Biology subjects

Gupta, K.

Publications and source records attributed to Gupta, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Stearoyl-CoA Desaturases regulate stem and progenitor cell metabolism and function in response to nutrient abundance

Dietary components and metabolites play a critical role in regulating intestinal stem and progenitor cell function and proliferation. Here we show that Stearoyl-CoA Desaturases (SCDs), which regulate intracellular saturated to monounsaturated fatty acids ratios, are induced in response to nutrient abundance, especially in the distal intestine, and regulate intestinal homeostasis. Genetic or pharmacological inhibition of SCDs altered lipid metabolism, increased ER stress, and reduced proliferative intestinal stem and progenitor cells in intestinal organoids. These effects were largely mitigated by oleic acid supplementation. Intestinal epithelium-specific deletion of Scd1 and Scd2 led to metabolic rewiring, leading to expansion of progenitor cell populations. DSS-induced epithelial damage revealed a dependence on SCD enzymes during regeneration, accelerating epithelial damage and inflammation in intestines lacking epithelial Scd1 and Scd2. These findings underscore key metabolic pathways and dependencies that enable intestinal stem and progenitor cells to adapt to nutrient fluctuations and support epithelial tissue regeneration following injury.

physiology↗

bioRxiv2025

Human Platelet-derived Lysates and Extracellular Vesicles Restore Mitochondrial Function and Redox Balance in Neuronal Models

Platelet-derived biomaterials are emerging as promising cell-free therapeutic platforms for regenerative medicine and neurorestoration. Among them, human platelet pellet lysates (HPPL) and platelet-derived extracellular vesicles (PEVs), prepared from clinical-grade allogeneic platelet concentrates, provide two complementary biomaterial formats: a soluble trophic factor-rich lysate and a vesicular formulation enriched in bioactive cargo. Because mitochondrial dysfunction and redox imbalance are hallmarks of neurodegeneration, we investigated whether these platelet-derived biomaterials could protect against rotenone-induced mitochondrial injury. HPPL and PEVs were bioprocessed from clinical-grade platelet concentrates and characterized for protein content, antioxidant capacity, vesicle morphology, size distribution, concentration and platelet and EVs markers. Differentiated N2A and SH-SY5Y neuronal cells were pretreated with 5% v/v HPPL or PEVs before rotenone (5 {micro}M) exposure, while zebrafish embryos received 20 g/mL HPPL or PEVs before rotenone (200 nM) challenge. Both biomaterials restored ATP production, reduced reactive oxygen species (ROS), preserved mitochondrial membrane potential and ultrastructure, and improved neuronal survival. They also normalized key markers of mitochondrial biogenesis and dynamics, including peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1), mitofusin-1 (MFN1), and dynamin-related protein 1 (DRP1). Proteomic analyses further showed enrichment of mitochondrial-associated antioxidant and metabolic proteins in HPPL and PEVs, and revealed restoration of oxidative phosphorylation, tricarboxylic acid cycle-related pathways, antioxidant defense, and mitochondrial dynamics in rotenone-injured cells following pretreatment. In zebrafish embryos, both biomaterials improved survival and hatching, reduced developmental abnormalities and oxidative stress, and preserved mitochondrial ultrastructure. These findings identify HPPL and PEVs as platelet-derived biomaterials with complementary mitochondrial protective activity, supporting their development as scalable cell-free biotherapeutic platforms for neurodegenerative disorders.

bioengineering↗

bioRxiv2025

Structures of Nucleotide-Bound Redondovirus Rep Protein Link Conformation and Function

