Biology subjects

Garcia, M. A.

Publications and source records attributed to Garcia, M. A..

18 recordsLinked to original sources

bioRxiv2026

High-Throughput Morphological Profiling of Patient-Derived Human Liver Organoids for Hepatotoxicity Hazard Identification and Compound Prioritization

Drug-induced liver injury (DILI) remains a major challenge in drug development because existing in vitro models poorly predict clinical hepatotoxicity. We coupled patient-derived human liver organoids (HLOs) with high-content imaging and automated machine learning (ML) to detect hepatotoxicity-associated phenotypes by morphological profiling. HLOs from six DILI patients and one healthy donor (n = 7) retained donor-specific baseline phenotypes in hepatocyte maturity, metabolism, and morphology. ML models trained per line on MAD-robustized, well-level-aggregated median features achieved compound-level AUROCs of 0.88 (95% CI 0.86-0.90) to 0.91 (95% CI 0.89-0.93) and balanced accuracies of 0.81-0.84 in distinguishing 54 hepatotoxicants from 20 non-hepatotoxic controls. Averaging predictions across all seven lines at an optimized threshold gave 94.4% sensitivity (95% CI 84.6-98.8%) and 85.0% specificity (95% CI 62.1-96.8%), substantially outperforming HLO cytotoxicity assays and cancer cell line profiling. Lines were moderately-to-highly concordant (Pearson r = 0.66-0.85), and benchmark concentration modeling revealed reproducible inter-line differences in phenotypic sensitivity (median 4.0-fold, several compounds exceeding 20-fold). Exposure anchoring of benchmark concentrations to human-equivalent plasma concentrations, using in vitro mass-balance dosimetry and curated plasma free fractions, placed organoid points of departure within 100-fold of clinical Cmax for 60 of 66 compounds (23-fold residual offset, 95% CI 12-45), and placed two of the three misclassified negative controls an order of magnitude above clinical Cmax. Sulindac injury signatures were significantly enhanced by TNF co-treatment, demonstrating capacity to detect pro-inflammatory-mediated DILI. This platform preserves inter-line variability while enabling reproducible, scalable multi-line consensus hazard classification; because compounds were tested over a common nominal concentration range, the readout defines hepatocellular hazard, which must be combined with exposure as an independent input to estimate clinical DILI risk.

pharmacology and toxicology↗

bioRxiv2026

Micro- and nanoplastics alter electrophysiological brain patterns and reshape human neurodevelopmental trajectories

Environmental microplastics and nanoplastics (MNPs) are emerging contaminants that accumulate in human tissues, including the brain, yet their mechanistic impact on the nervous system remains poorly understood. Converging postmortem evidence indicates increased MNP burden in brains from individuals with dementia, suggesting a potential link between plastic accumulation and disorders of the nervous system. However, whether MNPs directly contribute to brain aging and ensuing neurodegenerative processes, or disrupt neurodevelopment, is unknown. Here, we show that exposure to environmentally relevant MNPs induces cellular senescence and innate immune transcriptional programs in human brain organoids. Using region-specific cortical organoids, we demonstrate that synthetic polystyrene (PS), polyethylene terephthalate (PET), low-density polyethylene (LDPE), and high-density polyethylene (HDPE), as well as environmental ocean-derived MNPs (eMNPs) isolated from the coast of Hawaii, elicit a robust neuroinflammatory response. Transcriptomic analyses revealed activation of senescence-associated secretory phenotype (SASP) pathways. MNP exposure further disrupted neurodevelopmental trajectories, impairing neuroectodermal differentiation and redirecting lineage commitment toward mesoderm-like states, with concomitant enrichment of choroid plexus-like populations and a marked imbalance in neuronal and glial differentiation. These developmental alterations occurred in parallel with a pro-inflammatory and senescent microenvironment, suggesting coordinated disruption of developmental and aging programs. Importantly, high-density multielectrode array recordings revealed that MNP exposure altered electrophysiological activity and neuronal network dynamics, demonstrating that MNP-driven perturbations translate into dysfunctional neurological outcomes. Collectively, our findings identify MNPs as potent disruptors of human neurophysiology, linking plastic exposure to altered developmental trajectories, cellular senescence, neuroinflammation, and impaired neuronal network activity, providing mechanistic insights into how plastic accumulation may contribute to impaired brain dynamics.

