Biology subjects

Lee, M.

Publications and source records attributed to Lee, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Systematic analysis reveals an Mln1-dependent trade-off between metabolic flexibility and thermotolerance in Cryptococcus neoformans

During infection, fungal pathogens must adapt to multiple environmental challenges, including changes in temperature, nutrient availability, and immune stress. Although transcriptional regulators are central to these adaptations, how individual regulators contribute to distinct environmental responses and how these responses are functionally interconnected remain poorly understood. Here, we investigated the functions of infection-associated transcription factors under distinct host-relevant conditions in Cryptococcus neoformans. Integrating in vivo expression profiling with infectivity data from signature-tagged mutagenesis, we prioritized 24 transcription factors, 12 of which were induced under host-mimicking conditions. Initial functional analyses revealed distinct roles for these transcription factors in adaptation to temperature, carbon, and nitrogen availability. Further characterization of Mln1, a fungal basic helix-loop-helix transcription factor, uncovered an unexpected connection between carbon source adaptation and thermotolerance. Mig1-dependent repression limited MLN1 expression in the presence of glucose, whereas Mln1 accumulated in the nucleus in the presence of trehalose or ethanol, but not glucose. Loss of MLN1 impaired carbon-starvation gene expression and growth on non-preferred carbon sources, particularly trehalose. Conversely, mln1{Delta} cells accumulated more intracellular trehalose and exhibited enhanced thermotolerance, yet showed reduced melanization, titan cell formation, virulence in Drosophila, and brain fungal burden in mice. Together, these results suggest that Mln1 contributes to metabolic flexibility and fungal virulence while differentially influencing thermotolerance. More broadly, these findings highlight a potential physiological trade-off between nutrient utilization and stress resistance, suggesting that these adaptive processes may be interconnected in fungal pathogens.

microbiology↗

bioRxiv2026

A convergent proline biosynthetic step couples cell wall integrity to virulence in Cryptococcus neoformans

Metabolic adaptation is essential for the pathogenicity of the opportunistic fungus Cryptococcus neoformans. Although amino acid biosynthesis pathways are critical fitness determinants in fungal pathogens, the physiological contributions of proline biosynthesis remain incompletely understood, with most studies focusing on proline catabolism. Here, we characterized the proline biosynthetic enzymes Pro1, Pro2, and Pro3 in C. neoformans. Proline is produced by two routes that converge on {Delta}1-pyrroline-5-carboxylate (P5C): a de novo pathway from glutamate via Pro1 and Pro2, and an arginine-derived bypass via Car1 and Car2--both requiring Pro3 for the final reduction of P5C to proline. Although all biosynthetic mutants were strictly auxotrophic for proline, their requirements for virulence differed markedly: in a murine infection model, pro1{Delta} and pro2{Delta} mutants retained full virulence, whereas pro3{Delta} exhibited significantly attenuated virulence accompanied by growth defects and impaired cell wall integrity. Genetic disruption of P5C-generating routes partially restored distinct pro3{Delta} phenotypes, with PRO2 deletion alleviating delayed growth and combined PRO2 and CAR2 deletion partially restoring cell size, while cell wall defects remained unresolved. This dissociation indicates that growth requires proper balance of P5C-generating metabolic flux, whereas cell wall integrity depends on a distinct mechanism that is not resolved by blocking P5C production. Together, these findings identify Pro3 as a metabolic gatekeeper coupling proline biosynthesis to cell wall homeostasis, establishing the biosynthetic branch of proline metabolism as a previously unappreciated determinant of fungal pathobiology.

microbiology↗

bioRxiv2026

Differential value processing in the rostral and causal subthalamic nucleus during flexible and habitual decision-making in primates: Implications for cognitive dysfunction in Parkinson's disease