Circular Rep-encoding single-stranded DNA (CRESS-DNA) virus Rep proteins are multidomain enzymes that mediate viral DNA rolling-circle replication. Reps nick viral DNA to expose a 3 end for polymerase extension, provide an NTP-dependent helicase activity for DNA unwinding, and join nicked ends to form circular viral genomes. Here, we present the first structures of a Rep protein from the Redondoviridae family, a newly discovered family of human-associated CRESS-DNA viruses that replicates within the oral protozoan Entamoeba gingivalis. Using cryo-EM, we characterized the hexameric structures of a Redondovirus Rep helicase bound with ATP{gamma}S, representing the initial ATP-bound state, and with ADP, reflecting the protein state after hydrolysis. The ADP state, but not the ATP state of Rep shows a staircase arrangement of DNA-binding loops that plays a central role in current models for SF3 helicase function. Additionally, we determined a head- to-tail dodecameric structure of ATP{gamma}S-bound Rep, in which both the helicase and endonuclease domains are ordered. Conservation of residues involved in stabilizing the dodecamer suggest that this assembly may be functionally relevant for many CRESS-DNA viruses. The positioning of endonuclease domains in the Rep hexamer, combined with our biophysical analyses of Rep oligomerization, provide new insights into Rep function during viral replication.

microbiology↗

bioRxiv2025

LASSO: versatile and selective biomolecule pulldown with combinatorial DNA-crosslinked polymers

Current methods for sequence-selective biomolecule isolation suffer from high cost, off-target effects, and limited flexibility. Here, we introduce LASSO (crossLink-Assisted Sequence-Selective isOlation), a versatile platform using programmable polymer phase separation to capture biomolecules under native conditions. LASSO relies on combinatorial crosslinker libraries--diverse mixtures of DNA strands that collectively trigger the formation of highly swollen polymer agglomerates with near-zero background binding. We demonstrate >80% pulldown efficiency for diverse targets, including DNA, SARS-CoV-2 RNA, and human thrombin. LASSO provides 8-20x higher binding capacity (4 nmol/mg polymer) than commercial microbeads. In RNA-seq workflows, LASSO depleted ribosomal RNA with 86% efficiency while yielding up to 7x fewer off-target outliers (p<0.001) versus state-of-the-art magnetic beads (riboPOOLs) and RNase H (NEBNext) kits. Thrombin was captured via switchable aptamers with 92% efficiency, and a gentle release mechanism allowed the subsequent isolation of 72% enzymatically active proteins from the polymer. LASSOs cost-effectiveness ($0.96/sample vs. $46-$51 for commercial kits), long-term stability (7+ years), simple usage, and modularity position it to transform diagnostics, transcriptomics, and bionanotechnology workflows.

bioengineering↗

bioRxiv2025

MCWs (MiCroWire sorter): A new framework for automated and reliable spike sorting in human intracerebral recordings.

Efficient and accurate spike sorting is critical for isolating single neurons from extracellular recordings to distinguish neural activity of interest. However, while the electrodes and acquisition systems for non-human electrophysiology have been enhanced over the past decades to enable higher-yield single-neuron detections, those advances have not been translated into human electrophysiology. Single-wire electrodes are still ubiquitously used, and although acquisition systems have augmented their signal-to-noise ratio over the last 15 years, we are still limited by their low electrode count. Moreover, unlike non-human recordings, human recordings often take place in hospitals where different noise sources and subject breaks can compromise the recording quality during experimental sessions. To bridge this gap, this work presents an automatic, open-source spike sorting pipeline that leverages contemporary computational capabilities and is tailored to single-neuron recordings from humans acquired via microwires. The pipeline is implemented in both MATLAB and Python, ensuring accessibility and compatibility across computational environments. Its modular and comprehensive structure supports customization and even opportunities for new developments as per the requirements of the user and the application. One feature is a data-driven automatic module to remove narrow-band interference, besides electrical line noise, which can be an essential tool while recording in clinical settings, particularly for online processing implementations. Following spike detection, the pipeline implements an artifact rejection module that separates waveforms that are unlikely to be associated with actual spikes. Additionally, we introduce a configurable feature-extraction, clustering, and benchmarking framework that not only allows flexibility in employing user-defined or conventional algorithms, such as wavelet transform with superparamagnetic clustering, but can also evaluate multi-method agreement among the different sorters. The pipeline also utilizes established and novel quality metrics to support semiautomatic curation of isolated clusters. Furthermore, we can integrate the customized pipeline with experimental tasks by removing task-unrelated waveforms (e.g., during a break in a task) and prevent over-clustering with the aid of metrics for comparing response profiles. Thus, the presented pipeline addresses the three-pronged objectives of algorithm-adaptability, rigorous validation, and human single-neuron recording optimization to support clinical and cognitive neuroscience applications.

