strains exhibiting superior tolerance to extreme environmental conditions. These screening techniques not only provided clear differentiation among the closely related strains but also delivered reproducible outcomes suitable for scaling up to larger bioprospecting efforts. Furthermore, the affordability and simplicity of these methods facilitate their implementation in resource-limited laboratories, thereby broadening participation in algae bioprospecting endeavors. This study highlights the potential of low-cost, accessible screening techniques to significantly enhance the discovery and characterization of algal strains with extreme traits. Ultimately, these methods support the development of robust algae-based resources, driving innovation in diverse industrial processes and environmental solutions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/653396v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@6b179borg.highwire.dtl.DTLVardef@dadbfforg.highwire.dtl.DTLVardef@a1750dorg.highwire.dtl.DTLVardef@baffc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Identification and Overexpression of Endogenous Transcription Factors to Enhance Lipid Accumulation in the Commercially Relevant Species Chlamydomonas pacifica

Sustainable low-carbon energy solutions are critical to mitigating global carbon emissions. Algae-based platforms offer potential by converting carbon dioxide into valuable products while aiding carbon sequestration. However, scaling algae cultivation faces challenges like contamination in outdoor systems. Previously, our lab evolved Chlamydomonas pacifica, an extremophile green alga, which tolerates high temperature, pH, salinity, and light, making it ideal for large-scale bioproduct production, including biodiesel. Here, we enhanced lipid accumulation in evolved C. pacifica by identifying and overexpressing key endogenous transcription factors through genome-wide in-silico analysis and in-vivo testing. These factors include Lipid Remodeling Regulator 1 (CpaLRL1), Nitrogen Response Regulator 1 (CpaNRR1), Compromised Hydrolysis of Triacylglycerols 7 (CpaCHT7), and Phosphorus Starvation Response 1 (CpaPSR1). Under nitrogen deprivation, CpaLRL1, CpaNRR1, and CpaCHT7 overexpression enhanced lipid accumulation compared to wildtype. However, CpaPSR1 increased lipid accumulation compared to wildtype in normal media despite causing no effect under nitrogen depravation, highlighting the difference in function based on media conditions. Notably, lipid analysis of CpaPSR1 under normal media conditions revealed a 2.4-fold increase in triglycerides (TAGs) compared to the wild type, highlighting its potential for biodiesel production. This approach provides a framework for transcription factor-focused metabolic engineering in algae, advancing bioenergy and biomaterial production. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/651737v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@8ab995org.highwire.dtl.DTLVardef@19dff1forg.highwire.dtl.DTLVardef@1d865aborg.highwire.dtl.DTLVardef@1fafd12_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender.com.

systems biology↗

bioRxiv2025

Engineering microalgal cell wall-anchored proteins using GP1 PPSPX motifs and releasing with intein-mediated fusion

Harnessing and controlling the localization of recombinant proteins is essential for advancing synthetic biology, industrial biotechnology, and drug delivery. This study presents a dual system for protein anchoring and controlled release in Chlamydomonas reinhardtii. Using truncated variants of the GP1 glycoprotein fused to the plastic-degrading enzyme PHL7, we identified the PPSPX motif as critical for anchoring proteins to the cell wall. Constructs with increased PPSPX content exhibited reduced secretion but enhanced anchoring, revealing key anchor-signal sites within GP1. To enable controlled release, we incorporated a pH-sensitive intein from derived from Mycobacterium tuberculosis RecA. Under acidic conditions (pH 5.5), this intein efficiently cleaved and released mCherry and PHL7 from the GP1 anchor. Fluorescence kinetics demonstrated significant mCherry release from the mCherry-intein-GP1 construct within 4 hours at pH 5.5, while release was minimal at pH 8.0 and negligible for the mCherry-GP1 control. Western blot analysis confirmed efficient cleavage, showing a lower band corresponding to free mCherry at pH 5.5 and no release at pH 8.0. This anchor-release strategy integrates glycomodules with pH-sensitive inteins to enable precise spatial and temporal protein control. The system offers broad utility for targeted drug delivery, environmental biosensing, and biocatalyst deployment. Overall, we establish a versatile framework for optimizing protein localization and environmentally triggered release in C. reinhardtii, with broad implications for proteomics, biofilm engineering, and scalable therapeutic delivery systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/634604v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@8bdc2org.highwire.dtl.DTLVardef@1e213c7org.highwire.dtl.DTLVardef@1808559org.highwire.dtl.DTLVardef@b2c1e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production

Chlamydomonas incerta, a genetically close relative of the model green alga Chlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incertas capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii, C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii. For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil(R) DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incertas potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/618925v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@173d398org.highwire.dtl.DTLVardef@148ad36org.highwire.dtl.DTLVardef@63b5e9org.highwire.dtl.DTLVardef@3be081_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Description of a novel extremophile green algae, Chlamydomonas pacifica, and its potential as a biotechnology host

We present the comprehensive characterization of a newly identified microalga, Chlamydomonas pacifica, originally isolated from a soil sample in San Diego, CA, USA. This species showcases remarkable biological versatility, including a broad pH range tolerance (6-11.5), high thermal tolerance (up to 42{degrees}C), and salinity resilience (up to 2% NaCl). Its amenability to genetic manipulation and sexual reproduction via mating, particularly between the two opposing strains CC-5697 & CC-5699, now publicly available through the Chlamydomonas Resource Center, underscores its potential as a biotechnological chassis. The biological assessment of C. pacifica revealed versatile metabolic capabilities, including diverse nitrogen assimilation capability, motility and phototaxis. Genomic and transcriptomic analyses identified 17,829 genes within a 121 Mb genome, featuring a GC content of 61%. The codon usage of C. pacifica closely mirrors that of C. reinhardtii, indicating a conserved genetic architecture that supports a trend in codon preference with minor variations. Phylogenetic analyses position C. pacifica within the core-Reinhardtinia clade yet distinct from known Volvocales species. The lipidomic data revealed an abundance of triacylglycerols (TAGs), promising for biofuel applications and lipids for health-related benefits. Our investigation lays the groundwork for exploiting C. pacifica in biotechnological applications, from biofuel generation to synthesizing biodegradable plastics, positioning it as a versatile host for future bioengineering endeavors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/611117v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1ead226org.highwire.dtl.DTLVardef@18af631org.highwire.dtl.DTLVardef@291c2borg.highwire.dtl.DTLVardef@f860ac_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2021

Development of a Cell Surface Display System in Chlamydomonas reinhardtii

Cell-surface display systems are biotechnological techniques used to express heterologous proteins on the cell surface. Their application depends directly on the cell system used, as well as on the anchoring point for the surface displayed protein. To meet most application demands an inexpensive, safe, and scalable production platform, that reduces the economic barriers for large scale use is needed. Towards this goal, we screened three possible cell surface anchoring points in the green algae Chlamydomonas by fusing mVenus to prospective anchors moieties. The vectors harboring mVenus:anchor were screened for mVenus fluorescence and tested for cellular localization by confocal laser scanning microscopy. This strategy allowed the identification of two functional anchors, one for the cytoplasmic membrane using the MAW8 GPI-anchor signal, and one for the cell wall using the GP1 protein. We also exploited GP1 chemical and biological traits to release the fused proteins efficiently during cell wall shedding. Our work provides a foundation for surface engineering of C reinhardtii supporting both cell biology studies and biotechnology applications.

synthetic biology↗