Dopamine contributes to reward-related decision making, but its contributions to decision contexts that include explicit punishment are less well understood. To elucidate the role of ventral tegmental area (VTA) dopamine neurons in decision making under risk of punishment, we used fiber photometry to record activity in these neurons during a decision-making task in which rats choose between a small, "safe" reward and a large reward associated with varying probabilities of explicit punishment. Dopamine neuron activity exhibited phasic increases during risky "Wins" (reward without punishment) and phasic decreases during risky "Losses" (reward plus punishment), each of which scaled with punishment probability and intensity. Further analyses revealed that this outcome-evoked activity predicted choices on subsequent trials. To determine whether VTA dopamine neuron activity plays a causal role in these risk-based decisions, we used optogenetics to selectively inhibit these neurons during risky Wins and Losses. Inhibition of VTA dopamine neurons during Wins selectively reduced the frequency of risky choices after a Win, whereas inhibition during Losses selectively reduced the frequency of risky choices after a Loss. These data reveal that VTA dopamine neuron activity during outcome receipt is causally related to subsequent decision-making behavior. In addition, the fact that the effects of inhibition on choice behavior were specific to the outcome with which the inhibition was paired suggests that VTA dopamine neuron activity contributes to updating after Wins and Losses independently. Dopamine is implicated in movement, motivation, and learning, and has been strongly linked with substance use and risk-seeking behavior. Despite these associations, it is unclear how the activity of dopamine neurons influences decision making under risk of punishment. To elucidate this, we recorded from ventral tegmental area (VTA) dopamine neurons and found that their activity at the population level integrates reward alongside the probability and intensity of punishment experienced during risky outcomes. Further, inhibition of VTA dopamine neuron activity during risky outcomes decreased risk-seeking behavior in an outcome-specific manner. Our findings align with models suggesting that VTA dopamine neurons signal prediction errors, and provide insight into the role of these neurons in adaptive decision making.
Primary mitochondrial diseases (PMD) have limited disease-modifying therapies, currently applicable to only 3 of over 400 discrete gene disorders. Cycloheximide (CHX) is a global cytosolic translation inhibitor we previously reported to rescue PMD preclinical models, although its toxicity precluded clinical development. To identify specific mediators underlying CHX treatment benefit in PMD, SOMAscan-based proteomics was performed in complex I deficient and genetic disease fibroblast cell line models grown in galactose. Thrombopoietin (THPO) and insulin-like growth factor binding protein 5 (IGFBP5) were the only two differentially regulated proteins, together with ERK/MAPK pathway dysregulation, identified upon CHX treatment in PMD versus healthy control cells. THPO inhibition by siRNA or pharmacologic approaches rescued stress-induced viability loss in patient fibroblasts having diverse PMD gene etiologies, and significantly improved mitochondrial stress, linear growth, and neuromuscular function in a classical ndufs2 -/- C. elegans model. IGFBP5 overexpression by lentiviral or mRNA approaches rescued cell viability across distinct PMD gene etiologies, as did IGF1 pharmacologic inhibition across both PMD mutant and C. elegans models. MAPK pharmacologic inhibition rescued multiple distinct complex I disease cells' survival, as well as mitochondrial stress in SLC25A46 -/- C. elegans . Combination therapies targeting multiple of these glucose signaling pathway proteins, together with glucose and N-acetylcysteine, yielded superior therapeutic benefit in complex I disease cell and C. elegans models. Additionally, single or combined pharmacologic inhibition of THPO or IGF1 significantly enhanced primary and metastatic osteosarcoma cell death. Collectively, targeted small molecule and genetic modulation of THPO, IGF1, or MAPK recapitulated the significant therapeutic benefit of CHX in PMD, while avoiding global translation inhibition. These novel PMD therapies likely confer benefit by attenuating MAPK-driven autophagy and potentially promoting noncanonical glucose uptake, improving cellular energy balance. Overall, these glucose signaling cellular pathway targets hold broad therapeutic promise for PMD patients, warranting further clinical research development.
Inter-subunit communication and allosteric regulation are central to the function of oligomeric enzymes, yet these features remain difficult to characterize. Conventional kinetic and structural methods typically yield ensemble averages or static snapshots, thus making it difficult to uncover the dynamic cross-subunit cooperation obligatory for multi-site catalysis by oligomeric enzymes. Here, we investigate Salmonella FraB-a homodimeric deglycase and a potential drug target-to showcase the value of an integrated approach combining native mass spectrometry (nMS), surface-induced dissociation (SID), and kinetic studies to gain insights into catalytic intermediates and inter-subunit communication. By resolving substrate-, product-, and mixed-occupancy species, nMS revealed that both inter-subunit active sites in FraB bind substrate even though only one catalytic center generates the product at any given time. To characterize each active site independently, we designed heterodimers with a mutation that changes the general base or acid in only one active site. Kinetic studies with these mutants indicate that although the two active sites are likely coupled, they do not concomitantly perform cleavage. Consistent with the conformational asymmetry observed in apo -FraB crystal structures, our findings establish a half-site reactivity mechanism in which post-binding conformational changes across the dimer interface restrict substrate cleavage to one active site even though both protomers are able to bind substrate. Importantly, this nMS-based workflow offers a broadly applicable framework for resolving the catalytic states and inter-site communication of oligomeric enzymes that are otherwise difficult to uncover by conventional structural methods.