neuroscience↗

bioRxiv2025

An online dynamic experimental framework to study single neuron responses in humans

Single-neuron activity can be recorded from patients being evaluated for neurosurgical treatment of drug-resistant epilepsy who volunteer to have microwires implanted for single cell research that aims to study various aspects of the human brain. These recordings provide an unprecedented window into the human brain to observe how information is encoded and retrieved at the cellular level. Current single cell research paradigms require laborious offline analysis to identify stimuli eliciting neuronal responses and introduce new relevant stimuli in a follow up task. Any delays due to patient availability can compromise the follow up task, risking loss of experimental progress as recording electrodes may move away from previously recorded neurons. This paper introduces a novel experimental framework for online single cell data analysis and dynamic sampling of the stimulus space. It allows for real-time data acquisition and analysis to identify response-eliciting stimuli, introduce new relevant stimuli, and discard those that fail to elicit responses.

neuroscience↗

bioRxiv2025

Molecular mechanisms of 10-Butyl Ether Minocycline (BEM), a novel non-antibiotic tetracycline, as a potential treatment for inflammatory and neuroimmune-related disorders.

The pleiotropy of minocycline (MINO), including anti-inflammatory, antioxidant, anti-migratory, anti-MMP, and neuroprotective effects, has been extensively reported. A novel non-antibiotic minocycline derivative, 10-butyl ether minocycline (BEM), was synthesized to retain the pleiotropy of minocycline while minimizing side effects such as antibiotic resistance and gut dysbiosis. Previously, we showed that BEM reduced alcohol consumption in dependent murine and porcine models of Alcohol Use Disorder (AUD). In this study, we investigated the molecular mechanisms of BEM to determine its potential as a therapeutic agent for neuroimmune and inflammatory conditions such as AUD. Here, we report that BEM showed a nearly complete loss of antimicrobial activity against E. coli, S. typhi, and C. albicans. BEM showed a dose-dependent reduction in cell viability as measured by the MTT assay, similarly to MINO. BEM also suppressed LPS-induced microglial activation as shown by reduced Iba1 expression in immunohistochemistry and western blot analyses. Inhibition of MMP-9 by BEM (IC50 = 42.2 {micro}M) was improved compared to MINO (IC50 = 60.3 {micro}M) while MMP-8 inhibition was moderate (IC50: BEM = 69.4 {micro}M; MINO = 45.4 {micro}M). BEM was found to be effective in inhibiting VEGF-induced endothelial cell migration and L-glutamine-induced ROS levels. Limited inhibition of 15-LOX activity was observed (IC50: BEM = 92.6 {micro}M; MINO = 65.6 {micro}M). BEM was not toxic to mitochondria, even at high concentrations (200 {micro}M). By eliminating antimicrobial properties while preserving therapeutic pleiotropy, BEM presents an advancement in the development of a promising candidate with multimodal mechanisms to treat neuroimmune-inflammatory pathologies. ImpactOur current approach to correlate non-antibacterial actions of BEM to MINOs established mechanistic effects will enable the informed use of BEM for several medical indications including for inflammation and neuroimmune conditions. The focus on BEMs multimodal actions and long-term safety during drug discovery represents a paradigm shift toward complex therapeutic drug development and repositioning, improving upon traditional singular high-affinity target-based approaches. Such new drug discovery attempts could potentially enhance treatment relevance in complex disorders with multiple targets and theoretically guide the creation of second-generation analogs. Significance statementWe report mechanisms of action for BEM, a minocycline analog under evaluation for the treatment of Alcohol Use Disorder, which may also show efficacy for other complex disease processes that involve inflammatory or neuroimmune components. We show that BEM had a nearly complete loss of antimicrobial action, yet retained the pleiotropy of MINO, likely making it a better multimodal therapeutic for long-term treatment of complex diseases with neuroimmune-related components. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/661183v3_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1ade11corg.highwire.dtl.DTLVardef@961d1borg.highwire.dtl.DTLVardef@15101f8org.highwire.dtl.DTLVardef@158810e_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Orthogonal resistance mechanisms of classical- and induced-proximity inhibitors