neuroscience↗

bioRxiv2026

Actin disassembly triggers CNS myelin compaction and wrapping

Compact myelin enables rapid and precise impulse conduction in the vertebrate nervous system. During CNS development, oligodendrocytes wrap spirally around axons while compacting membranes, yet how cytoskeletal remodeling is coupled to these events remains unclear. Actin disassembly is required for wrapping, but whether wrapping is driven by iterative actin-based protrusion or follows a transition to actin-independent mechanisms is unresolved. Here we integrate tract-resolved developmental profiling, live-cell compaction mapping, and in vivo genetic perturbation to define how actin disassembly is coupled to myelin wrapping. We find that actin filaments undergo pronounced, sustained disassembly before active wrapping begins, with little evidence for persistent actin within sheaths during wrapping. We develop a live-cell assay that maps membrane compaction in cultured oligodendrocytes and show that compaction zones are depleted of actin filaments and expand when actin disassembly is promoted. Consistent with this model, oligodendrocyte-specific actin disassembly in vivo accelerates the appearance of thicker myelin early in development. Together, our results support a model in which actin disassembly promotes myelin wrapping by enabling membrane compaction, and provide a platform to dissect how compaction is regulated in development and disease.

neuroscience↗

bioRxiv2025

Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir

HIV-1 cure requires preventing viral rebound after treatment interruption, but quantitative criteria defining the rebound-competent reservoir are lacking. We studied individuals undergoing observational treatment interruption without confounding interventions to identify virologic and immunologic determinants of rebound. In 9 of 13 participants, rebound viruses were genetically identical or similar to proviruses in circulating resting CD4+ T-cells. We found no evidence of recombination among rebound sequences. Instead, resistance to autologous neutralizing antibodies (aNAbs) was a critical determinant of viral rebound. Increased suppression of viral outgrowth by contemporaneous IgG isolated from plasma was correlated with longer time to rebound. Using inhibitory potential (IP), the log reduction in single-round infection at physiologic IgG concentrations, we defined quantitative limits governing rebound-competency with respect to contemporaneous aNAbs. Contemporaneous IgG antibodies inhibited different reservoir variants with a wide range of IP values (0.4-8.2 logs), whereas rebound viruses were minimally inhibited (0.5-2.8 logs), indicating that inhibition by even up to 2.8 logs (631-fold) cannot prevent rebound. Longitudinal analyses revealed that waning aNAb potency over time on ART allows previously neutralized variants to gain rebound potential, consistent with the finding that rebound can come from variants deposited in the reservoir at different pre-ART time points. Thus, rebound competency is a dynamic, immune-governed property defined by quantitative immunologic constraints, including those exerted by aNAbs. SIGNIFICANCE STATEMENTPreventing viral rebound after treatment interruption is the goal of HIV-1 cure research, but the latent proviruses responsible remain undefined. Although rebound is initiated in lymphoid tissues, we found rebound viruses are genetically similar to proviruses in circulating resting CD4+ T-cells. Rebound is not explained by recombination and is not solely from proviruses seeded at treatment initiation. Instead, rebound potential is governed by autologous neutralizing antibodies (aNAbs). We define a quantitative threshold of aNAb-mediated inhibition identifying reservoir variants with rebound potential. During treatment, waning aNAb levels allow previously neutralized variants to become rebound-competent. Thus rebound-competency is not a static property, but a dynamic immune-governed feature. Durable aNAb responses against all rebound-competent reservoir variants may be required for functional HIV-1 cure.

immunology↗

bioRxiv2025

Deposition of microplastics associated with bioaccumulation of heavy metals in human lungs of smokers: Implications of adsorption and mobilization of metals via microplastics