Microbes closely interact with every living organism, including meiofauna (i.e., microbial eukaryotes 38 μm - 1 mm in length), and influence the development, life cycle, and evolution of diverse metazoans. Together, meiofauna and their microbiomes, collectively referred to as the holobiont, underpin biogeochemical cycles and drive decomposition of organic matter. However, our understanding of the ecological and evolutionary dynamics of meiofauna microbiomes are limited, typically owed to low-resolution 16S rRNA surveys, which cannot accurately delineate bacterial taxa. Single-specimen holobiont sequencing can help overcome the limitations of metabarcoding approaches by 1) generating metagenome-assembled genomes (MAGs) of the host microbiome and 2) recovering host single-copy genes (SCGs) to phylogenetically confirm the identity of the host organism. However, most bioinformatics pipelines for the assembly of metagenomic datasets have been developed for the assembly of high-complexity microbial communities of bulk sediment or soil samples (and cannot be used for the assembly of host genomes), rely on co-assembly approaches (which collapses strain-level genomic information of bacterial taxa), and focus on binning either prokaryotic or eukaryotic taxa. Therefore, there is a tremendous need for a computational workflow for the dual analysis of host genomes and their microbiomes. Here, we developed MeioBIOME, a modular Snakemake pipeline for the reproducible analysis of holobiont metagenomes obtained from individually sequenced microbial metazoa. We analyze publicly available single-specimen metagenomics datasets to show the utility of MeioBIOME and recover host-associated symbiont MAGs and host SCGs. Additionally, we integrate state-of-the-art binning algorithms which generate more MAGs than the DOE Joint Genome Institute metagenomic pipeline. We anticipate that MeioBIOME will facilitate studies of phylosymbiosis by generating high-quality host genome skims (to build well-supported host phylogenetic trees) and host-associated prokaryotic MAGs obtained from single specimens.
Hepatitis B virus (HBV) chronically infects approximately 250 million people worldwide, and reliable curative therapies are lacking. A broader understanding of viral-host interactions could accelerate efforts to find new host-centric therapeutic targets. However, inefficient cell culture systems and limited replication markers compatible with pooled screening have precluded the widespread use of genetic perturbation screens. Here, we performed the first pooled, genome-wide CRISPR-Cas9 knockout (KO) screen with authentic HBV infection and integrated these results with two orthogonal pooled screens to identify host factors. We selected 72 genes for a multi-step assessment that included arrayed validation assays using both HBV infection and pgRNA transfection. We then independently tested thirteen genes using high-efficiency bulk KO experiments to guide further investigations of both antiviral and proviral factors. In both KO and siRNA-mediated knockdown experiments, depletion of the top antiviral factor, EXOC1 , enhanced multiple HBV replication markers, and transcriptomic analysis revealed activation of hypoxia- and HIF-1 gene signatures. Three proviral factors, IRF2 , WDR48 , and ZCCHC14 , were investigated in vivo using a human liver chimeric mouse model, which demonstrated that ZCCHC14 KO greatly reduced HBV replication and spread. Together, these complementary in vitro and in vivo platforms expand the catalog of HBV host factors and provide a scalable framework for host target discovery.
Protease-activated receptor 4 (PAR4) is the only functional thrombin receptor on mouse platelets. Expression and activation of platelet PAR4 was shown to influence hemostatic plug stability and contributes to thrombosis in different murine arterial and venous thrombosis models. PAR4 activation by thrombin and other serine proteases occurs at the canonical activation site at Arginine (Arg) 59 in mice and Arg47 in humans. If murine PAR4 has functional non-canonical activation sites as shown for other PARs is unknown. To investigate canonical and potentially non-canonical PAR4 signaling in mice, we generated a mouse model expressing a functional, thrombin-cleavage resistant PAR4 by changing Arg59 to Alanine (Ala) 59 in murine PAR4 (PAR4 R59A ). PAR4 R59A mice were used to assess the impact of impaired canonical (thrombin)-dependent PAR4 signaling on hemostasis and thrombosis in mice. We analyzed platelet aggregation, platelet integrin activation and α-granule release ex vivo . Hemostasis and thrombosis in PAR4 R59A and their control mice was compared using the jugular vein needle puncture injury-induced hemostasis model, and the ferric chloride-induced carotid artery and electrolytic injury-induced femoral vein thrombosis models. Platelets of PAR4 R59A mice did not response to thrombin but responded normally to PAR4 agonist peptide (PAR4AP) stimulation in aggregation assays. Platelets of PAR4 R59A and their control mice exhibited comparable responses to ADP, convulxin or PAR4AP regarding integrin activation and α-granular release. PAR4 R59A mice exhibited impaired hemostasis in the jugular vein needle puncture model, and were protected from ferric chloride-induced arterial thrombosis and from electrolytic injury induced thrombosis in the femoral vein. The novel PAR4 R59A mouse expresses a thrombin-insensitive but still functional PAR4. We propose that the new mouse line will increase the in vivo investigation of canonical PAR4 signaling pathways and may reveal unknown non-canonical PAR4 signaling in different pathologies.