The subthalamic nucleus (STN) is a major target region of deep brain stimulation (DBS) for Parkinson's disease (PD), effectively alleviating motor symptoms. However, STN-DBS can also induce cognitive impairments, suggesting that the STN contributes to cognitive functions beyond simple motor control. Furthermore, anatomical organization supports functional segregation of cognitive processes, as basal ganglia subregions encoding flexible and stable values topographically project to distinct STN subregions. Yet, whether the STN contributes to cognitive functions and, if so, how subregions of the STN differentially process cognitive flexibility and habitual stability remains unknown. Here, we reveal a rostral-caudal functional organization of the STN that differentially supports flexible value updating and long-term value memory, enabling adaptive and habitual decision-making, respectively. Neurons in the rostral STN (rSTN) preferentially encode flexibly updated values, whereas neurons in the caudal STN (cSTN) preferentially encode stable long-term value memories. Notably, inactivation of the STN along the rostral-caudal axis produced dissociable behavioral deficits: rSTN inactivation selectively impaired goal-directed behavior requiring flexible value updating, whereas cSTN inactivation selectively disrupted habitual behavior guided by long-term value memories. Together, these results reveal a rostral-caudal functional segregation of the STN, with the rSTN supporting goal-directed behavior and the cSTN supporting habitual behavior. These findings further provide a framework for understanding how STN dysfunction or STN-DBS in Parkinson's disease may differentially affect cognitive processes beyond motor control.

neuroscience↗

bioRxiv2026

Automated compartment-based quantification reveals distinct patterns of cutaneous innervation with aging and microbial colonization

Cutaneous innervation is commonly quantified using measures such as intraepidermal nerve fiber density, but these approaches capture only selected anatomical regions or features of nerve structure. We developed an automated image-analysis pipeline to quantify nerve area and nerve-skeleton length across defined epidermal and dermal compartments in whole, stitched mouse plantar skin sections. Automated epidermis-dermis boundary detection showed agreement with manual tracing within the range of inter-observer variation and produced similar downstream measurements. We then applied the pipeline to skin from mice across age and distinct microbial environmental exposures. Conventionalized mice exposed to microbes had greater nerve area and length than germ-free mice in both the epidermis and dermis. In the aging cohort, aged mice had greater dermal nerve area and length than young mice, while epidermal measurements did not differ significantly. We also analyzed the epidermis and dermis together to assess whole-skin innervation. Whole-skin nerve area and length were higher in conventionalized mice than in germ-free mice but did not differ significantly between young and aged mice. These findings show that analyzing the epidermis and dermis separately helps identify where nerve measurements differ, including differences that may not be detected when both skin layers are analyzed together. Compartment-resolved quantification therefore provides a complementary approach for studying changes in cutaneous innervation across biological conditions and behavioral outcomes.

neuroscience↗

bioRxiv2026

Distinct Aβ40 and Aβ42 co-assembly pathways with free lipids: Experiment-driven MD study

Amyloid {beta}-protein (A{beta}), which is known for its role in Alzheimer's disease (AD), is also involved in several physiological functions. Of the two predominant isoforms, A{beta}40 and A{beta}42, the latter is more aggregation-prone and more active in health and disease. In the brain, A{beta} interacts with free lipids, which may affect its assembly pathways. Here, thioflavin T (ThT) fluorescence spectroscopy is combined with molecular dynamics (MD) to investigate A{beta}40 and A{beta}42 co-assembly with free dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (CHOL) lipids. ThT measurements at several protein-to-lipid (PL) ratios show isoform- and lipid composition-dependent kinetics of A{beta} fibril formation with clear isoform-dependent lipid effects at a PL ratio of 1:5. Early co-assembly stages of six A{beta}40/A{beta}42 with DMPC and DMPC/CHOL lipids at this PL ratio are then examined by MD. MD results indicate that lipids alter the secondary structure more and interact more avidly with A{beta}40 than with A{beta}42. In turn, A{beta}40 increases the planar order of lipid bicelles more than A{beta}42. Unlike A{beta}40, A{beta}42 decreases the nematic order of CHOL. In DMPC/CHOL lipids, A{beta}42 but not A{beta}40 exhibits two A{beta}-lipid interaction modes associated with mutually exclusive A{beta}42-lipid and A{beta}42-A{beta}42 interactions. Our MD findings provide a plausible explanation for an experimentally observed increase in the lag time for A{beta}40 fibril formation in DMPC lipids and even more so in DMPC/CHOL lipids that contrasts with much smaller effects of lipids on A{beta}42 fibril formation and offer additional atomistic-level insights into A{beta} co-assembly with lipids amenable to experimental testing.