BackgroundAirborne micro- and nano plastics (MNPs), generated from synthetic textiles, vehicle tires and plastic wastes, are omnipresent in the environment and known to deposit in human lungs. Bioaccumulation of MNPs in lung tissues has been studied, but effect of tobacco smoking on deposition is not well understood. Tobacco smoke is known to contain heavy metals which can get adsorbed to the MNP surface, leading to increased environmental bioavailability. We hypothesized that smoking leads to enhanced chelation of heavy metals with airborne MNPs leading to accentuated lung bioaccumulation, thereby leading to lung pathologies. MethodsAge (20-75 years) and sex-matched archival lung tissue samples from smokers (n = 9) and non-smokers (n = 10) were procured and quantified to assess: (a) the levels of MNPs by pyro-GC/MS and (b) heavy metals by ICP-MS. Furthermore, to establish if there exists a correlation between MNP deposition and disease occurrence in current smokers, tissue samples from age-matched patients with COPD (n = 4) and IPF (n = 4) underwent pyro-GC/MS analyses. ResultsA relatively higher level of MNPs were found in the lungs of smokers (43.67 mcg/gm) as compared to non-smoker (34.53 mcg/gm) upon pyro-GC/MS analyses. We observed a marked increase in the levels of polyethylene (PE; p< 0.0076), polycarbonate (PC; p< 0.0947) and nylon6 (N6; p < 0.0653) polymers in the lungs of smokers as compared to non-smokers. Metal analyses showed a significant (p <0.0001) increase in the levels of cadmium (Cd) in the lungs of smokers with the levels of deposited Cd being more pronounced for female smokers. Importantly, we observed a strong correlation (p < 0.05) between the amounts of accumulated PE and levels of Cd in the lungs. Pyro-GC/MS analyses did not reveal any relation between MNP deposition and disease occurrence in our study. Furthermore, little to no change in the level of deposited MNPs was observed in the lungs of current versus ex-smokers in disease cohorts. ConclusionsOverall, our results show that smoking promotes bioaccumulation of certain MNPs (PE) with heavy metals (Cd) which may lead to adverse lung effects in smokers.

pharmacology and toxicology↗

bioRxiv2025

Metabolomic, Lipidomic, and Enterohormone Changes in the Progression from MASLD to MASH

Background & AimsMetabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) and Metabolic Dysfunction-Associated Steatohepatitis (MASH) represent progressive stages of liver disease, with distinct metabolic and cellular alterations. This study investigates the progression from MASLD to MASH through metabolomics, lipidomics, and assessment of hormones. MethodsMale C57BL/6NTac mice were fed a high-fat diet for 16 weeks to induce MASLD and for 29 weeks to develop MASH. Aged-matched controls on a normal diet were used for comparison. Histology confirmed the progression of MASLD to MASH. We performed metabolomic and lipidomic profiling of liver, colon, and stool samples to identify metabolic and lipid alterations. Plasma enteroendocrine hormones and cytokines were quantified. Immunofluorescence was performed to assess enteroendocrine cells changes in the colon and the association of serotonin (5-HT) with fibronectin in the liver. ResultsMetabolomic and lipidomic analysis revealed significant alterations at different stages of the disease. Specifically, cholic acid was increased across the liver, colon, and stool in both MASLD and MASH mice compared to controls. Compared to the control group, MASLD mice exhibited an increase in enteroendocrine hormones, GLP-1, GIP, and PYY, whereas no changes were observed in MASH mice. Comparing MASLD to MASH livers, we found hepatic 5-HT levels were increased in MASH mice compared to MASLD mice. The MASH liver also exhibited a colocalization between fibronectin and 5-HT, suggesting a potential role of 5-HT in liver fibrosis. ConclusionsOur study provides novel insights into the progressive metabolic and hormonal changes from MASLD to MASH. The increase in cholic acid and differential enteroendocrine hormone responses highlight the complex interactions between the gut and liver in metabolic liver diseases. These findings suggest that enteroendocrine hormones may play a role in the progression of MASLD to MASH as well as liver fibrosis, offering potential therapeutic avenues for targeting the gut-liver axis in metabolic liver diseases.

cell biology↗

bioRxiv2025

Chronic Cadmium Exposures and Hyperglycemia Additively Drive Mitochondrial Dysfunction in Hepatic Cells: Key Implications for MASLD Etiopathogenesis