Resistance development is an inevitable failure mode of many drugs, pointing to the need to develop agents with orthogonal resistance mechanisms. Induced-proximity modalities, an emergent class of therapeutics, operate by forming a ternary complex with the protein-of-interest (POI) and effectors, unlike classical inhibitors that form binary complexes with the POI. Using KRAS as a model system, we employed base editor tiling mutagenesis screening to show that induced-proximity inhibitors exhibit orthogonal resistance mechanisms to classical inhibitors despite overlapping binding sites, offering an opportunity to circumvent resistance mechanisms of classical inhibitors. These findings highlight the use of base editor mutagenesis screens to prioritize inhibitors with orthogonal resistance mechanisms and the potential of induced-proximity inhibitors to overcome the drug resistance of classical inhibitors.

biochemistry↗

bioRxiv2025

Microbes regulate glomerular filtration rate in health and chronic kidney disease in mice

Microbes are implicated in a variety of host physiological and pathophysiological processes. In this study, we tested the hypothesis that microbes modulate glomerular filtration rate (GFR). Microbiota were depleted in mice using oral antibiotics (ABX; a mixture of ampicillin, neomycin, and vancomycin). GFR was significantly increased in ABX-treated mice. To confirm that the increase in GFR was due to decreased microbes, we also measured GFR in germ-free (GF) mice. GFR was increased in GF mice as compared to both conventional and conventionalized GF (CGF) mice. We next used the murine adenine diet model to ask if suppressing gut microbes with ABX also increases GFR in a setting of chronic kidney disease (CKD), where GFR is impaired. In females on an adenine diet, ABX increased GFR versus adenine alone on weeks 4 and 6. In males, ABX elevated GFR on week 2. Adenine diet significantly increased plasma creatinine and kidney fibrosis; this was suppressed by ABX in both sexes. To explore the mechanism of this increase, we tested the hypothesis that altered tubuloglomerular feedback (TGF) contributes to elevated GFR using the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA); EMPA impairs Na+ reabsorption in the proximal tubule, altering TGF. We found that EMPA impaired ABX-induced GFR increases on week 3 but not week 5, suggesting that altered TGF contributes to the initial increase in GFR. In conclusion, the microbiome plays a key role in setting baseline GFR by a mechanism which partially involves TGF, and, suppressing gut microbes can elevate GFR even in CKD. Translational StatementThis study reports that GFR is elevated when gut microbes are absent or suppressed in mice, indicating a role for commensal microbes to help establish baseline GFR in health. Likewise, suppressing gut microbes also elevates GFR in a chronic kidney disease model. These data suggest a future possibility of modulating the commensal microbes to elevate GFR in a clinical setting.

physiology↗

bioRxiv2025

Kinetic organization of the genome revealed by ultra-resolution, multiscale live imaging