biophysics↗

bioRxiv2026

Native oligomerisation of a small type I antifreeze protein from winter flounder

Antifreeze proteins and peptides adhere to ice crystals and inhibit their growth, resulting in freezing point depression. AFP6 is an abundant antifreeze peptide in the blood serum of winter flounder (Pseudopleuronectes americanus). This 37-residue peptide forms an amphiphilic -helix with a hydrophobic ice-binding surface. In this study, prediction of the peptide structure suggested the formation of an antiparallel dimer burying the hydrophobic surfaces of both subunits. The oligomerisation of AFP6 was therefore examined in aqueous solutions using size-exclusion chromatography and chemical crosslinking along with ice-binding evaluation. Crosslinking of non-amidated AFP6, followed by denaturing electrophoresis, revealed bands consistent with monomers and dimers, whereas results for the amidated form and a recombinant mutant suggested additional larger forms. Bands consistent with the presence of monomers and dimers were also predominant following non-amidated AFP6 crosslinking under a variety of solution conditions, with smaller proportions of peptide assembled up to tetrameric sizes; however, the larger oligomers appeared to be absent from AFP6 treated with sodium bicarbonate. Isolated oligomers of non-amidated AFP6 produced ice crystal morphologies and thermal hysteresis comparable to those for the unfractionated peptide suggesting similar interaction with ice. Oligomerisation of AFP6 may allow greater solubility of this amphiphilic -helical peptide in aqueous solutions.

biochemistry↗

bioRxiv2026

Microfluidic Capture-Enrichment of individualized microbes from urban wastewater reveals a hidden reservoir of potential pre-emergent pathogens

Current environmental biosurveillance infrastructure excels at tracking known biological threats but is limited in its ability to proactively identify potential pre-emergent pathogens (PEPs). To address this gap, we developed a microfluidics-based Capture-Enrichment pipeline that isolates individual environmental cells via microdroplet encapsulation, preserving community biodiversity while enabling iterative selection against human sera. When applied to urban wastewater, this approach successfully recovered pathogens, commensals, and PEPs that fell below the detection limits of conventional amplicon sequencing. Genomic analysis revealed that wastewater-derived ESKAPE pathogens cluster closely with clinical isolates, showing that the pipeline captures clinically relevant threats. Furthermore, isolated PEPs exhibited known and emerging phenotypes associated with early-stage pathogenesis and antimicrobial resistance. These findings demonstrate that targeting PEPs can fundamentally expand the scope and quality of biosurveillance by providing a scalable strategy to identify, characterize, and close important gaps in our understanding of emerging microbial pathogens.

microbiology↗

bioRxiv2026

HIGH DOSE GAMMAHERPESVIRUS INFECTION IN MACROPHAGES RESULTS IN INFLAMMATORY MULTIMODAL CELL DEATH

Human gammaherpesviruses (gHVs) including Epstein-Barr Virus and Kaposis sarcoma associated herpesvirus are linked to cancer and inflammatory disease development. The outcome of gHV infection is highly regulated by cell type, with macrophages identified as an important early infection target that can also serve as a latency reservoir. Here, we used the mouse gHV model, murine gammaherpesvirus (MHV68), to identify a dose-dependent outcome of MHV68 infection of macrophages, with high dose infection resulting in extensive morphological changes, viral gene expression, and a pronounced inflammatory cell death not observed under conditions of lytic replication occurring at lower infectious dose. Virion envelope and tegument components were not sufficient for this dose-dependent cell death, which required intact viral DNA and virus transcription, yet occurred independently of viral DNA replication and late gene transcription. Necrostatin-1 (Nec-1) treatment, an anti-inflammatory therapeutic, limited cell death and reduced cell morphology alterations, with no impact on virus replication, uncoupling cell death from lytic replication. Activation of autophagy profoundly limited high dose infection outcomes, with increased cell survival and reduced viral gene expression. Despite the improved cell survival observed in cells treated with Nec-1, biochemical analysis of infected cultures identified robust apoptosis induction with subsequent activation of gasdermin E, suggesting a multi-modal cell death mechanism culminating in pyroptosis. Our findings suggest that high dose macrophage infection byMHV68 is a major driver of inflammatory cell death to potentially shape downstream inflammation and immune responses.