Effects of chronic heavy metal stress on hepatocellular pathophysiology remains ill-understood. Human livers are a long-term accumulative site for many toxic heavy metals (e.g., cadmium and arsenic) whose effects are unknown. In the current study, we studied effects of chronic, low-dose exposures of cadmium (CLEC) modulated by normoglycemic (5.6 mM) and hyperglycemic (15 mM) exposures, focusing on hepatocellular mitochondrial function. HepG2 and HUH7 cell lines were exposed to CLEC and glucose for 24 weeks, mimicking a chronic heavy metal exposure paradigm seen in normal and type II diabetic individuals. We observe that CLEC exposures significantly affect the long-term health of mitochondria, including decreased mitochondrial mass, increased superoxide production, and loss of mitochondrial membrane potential (MMP) in a CLEC and glucose-dependent manner. Furthermore, the Seahorse MitoStress assay revealed CLEC induced significant chronic oxidative stress. In particular, CLEC cells showed altered levels of basal and non-mitochondrial respiration, causing dysregulation in mitochondrial oxygen consumption rates (OCRs). Lastly, we identified significant impacts of CLEC and glucose exposures on the mitochondrial dynamics (fission/fusion) of the CLEC cells, which showed enhanced mitochondrial fragmentation and turnover rates. We also identified novel cell compensatory mechanisms that may mask the true extent of chronic Cd exposure induced damage in liver cells. CLEC and glucose work additively to damage hepatocellular mitochondrial function. New approach methodologies (NAMs), such as the current vitro toxicology study, establish the insidious effects of chronic heavy metal pollutant exposures on human hepatocellular function.

pharmacology and toxicology↗

bioRxiv2025

Environmental NaCl affects C. elegans development and aging

Sodium is an essential nutrient, but is toxic in excess. In humans, excessive dietary sodium can cause high blood pressure, which contributes to age-related diseases including stroke and heart disease. We used C. elegans to elucidate how sodium levels influence animal aging. Most experiments on this animal are conducted in standard culture conditions: Nematode Growth Medium (NGM) agar with a lawn of E. coli. Here, we report that the supplemental NaCl in standard NGM, 50 mM, accelerates aging and decreases lifespan. For comparison, we prepared NGM with reduced NaCl or excess NaCl. Considering reduced NaCl as a baseline, wild-type worms on standard NGM displayed normal development and fertility but reduced lifespan and health span, indicating toxicity in old animals. The long-lived mutants daf-2, age-1, and nuo-6, cultured on standard NGM, also displayed reduced lifespan. Thus, NaCl in standard NGM accelerates aging in multiple genetic backgrounds. Wild-type worms on excess NaCl displayed delayed development and reduced fertility, and reduced lifespan and health span, indicating toxicity in both young and old animals. These results suggest that young animals are relatively resistant to NaCl toxicity, but that aging causes progressive sensitivity, such that old animals display toxicity to both standard and excess NaCl. We investigated pathways that respond to NaCl. Young animals cultured with excess NaCl activated gpdh-1, a specific response to NaCl stress. Old animals cultured with excess NaCl activated gpdh-1 and hsp-6, a reporter for the mitochondrial unfolded protein response. Thus, excess NaCl activates multiple stress response pathways in older animals.

developmental biology↗

bioRxiv2024

White matter hyperintensities and microplastics

SynopsisWhite matter hyperintensities are abnormalities that appear in MRI scans of living patients but are not apparent in MRI post-mortem. GoalOur goal is to understand the cellular/biological basis of white matter hyperintensities (WMH). ApproachWe aligned post-mortem MR scans with those collected ante-mortem and performed histopathology and pyrGC/MS for plastics on regions with WMH. ResultPyrGC/MS detected large amounts of plastics and we determined their cellular locations with a novel optical imaging approach in regions with small vessel disease and Abeta plaques. ImpactMicroplastics in the brains of people with cognitive impairment may be due to pre-existing vascular injury or contribute to it. Many questions remain: Where do they come from, do they impair function? Can they be diagnosed by MRI ante-mortem?

neuroscience↗

bioRxiv2024

Cytogenetic comparison of Cuscuta psorothamnensis and C. veatchii (Convolvulaceae), two species originated from recurrent hybridization between the same diploid parents