In the last decade, sequencing methods like Hi-C have made it clear the genome is intricately folded, and that this organization contributes significantly to the control of gene expression and thence cell fate and behavior. Single-cell DNA tracing microscopy and polymer physics-based simulations of genome folding have proposed these population-scale patterns arise from motor- driven, heterogeneous movement, rather than stable 3D genomic architecture, implying that motion, rather than structure, is key to understanding genome function. However, tools to directly observe this motion in vivo have been limited in coverage and resolution. Here we describe TRansposon Assisted Chromatin Kinetic Imaging Technology (TRACK-IT), which combines a suite of imaging and labeling improvements to achieve ultra-resolution in space and time, with self-mapping transposons to distribute labels across the chromosome, uncovering dynamic behaviors across four orders of magnitude of genomic separation. We find that sequences separated by sub-megabase distances, typically 200-500 nm of nanometers apart, can transition to close proximity in tens of seconds - faster than previously hypothesized. This rapid motion is dependent upon cohesin and is exhibited only within certain genomic domains. Domain borders act as kinetic impediments to this search process, substantially slowing the rate and frequency of the transition to proximity. The genomic separation-dependent scaling of the search time for cis-interactions within a domain violates predictions of diffusion, suggesting motor driven folding. This distinctive scaling is lost following cohesin depletion, replaced with a behavior consistent with diffusion. Finally, we found cohesin containing cells exhibited rare, processive movements, not seen in cohesin depleted cells. These processive trajectories exhibit extrusion rates of [~]2.7 kb/s across three distinct genomic intervals, faster than recent in vitro measurements and prior estimates from in vivo data. Taken together, these results reveal a genome in motion across multiple genomic and temporal scales, where motor-dependent extrusion divides the sequence, not into spatially separate domains, but into kinetically separated domains that experience accelerated local search.

biophysics↗

bioRxiv2025

Solution conformational differences between conventional and CENP-A nucleosomes are accentuated by reversible deformation under high pressure

Solution-based interrogation of the physical nature of nucleosomes has its roots in X-ray and neutron scattering experiments, including those that provided the initial observation that DNA wraps around core histones. In this study, we performed a comprehensive small-angle scattering study to compare canonical nucleosomes with variant centromeric nucleosomes harboring the histone variant, CENP-A. We used nucleosome core particles (NCPs) assembled on an artificial positioning sequence (Widom 601) and compared these to those assembled on a natural -satellite DNA cloned from human centromeres. We establish the native solution properties of octameric H3 and CENP-A NCPs using analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and contrast variation small-angle neutron scattering (CV-SANS). Using high-pressure SAXS (HP-SAXS), we discovered that both histone identity and DNA sequence have an impact on the stability of octameric nucleosomes in solution under high pressure (300 MPa), with evidence of reversible unwrapping in these experimental conditions. Both canonical nucleosomes harboring conventional histone H3 and their centromeric counterparts harboring CENP-A have a substantial increase in their radius of gyration, but this increase is much less prominent for centromeric nucleosomes. More broadly for chromosome-related research, we note that as HP-SAXS methodologies expand in their utility, we anticipate this will provide a powerful solution-based approach to study nucleosomes and higher-order chromatin complexes.

biophysics↗

bioRxiv2024

Nectin-4 PET For Optimizing Enfortumab Vedotin Dose-Response In Urothelial Carcinoma

The optimization of dosing strategies is critical for maximizing efficacy and minimizing toxicity in drug development, particularly for drugs with narrow therapeutic windows such as antibody-drug conjugates (ADCs). This study demonstrates the utility of Nectin-4-targeted positron emission tomography (PET) imaging using [68Ga]AJ647 as a non-invasive tool for real-time assessment of target engagement in enfortumab vedotin (EV) therapy for urothelial carcinoma (UC). By leveraging the specificity of [68Ga]AJ647 for Nectin-4, we quantified dynamic changes in target engagement across preclinical models and established its correlation with therapeutic outcomes. PET imaging revealed dose-dependent variations in Nectin-4 engagement, with suboptimal EV doses resulting in incomplete Nectin-4 engagement and reduced tumor growth. Importantly, target engagement measured by PET emerged as a more reliable predictor of therapeutic efficacy than dose or baseline Nectin-4 expression alone. Receiver operating characteristic (ROC) analysis identified a target engagement threshold that is determinant of response, providing a quantitative benchmark for dose optimization. Furthermore, PET imaging measures provide a promising framework to account for key challenges in ADC development, including tumor heterogeneity, declining drug-to-antibody ratios over time, and limitations of systemic pharmacokinetic measurements to account for tumor-drug interactions. These findings underscore the transformative potential of integrating PET pharmacodynamic measures as early biomarkers to refine dosing strategies, improve patient outcomes, and accelerate the clinical translation of next-generation targeted therapeutics.