Mutations in the molecular motor protein KIF1A result in a spectrum of neurodevelopmental and neurodegenerative disorders termed KIF1A-Associated Neurological Disorder (KAND). KIF1A mutations variably disrupt synaptic vesicle trafficking, but the effects of KIF1A mutations on other trafficking pathways remain unexplored. Autophagy is a conserved pathway required for neuronal homeostasis. We investigated the role of KIF1A in autophagy using gene-edited human IPSC-derived neurons. KIF1A loss inhibited the trafficking of ATG9, a transmembrane lipid scramblase necessary for autophagosome biogenesis. This deficit significantly reduced autophagosome biogenesis and the density of axonal autophagosomes. KIF1A loss also depleted lysosomes from the axon, inhibiting autophagosome maturation. In neurons gene-edited to heterozygously express a pathogenic variant linked to a Rett-like syndrome in KAND patients, we also noted significant deficits in autophagy and lysosomal trafficking. Together, these results suggest that KIF1A-mediated transport is critical to neuronal autophagy and that deficits in autophagy may contribute to pathogenesis in KAND.
The dynamics of calcium ions (Ca 2+ ) in skeletal muscles link electrochemical activation and contractile force generation. Recent experimental data suggest that store-operated Ca 2+ entry (SOCE), the process of extracellular Ca 2+ influx upon depletion of Ca 2+ from the sarcoplasmic reticulum (SR), helps delay the onset of muscle fatigue during exercise. We hypothesize that SOCE regulates force generation during prolonged muscle activity by allowing for sustained Ca 2+ release from the SR. We test this hypothesis with a quantitative biophysical model that simulates the biochemical events of muscle contraction, from depolarization at the T-tubules to Ca 2+ release from the SR to Ca 2+ binding and force generation throughout the myoplasm. We also consider the balance between Ca 2+ removal from the myoplasm and SOCE through the T-tubule membrane, along with mitochondrial uptake of free Ca 2+ and phosphate. We use the model to test the effects of SOCE inhibition on force production. The magnitude of myoplasmic Ca 2+ and force are lower in SOCE knockout cells, especially when SOCE reduction is combined with impaired uptake of phosphate by mitochondria. We then test the effects of SOCE during resistance exercise or high-intensity interval training. These simulations predict a context-dependent relationship between force generation and SOCE - increased SOCE is associated with greater force production during resistance exercise, but worsens the effects of fatigue in certain cases of high-intensity training. Such SOCE-induced fatigue is attributed to phosphate accumulation in the myoplasm and can be mitigated by increased rates of mitochondrial phosphate uptake. Store-operated calcium entry (SOCE) provides a mechanism for calcium ion (Ca 2+ ) influx following depletion of Ca 2+ from intracellular stores such as the sarcoplasmic reticulum (SR). Recent experiments suggest that SOCE is an important modulator of contractile force generation in skeletal muscle. Here, we develop a computational model of Ca 2+ handling in the myoplasm, SR, and mitochondria and the resulting effects on force generation in skeletal muscle fibers to examine the role of SOCE during extended periods of activity. Our model predicts that increasing SOCE leads to enhanced force over periods of repeated stimuli during resistance exercise due to sustained Ca 2+ release. Our simulations show a complex relationship between SOCE and force production during high-intensity interval training, with exacerbated phosphate accumulation in the myoplasm leading to force reduction for very high levels of SOCE. This effect can be mitigated by enhanced mitochondrial phosphate uptake. Emmet Francis is a K99/R00 awardee in the Rangamani Lab at UC San Diego whose research explores the intersection between cell signaling and mechanics. His doctoral research in the Heinrich Lab at UC Davis examined the role of calcium bursts in neutrophil chemotaxis and phagocytosis. More recently, he has used spatial modeling approaches to shed light on the role of nanoscale membrane curvature and nuclear deformation in YAP/TAZ mechanotransduction. In his own research lab, he plans to use both experiments and computational models to probe the mechanisms of bidirectional mechanotransduction in neutrophils. This study uses systems modeling to demonstrate a role for SOCE in sustained force generation during exercise. SOCE leads to two competing effects on contractile force in myofibers - increased crossbridge cycling due to elevated myoplasmic Ca 2+ enhances force, whereas increased accumulation of myoplasmic phosphate (due to increased ATP hydrolysis) can lead to force reduction (fatigue). The tradeoff between these two effects is modulated by phosphate uptake into mitochondria via the phosphate carrier PiC. Figure created in BioRender.