Genus Cuscuta L. (Convolvulaceae) exhibits cases of hybridization and allopolyploidy. Section Denticulatae, subg. Grammica, includes four species: the allopolyploids, C. veatchii and C. psorothamnensis (2n = 60), which originated from two independent reticulation events between the diploids, C. denticulata and C. nevadensis (2n = 30). The allopolyploids are morphologically similar, but are differing in their geographical distribution and host specificity. While cytogenetic data have been reported for C. veatchii, this study aims to provide a comparative analysis with C. psorothamnensis. To characterize the chromosomal complement of C. psorothamnensis and compare it with C. veatchii, we used CMA/DAPI banding, FISH, and GISH. The karyotypes of both species displayed similarity in chromosome number, size, and symmetry, and interphase nucleus organization. Both species exhibited a pair of 5S and 35S rDNA sites adjacent on the same chromosome. The number of 5S rDNA sites in C. psorothamnensis is variable, with some individuals displaying four, five, and six sites. Our results show: 1) the chromosomal pair carrying adjacent 5S and 35S rDNA in C. denticulata is retained in the polyploids; 2) the loss of C. nevadensis rDNA sites occurred in both tetraploids; 3) C. psorothamnensis and C. veatchii are allopolyploids part of a species complex, originated from successive independent hybridization events between C. denticulata and C. nevadensis; and 4) C. psorothamnensis is probably more recent in origin than C. veatchii based on the degree of diploidization. This cytogenetic comparison allows us to understand the processes involved in the emergence of new polyploid species by hybridization. Main ConclusionCuscuta psorothamnensis and C. veatchii form a complex of allopolyploid species originating from independent, successive hybridization events between C. denticulata and C. nevadensis.

plant biology↗

bioRxiv2023

A High-Throughput Microphysiological Liver Chip System to Model Direct and Idiosyncratic Drug-Induced Liver Injury Using Human Liver Organoids

1.ObjectiveDrug-induced liver injury (DILI) is a major failure mode in pharmaceutical development. This study aims to address the limitations of existing preclinical models by introducing a high-throughput, microfluidic liver-on-a-chip system, termed as "Curio Barrier Liver Chips," seeded with human liver organoids to enable metabolic and phenotypic morphologic characterization. MethodsCurio Barrier liver chips, fabricated in an 8x2 well configuration, were utilized to establish 3D liver organoid cultures. Human-induced pluripotent stem cells (iPSCs) were differentiated into hepatic-like organoids (HLOs), and their viability, liver-specific functions, and pharmacological responses were assessed over 28 days. ResultsThe Curiochips successfully maintained liver physiology and function, showing strong albumin secretion and cytochrome P450 (CYP) activities through 28 days. Unlike traditional models requiring millimolar drug concentrations to detect hepatotoxicity, this platform showed increased sensitivity for APAP and FIAU at micromolar concentrations. In situ differentiation of foregut spheroids to liver organoids was also achieved, further simplifying the establishment of liver chips. Furthermore, the chips demonstrated viability, function and DILI responsiveness for 28 days making this an improved model for studying idiosyncratic DILI with prolonged drug exposure and high-throughput capabilities compared to other available systems or primary human hepatocytes. ConclusionsThe Curiochips offer an advanced, miniaturized in vitro model for early-stage drug development and a sensitive, responsive and cost-effective means to detect direct hepatotoxicity. The high-throughput capability, coupled with robust functionality and pharmacological responses make it a promising tool for improving the prediction and understanding of DILI mechanisms in general and those that required prolonged drug exposure. The model also opens new avenues for research in other chronic liver diseases.

pharmacology and toxicology↗

bioRxiv2023

Preclinical Characterization of the Omicron XBB.1.5-Adapted BNT162b2 COVID-19 Vaccine

As SARS-CoV-2 continues to evolve, increasing in its potential for greater transmissibility and immune escape, updated vaccines are needed to boost adaptive immunity to protect against COVID-19 caused by circulating strains. Here, we report features of the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine, which contains the same mRNA backbone as the original BNT162b2 vaccine, modified by the incorporation of XBB.1.5-specific sequence changes in the encoded prefusion-stabilized SARS-CoV-2 spike protein (S(P2)). Biophysical characterization of Omicron XBB.1.5 S(P2) demonstrated that it maintains a prefusion conformation that adopts a flexible and predominantly open one-RBD-up state, with high affinity binding to the human ACE-2 receptor. When administered as a 4th dose in BNT162b2-experienced mice, the monovalent Omicron XBB.1.5 vaccine elicited substantially higher serum neutralizing titers against pseudotyped viruses of Omicron XBB.1.5, XBB.1.16, XBB.1.16.1, XBB.2.3, EG.5.1 and HV.1 sublineages and the phylogenetically distant BA.2.86 lineage than the bivalent Wild Type + Omicron BA.4/5 vaccine. Similar trends were observed against Omicron XBB sublineage pseudoviruses when the vaccine was administered as a 2-dose primary series in naive mice. Strong S-specific Th1 CD4+ and IFN{gamma}+ CD8+ T cell responses were also observed. These findings, together with prior experience with variant-adapted vaccine responses in preclinical and clinical studies, suggest that the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine is anticipated to confer protective immunity against dominant SARS-CoV-2 strains. ONE-SENTENCE SUMMARYThe monovalent Omicron XBB.1.5-adapted BNT162b2 mRNA vaccine encodes a prefusion-stabilized spike immunogen that elicits more potent neutralizing antibody responses against homologous XBB.1.5 and other circulating sublineage pseudoviruses compared to the bivalent Wild Type + Omicron BA.4/5 BNT162b2 vaccine, thus demonstrating the importance of annual strain changes to the COVID-19 vaccine.