microbiology↗

bioRxiv2026

Aβ42-Driven α-synuclein Fibril Polymorphism and Distinct Intracellular Aggregation

The frequent coexistence of -synuclein (-syn) and amyloid-{beta} (A{beta}) aggregates in neurodegenerative diseases suggests that heterotypic interactions between these amyloidogenic proteins may influence disease progression, yet their molecular consequences remain poorly understood. Here, we investigated how distinct aggregation states of A{beta}42, monomers and preformed fibrils (PFFs), modulate -syn fibril formation, structure, and downstream neuronal pathology. Thioflavin T kinetics showed that A{beta}42 monomers delayed -syn fibril formation, whereas A{beta}42 PFFs exhibited a trend toward accelerated aggregation, indicating aggregation state-dependent effects on -syn aggregation. Negative-stain TEM, proteinase K digestion, and solid-state NMR spectroscopy further demonstrated that both A{beta}42 monomers and PFFs altered -syn fibril structure, generating distinct fibril conformations depending on the A{beta}42 concentration and aggregation state. To determine whether these conformational differences influence pathological activity, -syn PFF variants generated in the presence of different concentrations of A{beta}42 monomers or PFFs were applied to dopaminergic neuronal cells. -Syn fibrils formed in the presence of A{beta}42 PFFs showed greater capacity to induce intraneuronal -syn aggregation than -syn PFFs, whereas fibrils formed in the presence of A{beta}42 monomers exhibited similar or reduced seeding capacity relative to -syn PFFs. Together, our findings demonstrate that heterotypic interactions with A{beta}42 reshape -syn aggregation pathways and fibril conformations, generating structurally distinct -syn fibril populations with different neuronal seeding activities. These results provide a molecular framework for understanding how cross-talk between amyloidogenic proteins may contribute to structural and pathological heterogeneity in mixed neurodegenerative diseases.

molecular biology↗

bioRxiv2026

Metabolic responses to physiological stressors assessed using continuous glucose monitoring integrated with indirect calorimetry in mice

Continuous glucose monitoring (CGM) in rodents has provided unprecedented temporal resolution of glycemic dynamics in vivo. Even in the absence of deliberate perturbation, glucose levels in mice are dynamic, fluctuating in response to the timing and duration of feeding events, changes in neurological and hormonal states, physical activity, and photoperiod. To obtain a comprehensive view of metabolic adaptations under common experimental conditions, we monitored freely moving mice simultaneously using CGM and indirect calorimetry to quantify glucose, food intake, physical activity and metabolic rate. We characterized glycemic and metabolic responses to routine laboratory interventions, including short-term and overnight fasting, refeeding, tail blood sampling during glucose tolerance tests, changes in ambient temperature to cold or thermoneutral conditions, and access to running wheels. We found that food removal induced a robust, transient stress response characterized by increased blood glucose, body temperature, energy expenditure, and physical activity. However, prolonged fasting ultimately led to hypoglycemia and torpor. The magnitude and variability of glycemic responses to insulin tolerance tests were strongly influenced by fasting duration, and tail-tip blood collection itself elicited substantial hyperglycemia. In contrast to prolonged fasting, refeeding produced relatively modest and transient effects on glucose and energy expenditure. Cold exposure elicited increased energy expenditure along with a sustained hyperglycemic response. Voluntary wheel running induced transient increases in glucose and metabolic activity and promoted a shift toward increased fatty acid oxidation. Together, these findings demonstrate that common laboratory manipulations exert dynamic, often substantial effects on glycemia and whole-body metabolism that are readily revealed by CGM and indirect calorimetry.

physiology↗

bioRxiv2026

An overlooked microbial pathway links organic nitrogen turnover in composts to nitrous oxide formation