cancer biology↗

bioRxiv2024

Solution biophysics identifies lipid nanoparticle non-sphericity, polydispersity, and dependence on internal ordering for efficacious mRNA delivery

Designing lipid nanoparticle (LNP) delivery systems with specific targeting, potency and minimal side effects is crucial for their clinical use. However, traditional characterization methods, such as dynamic light scattering, cannot accurately quantify physicochemical properties of LNPs and how they are influenced by the lipid composition and mixing method. Here we structurally characterize polydisperse LNP formulations by applying emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation, field-flow fractionation followed by multi-angle light scattering, and size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering. We show that LNPs have intrinsic polydispersity in size, RNA loading, and shape, which depends on both the formulation technique and lipid composition. Lastly, we predict LNP transfection in vitro and in vivo by examining the relationship between mRNA translation and physicochemical characteristics. Solution-based biophysical methods will be essential for determining LNP structure-function relationships, facilitating the creation of new design rules for LNPs.

bioengineering↗

bioRxiv2024

mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

In metazoans, epithelia perform functions of absorption, diffusion, and secretion. The actin-based apical projections on the epithelial cells, contribute to these functions and are formed via cell-autonomous mechanisms that control cell polarity, intracellular transport, and the cytoskeleton. However, the cues that function upstream of these cell-autonomous regulators remain poorly known. Using microridges on zebrafish epithelial cells as a paradigm, we show that mTOR, a metabolic sensor, regulates the formation of apical projections. Mechanistically, mTORC1 controls the RhoA-ROCK activity via S6K1 to prevent the overactivation of Non-muscle Myosin II (NMII) to restrict microridge elongation. Furthermore, genetic, biochemical, and molecular dynamics simulation analyses reveal that mTORC2 regulates microridge pattern by modulating the activity of aPKC via its differential phosphorylation at two conserved sites. We propose that mTOR integrates the developmental and/or metabolic status of epithelial cells with cell autonomously acting RhoA and aPKC to regulate tissue wide formation of apical projections.

cell biology↗

bioRxiv2024

Tetanus Toxoid Utilizes Dual-Faceted Anticancer Mechanism Through Targeting Tumoral Sialic Acids and Enhancing Cytotoxic CD4+ T cell Responses Against Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) remains profoundly resistant to conventional chemotherapy and immunotherapeutic interventions. Innovative therapeutic modalities, particularly microbe-derived immunotherapies, have demonstrated durable anti-tumor efficacy in preclinical PDAC models. This study elucidates that administration of the FDA-approved Haemophilus influenzae type b (H Flu - Hiberix) vaccine attenuates tumor progression and enhances survival outcomes in murine PDAC. H Flu treatment significantly augmented CD4+ T cell, CD8+ T cell, and natural killer (NK) cell infiltration within the tumor microenvironment, concurrently inducing a cytotoxic T cell phenotype, evidenced by upregulation of CD69, granzyme B, and perforin. Additionally, H Flu therapy promoted the accumulation of CD44+ CD62L-memory T cells within tumors of pre-immunized mice. Mechanistic investigations revealed that depletion of CD4+ T cells or NK cells, but not CD8+ T cells, negated the anti-tumor efficacy of H Flu, suggesting that CD4+ T cells and NK cells are critical mediators of H Flu-induced anti-tumor immunity. To further elucidate the mechanistic basis of H Flus anti-tumor activity, we assessed the individual constituents of the H Flu vaccine: tetanus toxoid (TT) and polyrobosyl ribitol phosphate (PRP). Notably, TT administration achieved superior tumor growth suppression, characterized by enhanced CD4+ T cell cytotoxicity and increased NK cell infiltration, relative to PRP or PBS-treated controls. Furthermore, TT induced apoptosis in PDAC cells and reduced their proliferation, potentially by targeting tumor-associated sialic acids. This disruption might interfere with the interaction between sialic acids and siglec receptors, thereby impairing mechanisms of immune evasion.TT-mediated modulation of sialic acid expression in cancer cells underscores its potential to augment immunotherapeutic efficacy in PDAC. Collectively, these findings reveal a novel anti-cancer mechanism for TT, leveraging both immunostimulatory and sialic acid-targeting pathways to suppress PDAC progression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/624337v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@14e7726org.highwire.dtl.DTLVardef@708042org.highwire.dtl.DTLVardef@1f48a00org.highwire.dtl.DTLVardef@190383d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Multi-stage-mixing to create a core-then-shell structure improves DNA-loaded lipid nanoparticles transfection by orders of magnitude