immunology↗

bioRxiv2023

A tough nutlet to crack: resolving the phylogeny of Thesium (Thesiaceae), the largest genus in Santalales

With over 350 species, Thesium is the largest genus in Santalales. It is found on all continents except Antarctica; however, its highest diversity is in the Cape Floristic Region of South Africa where approximately half the species occur. Thesium samples of ca. 590 collections from throughout its entire geographic range were obtained and nuclear ribosomal ITS sequenced from 396 accessions representing 196 named taxa and 30 currently unnamed taxa for a total of ca. 230 species. In addition, two chloroplast genome spacers (trnDT and trnLF) were sequenced from 269 and 315 accessions, respectively. Maximum parsimony, maximum likelihood and Bayesian methods were employed to generate gene trees and infer phylogenies. The value of the morphological characters traditionally used in the taxonomy of the genus and previous infrageneric classifications are discussed. Broad scale relationships were generally congruent among the ITS and the chloroplast trees. For example, both the nuclear and chloroplast trees support the presence of Eurasian and African clades. In contrast, major incongruence was detected between nuclear and chloroplast trees for a number of taxa including the recently described T. nautimontanum that is sister to the entire African clade on the ITS tree. Although the causes of this incongruence are currently unknown, a novel form of chloroplast capture is hypothesized. A hypothesis of the biogeographical history of the genus based on our molecular phylogeny is presented.

evolutionary biology↗

bioRxiv2023

Cell growth dilutes YAP/TAZ influencing its subcellular distribution and phenotypic heterogeneity

The concentration of many transcription factors exhibit high cell-to-cell variability due to differences in synthesis, degradation, and cell size. How these factors are robust to fluctuations in concentration is poorly understood. Here we quantified the single cell levels of the YAP/TAZ transcriptional co-activators in parallel with cell morphology for over 400,000 single cells across 17 cell lines. We show the whole cell concentration of YAP/TAZ sub-scales with respect to size as cells grow during proliferation. However, the mean nuclear concentration of YAP/TAZ remains constant during the cell cycle. Theoretical modelling demonstrates that the extent to which whole cell YAP/TAZ dilutes in single cells during proliferative growth dictates the variability of YAP/TAZ levels across the population. Integrative analysis of imaging and proteomic data show the average nuclear YAP/TAZ concentration is predicted by differences in RAS/MAPK signalling, focal adhesion maturation, and nuclear transport processes. We developed a statistical framework capable of discriminating between perturbations that affect YAP/TAZ directly, or via changes in morphology. Deployment of these models on genetic screening data or small-molecule treatments reveal that inhibition of MEK, CDK4/6, LATS and RhoGTPases decouple nuclear YAP/TAZ from cell morphology by regulating nuclear translocation. Thus signalling activity couples size changes to YAP/TAZ translocation; leading to a stable pool of nuclear YAP/TAZ during proliferation. Significance StatementMany proteins dilute/concentrate with changes in cell size. It is unclear how robustness in cell signalling emerges across differently sized cells, with varying intracellular protein concentrations, over generations. Here, we have shown that despite whole cell dilution of the transcriptional co activators YAP/TAZ with increasing size, a steady-state nuclear concentration distribution is maintained across the population. Thus nuclear transport promotes robustness of signal response in the face of a dwindling cytoplasmic YAP/TAZ levels. An integrative approach revealed that focal adhesions, RAS/MAPK and nuclear import contributes to the the maintenance of YAP/TAZ nuclear levels. Cells appear to have evolved systems to ensure robustness against alterations to cell size during the cell cycle.