Biological N2O production from organic nitrogen is generally assumed to require canonical nitrification, which generates oxidized nitrogen that subsequently fuel denitrification. Whether this paradigm universally applies to nitrogen-rich microbial communities remains unclear. Here, we investigated N2O production across an industrial poultry manure composting process and found that substantial N2O formation occurred despite the apparent absence of canonical ammonia oxidation. Neither allylthiourea inhibition nor metagenomic analyses provided evidence for ammonia-oxidizing microorganisms or their activity. Instead, metagenomic analyses identified abundant bacterial nitric oxide synthase (bNos) genes, many of which were phylogenetically affiliated with Bacilli, the dominant bacterial group throughout composting. Physiological experiments with Bacillus isolates demonstrated a nitrification-independent route in which L-arginine was oxidized to NO2-/NO3-, consistent with bNOS-mediated NO formation followed by abiotic oxidation. Recovery of 15N-labelled N2O following 15NO2- addition established NO2- as an immediate precursor of aerobically produced N2O, confirming that the oxidized nitrogen generated through this alternative route subsequently fueled denitrification. Metagenomic analyses further revealed extensive denitrification potential but comparatively low nosZ abundance. Together, these findings identify a previously overlooked route linking organic nitrogen turnover to denitrification independently of canonical nitrification, thereby expanding current models of microbial N2O production in composts and potentially other protein-rich thermophilic environments.

microbiology↗

bioRxiv2026

Redundant contacts and force redistribution stabilize limbless vertical climbing

Animals navigating complex vertical environments must secure stable footholds, a challenge for species without feet. While arboreal climbing has evolved repeatedly in snakes, the physical mechanisms they use to scale broad, nearly flat surfaces remain poorly understood. By measuring three-dimensional body kinematics and per-contact forces on a smooth vertical wall with protruding posts, we show that cornsnakes climb by dynamically balancing forces across a highly redundant network of 5 to 16 simultaneous contacts--far exceeding the three contacts minimally required for physical stability. Using a computational model and a robotic climber, we demonstrate that while simple body undulations and passive friction are mechanically sufficient to climb this terrain, snakes systematically deviate from this passive baseline. While downward climbing relies primarily on friction, ascending snakes actively generate positive mechanical work at their contacts to propel themselves. Furthermore, we found that whenever a snake engages a new contact, it triggers a stereotyped, system-wide redistribution of force that seamlessly integrates the new foothold without disrupting whole-body balance. These results reveal how a continuous, flexible body can transform sparse environmental features into a robust, fault-tolerant network. This mechanism provides a biomechanical framework for understanding the repeated evolution of limbless climbing and offers physical principles for designing agile robots for unstructured terrain.

biophysics↗

bioRxiv2026

In situ identification of substrates of the protein tyrosine phosphatase PTP1B using site-specific photo-crosslinking

Protein tyrosine phosphorylation is critical for cellular function, and aberrant phosphorylation is tied to a wide range of human diseases. Identifying the substrates of protein tyrosine phosphatases, the enzymes that erase this modification, is critical to understanding human biology and disease states. The state-of-the-art method for tyrosine phosphatase substrate identification requires the use of mutations that modestly increase the lifetime of enzyme-substrate complexes by kill catalytic activity. While these "substrate-trapping" mutants are useful tools, they work best for high-affinity or abundant substrates that remain phosphatase-bound through cell lysis and enrichment. Here, we use site-specific photo-crosslinking to covalently capture the substrates of tyrosine phosphatases in situ. We identify eight different positions around the active site of the phosphatase PTP1B where photo-crosslinker amino acids can be incorporated via amber codon suppression without dramatically disrupting catalytic activity. We then conduct photo-crosslinking experiments in mammalian cells and identify crosslinked proteins by mass spectrometry proteomics, revealing that our approach can capture known PTP1B interactors and substrates. We then show that PTP1B photo-crosslinking in situ is sensitive to enzyme localization and identify new PTP1B substrates that regulate contacts between the endoplasmic reticulum and plasma membrane. We also demonstrate that photo-crosslinking can capture signal-dependent interactions. For example, we observe PTP1B crosslinking to the epidermal growth factor (EGF) receptor, a known substrate, in an EGF-dependent manner, and we identify other potential EGF-dependent substrates. Overall, our approach reveals previously unknown roles of PTP1B in signaling systems and could be readily extended to other tyrosine phosphatases in the same family.