As they became the dominant gene therapy platform, lipid nanoparticles (LNPs) experienced nearly all their innovation in varying the structure of individual molecules in LNPs. This ignored control of the spatial arrangement of molecules, which is suboptimal because supramolecular structure determines function in biology. To control LNPs supramolecular structure, we introduce multi-stage-mixing (MSM) to successively add different molecules to LNPs. We first utilize MSM to create a core-then-shell (CTS) synthesis. CTS-LNPs display a clear core-shell structure, vastly lower frequency of LNPs containing no detectable mRNA, and improved mRNA-LNP expression. With DNA-loaded LNPs, which for decades lagged behind mRNA-LNPs due to low expression, CTS improved DNA-LNPs protein expression by 2-3 orders of magnitude, bringing it within range of mRNA-LNPs. These results show that supramolecular arrangement is critical to LNP performance and can be controlled by mixing methodology. Further, MSM/CTS have finally made DNA-LNPs into a practical platform for long-term gene expression.

bioengineering↗

bioRxiv2024

Restoring the Multiple Sclerosis Associated Imbalance of Gut Indole Metabolites Promotes Remyelination and Suppresses Neuroinflammation

In multiple sclerosis (MS) the circulating metabolome is dysregulated, with indole lactate (ILA) being one of the most significantly reduced metabolites. We demonstrate that oral supplementation of ILA impacts key MS disease processes in two preclinical models. ILA reduces neuroinflammation by dampening immune cell activation as well as infiltration; and promotes remyelination and in vitro oligodendrocyte differentiation through the aryl hydrocarbon receptor (AhR). Supplementation of ILA, a reductive indole metabolite, restores the gut microbiomes oxidative/reductive metabolic balance by lowering circulating indole acetate (IAA), an oxidative indole metabolite, that blocks remyelination and oligodendrocyte maturation. The ILA-induced reduction in circulating IAA is linked to changes in IAA-producing gut microbiota taxa and pathways that are also dysregulated in MS. Notably, a lower ILA:IAA ratio correlates with worse MS outcomes. Overall, these findings identify ILA as a potential anti-inflammatory remyelinating agent and provide insights into the role of gut dysbiosis-related metabolic alterations in MS progression. One Sentence SummaryIndole lactate, a postbiotic metabolite reduced in MS, corrects gut microbiome metabolic imbalances associated with remyelination and neuroinflammation.

neuroscience↗

bioRxiv2024

Early zygotic engineering promotes targeted large transgene integration and direct production of fully transgenic animals

Genetic engineering has become increasingly feasible with the advent of site-specific nucleases. This technology is highly effective for the generation of small mutations, however targeted insertion of large sequences to generate transgenic animals remains challenging, time consuming, and laborious. Following several failed attempts using the same reagents, we identified a protocol that achieved very high targeted integration of a 3.2 kb transgene into a single locus of a humanized mouse model of Down syndrome. Moreover, we demonstrate in multiple ways that this procedure directly generates numerous non-mosaic founder animals, which bred true to generate all transgenic progeny. In vitro fertilization of oocytes was followed by AAV-mediated delivery of donor sequence and electroporation of Cas9 ribonucleoprotein, all parameters designed to promote early zygotic engineering (EZE). This strategy can obviate the need to breed mosaic animals for germline transmission and can enable engineering of difficult to breed animals. While efforts to improve genome engineering focus on reagents and delivery techniques, findings here suggest a narrow window of time in the early fertilized oocyte, likely before genome replication, is key to achieve both high integration efficiency and one-step generation of non-mosaic, engineered mice.

developmental biology↗