cell biology↗

bioRxiv2022

SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination

Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)--a transcription factor known to regulate expression of actin and actin regulators in other cell types--as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the fundamental role of SRF in oligodendrocyte lineage cells. Here we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in OPCs and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies a novel pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease. HighlightsO_LIDevelopmental CNS myelination requires the transcription factor SRF in oligodendrocytes. C_LIO_LISRF targets actin and actin-regulatory but not myelin related genes. C_LIO_LISRF drives oligodendrocyte actin cytoskeleton dynamics during early stages of myelination. C_LI

neuroscience↗

bioRxiv2022

Identifying Optimal Neuroinflammation Treatment Using Nanoligomer Discovery Engine

Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leads to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and is causally linked to a range of neurodegenerative diseases such as Alzheimers diseases (AD), Multiple Sclerosis (MS), Parkinsons diseases (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier (BBB), making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our Nanoligomer discovery engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1{beta} or IL-1{beta}, tumor necrosis factor-alpha or TNF-, TNF receptor 1 or TNFR1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-{kappa}{beta}) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification via a bioinformatics and AI-based ranking method to design sequence-specific peptide molecules to up-or down-regulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of Nanoligomers targeted to NF-{kappa}{beta} (SB.201.17D.8_ NF-{kappa}{beta}1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes, and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low-dose (5mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy.

neuroscience↗

bioRxiv2021

Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

The establishment of macromolecular complexes by scaffolding proteins such as A-kinase anchoring proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for many cardiac functions. We have identified herein a novel AC scaffold, the Popeye domain-containing (POPDC) protein. Unlike other AC scaffolding proteins, POPDC1 binds cAMP with high affinity. The POPDC family of proteins are important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. TREK-1 binds the AC9:POPDC1 complex and co-purifies in a POPDC1-dependent manner with AC9-associated activity in heart. Although the interaction of AC9 and POPDC1 is cAMP independent, TREK-1 association with AC9 and POPDC1 is reduced in an isoproterenol-dependent manner, requiring an intact cAMP binding Popeye domain and AC activity within the complex. We show that deletion of Adcy9 (AC9) gives rise to bradycardia at rest and stress-induced heart rate variability. The phenotype for deletion of Adcy9 is milder than previously observed upon loss of Popdc1, but similar to loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel scaffolding protein for AC9 to regulate heart rate control. ONE-SENTENCE SUMMARYAdenylyl cyclase type 9 binds in an isoproterenol-dependent manner to the POPDC1:TREK-1 complex regulating heart rate.

biochemistry↗

bioRxiv2021

Karyotype asymmetry in Cuscuta L. subgenus Pachystigma reflects its repeat DNA composition

Cuscuta is a cytogenetically diverse genus, with karyotypes varying 18-fold in chromosome number and 89-fold in genome size. Each of its four subgenera also presents particular chromosomal features, such as bimodal karyotypes in Pachystigma. We used low coverage sequencing of the Cuscuta nitida genome (subgenus Pachystigma), as well as chromosome banding and molecular cytogenetics of three subgenus representatives, to understand the origin of bimodal karyotypes. All three species, C. nitida, C. africana (2n = 28) and C. angulata (2n = 30), showed heterochromatic bands mainly in the largest chromosome pairs. Eighteen satellite DNAs were identified in C. nitida genome, two showing similarity to mobile elements. The most abundant were present at the largest pairs, as well as the highly abundant ribosomal DNAs. The most abundant Ty1/Copia and Ty3/Gypsy elements were also highly enriched in the largest pairs, except for the Ty3/Gypsy CRM, which also labelled the pericentromeric regions of the smallest chromosomes. This accumulation of repetitive DNA in the larger pairs indicates that these sequences are largely responsible for the formation of bimodal karyotypes in the subgenus Pachystigma. The repetitive DNA fraction is directly linked to karyotype evolution in Cuscuta. HighlightsCuscuta subgenus Pachystigma contains species with strikingly bimodal karyotypes. The emergence of these karyotypes is linked to the enrichment of varied repetitive sequences in the largest chromosomal pairs.

plant biology↗