biochemistry↗

bioRxiv2026

Multi-Site Reproducibility Study of 3D High-Content Analysis with Dual-View Oblique Plane Microscopy

High content imaging is being applied to achieve quantitative fluorescence readouts in increasingly complex 3-dimensional (3D) cell culture models such as spheroids and organoids. Compared to conventional 2D assays, 3D assays better represent biological heterogeneity but require more complex sample preparation, 3D imaging and 3D image analysis that can affect the accuracy and precision of such assays. We used spheroids formed from the NRAS-activated melanoma cell line 19161 modified to express an ERK kinase translocation reporter (KTR) as an exemplar 3D phenotypic assay carried out in 96-well plates. The spheroids were treated with the ERK activator TPA and a range of concentrations of the MEK inhibitor Binimetinib. 3D live-cell imaging with sub-cellular spatial resolution was performed using a dual-view oblique plane microscope (dOPM) - a form of single-objective light-sheet microscope - and the experiment was performed separately at 4 different institutes. The results were analysed using an identical 3D analysis pipeline and parameters. We assessed the variation in assay readout using a linear mixed effects model. Random variance at the well level was negligible (SD = 0.0048 relative to range of KTR biosensor readout at reference site of 0.17), indicating low technical noise. Treatment effects were dose-dependent and highly statistically significant compared to DMSO control across all sites (Dunnett-corrected p < 0.001). The range in KTR readout between the minimum (3.5 M Binimetinib) and maximum (100 nM TPA) treatments varied between 59 to 96% relative to the reference site. Measured bias in KTR readout between sites was between 6 and 12% of the range of the reference site. This study quantifies the reproducibility of a 3D live spheroid-based assay employing a fluorescence biosensor requiring readout out at the per-cell level using the dOPM platform and discusses areas where experimental protocol could be improved in the future to further improve reproducibility.

bioengineering↗

bioRxiv2026

The metastasis susceptibility gene RRP1B is a stress-responsive regulator of nuclear RNA processing and splicing in breast cancer

The tumor microenvironment exposes cancer cells to mechanical, thermal, hypoxic, and acidic stresses, yet how cells integrate these signals to remodel RNA processing remains poorly understood. Here, we show that ribosomal RNA Processing 1B (RRP1B), previously characterized as a nucleolar ribosome biogenesis factor and metastasis modifier, functions as a broad-spectrum stress sensor that dynamically repositions among the nuclear envelope, nucleolus, and nuclear speckles (NS). Relocalization is governed by multi-site phosphorylation within intrinsically disordered regions (IDRs): phosphomimetic substitutions promote NS-proximal condensate formation in an RNA-dependent manner, while unphosphorylatable substitutions confine RRP1B to the nucleolus. Under stress, the RRP1B interactome shifts globally, with ribosomal processing partners replaced by pre-mRNA splicing components enriched for NS-resident proteins. RNA immunoprecipitation sequencing (RIP-seq) demonstrates that under basal conditions RRP1B associates with long, intron-rich, nuclear periphery-proximal transcripts, whereas heat shock redirects binding toward shorter, exon-dense transcripts enriched for motifs of serine/arginine-rich (SR) proteins near the NS. RRP1B overexpression nearly abolishes cytoplasmic retained intron accumulation and drives preferential export of specific transcript isoforms in a compartment- and temperature-dependent manner, establishing RRP1B as a regulator of RNA localization fidelity rather than transcriptional output. An RRP1B overexpression signature is most highly activated in basal-like and claudin-low breast tumors, and the RRP1B-associated retained intron splicing program correlates with reduced survival in a tumor-grade-dependent manner. These findings reframe RRP1B as a microenvironmentally sensitive regulator of nuclear RNA processing with direct implications for aggressive breast cancer biology.

Cancer Biology↗

bioRxiv2026

First Usutu virus detections in wild birds in Scotland, 2025

In summer 2025, several Eurasian Blackbird (Turdus merula) deaths were reported on the Isle of Arran in Scotland. Initial investigation included post-mortem examination, where no diagnosis was achieved. Following Orthoflavivirus and avian paramyxovirus testing, Usutu virus RNA was detected in two Blackbirds by reverse transcription-PCR. Phylogenetic analysis identified Usutu virus Africa 3.2 lineage which clustered closely with existing UK detections, indicating geographic expansion rather than a new incursion. Subsequent surveillance confirmed the presence of several potential mosquito vector species.

microbiology↗

bioRxiv2026

Duplication of superoxide dismutase and a mutation in aquaglyceroporin mediates the sensitivity of Plasmodium falciparum to cryptosporin, a natural product derived from Acaromyces ingoldii

Cryptosporin, a fungal metabolite, exhibited potent antimalarial activity against both asexual blood stage Plasmodium falciparum and liver-stage Plasmodium berghei with minimal human HepG2 toxicity. Unlike atovaquone, cryptosporins mechanism is independent of mitochondrial electron transport. Minimum inoculum of resistance showed a low risk of resistance development. RNA-Seq analysis revealed the upregulation of genes associated with sexual development including many canonical markers such as Pfs25, and PfCCp3, suggesting a stress response that is also seen when parasites are treated with artemisinin. In vitro evolution and whole genome sequencing analysis identified a mutation (F138Y) in PfAQP (PF3D7_1132800) and duplications of the two superoxide dismutase genes, PfSOD-1 (PF3D7_0814900) and PfSOD-2 (PF3D7_0623500). CRISPR/Cas9 editing confirmed that the F138Y mutation in PfAQP was sufficient to confer resistance to cryptosporin. Alignment of the P. falciparum structure with that of HsAQP3 suggests the mutation may impact transport of hydrogen peroxide and the transition between open and closed conformations. Indeed, studies with BY4742 {Delta}fps1 yeast expressing PfAQP showed that the permeability of PfAQP was not affected by cryptosporin and that it is likely not a direct target. Taken together, this study highlights the role of PfAQP in the resistance development of cryptosporin. In addition, cryptosporin likely induces high levels of oxidative stress which results in the duplications of oxidative dismutase genes as part of the parasites defense response. These findings highlight the role of PfAQP in mediating drug resistance, the mechanism of which warrants further research.

genetics↗

bioRxiv2026

Miniaturized subcutaneous cellular implants for sustained therapeutic protein delivery in resource-limited settings

Cell encapsulation offers a promising strategy for sustained therapeutic protein delivery, obviating the need for repeated injections. Among potential implantation sites, the subcutaneous space is particularly attractive for its accessibility and amenability to minimally invasive procedures. However, performance of subcutaneous devices reported to date has been limited due to various challenges including foreign body response (FBR) and inadequate mass transfer. Moreover, typical encapsulation devices require surgeries for implantation and retrieval, limiting their potential use in resource-limited settings. Here we present a miniaturized cell encapsulation platform comprising cells engineered to produce therapeutic proteins and an FBR-mitigating zwitterionic polyurethane nanofibrous membrane, in a thin cylindrical form factor compatible with applicator-based minimally invasive implantation and retrieval. Clonal mesenchymal stromal cells engineered to produce PGT121, a broadly neutralizing anti-HIV-1 antibody, were encapsulated and inserted subcutaneously, achieving long-term cell survival and sustained serum PGT121 concentrations for up to 36 weeks across multiple murine models. Cell-loaded devices retained therapeutic function after cryopreservation, supporting their potential use as an off-the-shelf product that can be centrally manufactured and implanted on-site without specialized infrastructure. The custom-designed applicator-based implantation and minimally invasive retrieval procedures were demonstrated in a more clinically relevant minipig model. These mini-"cellular factories" represent a translatable strategy for sustained delivery of biologic drugs in resource-limited settings. One Sentence SummaryAn insertable and retrievable mini cellular construct enables sustained protein delivery, supporting its potential use in resource-limited settings.

bioengineering↗