Music, which is organized hierarchically (notes, phrases, sections), provides an ideal model for studying fine-grained motor sequence production. However, it is unknown how musicians' brains integrate tonal structure over multiple timescales during real-life performance. Here, we scrambled an unfamiliar Tchaikovsky piano suite at four timescales (every 1/2/8 measures, or fully intact) and asked expert pianists to play (sightread) all four versions in the fMRI scanner. Responses in the motor network, default mode network, and hippocampus were strongly impacted by scrambling, indicating that they integrate tonal structure over relatively long timescales. Additionally, the emergence of functionally connected sub-networks between auditory, visual, motor, and default mode network regions across scramble levels supported this hierarchical integration process. Our results cannot be explained by lower-level cues (tempo, timbre, dynamics; local pitch height or rhythmic density) and instead reflect processing of high-level tonal structure. Our study highlights novel mechanisms of complex auditory-motor action planning during live music performance.
Genome size variation in eukaryotes is driven largely by transposable elements (TEs), yet the biological mechanisms that initiate their proliferation remain understudied. Here, we identify a recurrent association between bacterial horizontal gene transfer (HGT) and bursts of TE activity that contribute to genome expansion. By leveraging comparative genomics and genus-level pangenome analyses across three species of the nut weevil, Curculio , we detect extensive bacterially derived DNA sequences embedded within structurally dynamic genomic regions. These HGT-associated regions are dominated by a small number of young, proliferating TE families, particularly DNA type II Mavericks, which encapsulate transferred bacterial sequences and comprise a substantial fraction of recent genomic DNA in derived lineages. Analyses of codon usage bias, intron length, and functional enrichment suggest that most transferred genes undergo progressive pseudogenization over evolutionary time, whereas a subset of selectively advantageous HGTs persist. Together, our findings support a model linking foreign DNA invasion with TE proliferation, genome size variation, and molecular innovation.
In humans, 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) induces chloracne, a skin condition that presents with acanthosis, hyperkeratosis, comedones, and sebaceous gland (SG) atrophy (seboatrophy). Although chloracne-like phenotypes have been reported in TCDD-treated mice, the underlying mechanisms remain poorly understood. Previous studies showed that TCDD-induced CYP1A1 protein is expressed in LRIG1+ progenitor cells in hair follicles, suggesting that TCDD targets specific cell populations within the pilosebaceous unit. To explore the effects of TCDD on the epidermis and pilosebaceous unit, we analyzed single-cell RNA expression in wild-type and Ahr -null mice at postnatal day 21 (P21) following in utero and lactational exposure. The results showed that TCDD preferentially induced the AHR target genes Cyp1a1 and Cyp1b1 in the lower infundibulum and subjacent junctional zone overlapping the LRIG1+ progenitor cell niche. TCDD also caused Ahr -dependent seboatrophy, accompanied by increased expression of Blimp1 , a transcriptional repressor that regulates SG size. A second site of Cyp1a1 induction was the SG, where Cyp1a1 was markedly elevated in the basal proliferating cells and immature sebocytes. In a 3-day topical exposure study of early effects, TCDD produced a dose-dependent increase of Cyp1a1 expression in the SG that included the more differentiated sebocytes. This response was accompanied by expansion of the Scd1 -positive area, elevated Nile Red lipid staining, and an increased number of Blimp1 -high sebocytes, demonstrating that TCDD enhanced SG differentiation and lipid production in vivo . These changes preceded the onset of Ahr -dependent seboatrophy, providing new insight into the cellular and molecular events underlying chloracne pathogenesis.
Recent experimental evidence suggests that aging may arise from the progressive deterioration of the epigenetic landscape, while reversing the trend can result in cell and tissue rejuvenation. A mechanistic understanding of how restoration of a key component of this landscape - the 3D structure of the genome - can be accomplished is lacking. Here we investigate lamina-dependent disruption and recovery of the 3D architecture of the Drosophila melanogaster genome at TAD resolution (∼ 100kb), using a model of the entire nucleus; weakening of chromatin-lamina interactions mimics an aging-associated loss of chromatin organization. We characterize this loss using the Shannon entropy of appropriately normalized Hi-C contact matrices. Our main finding is that lamina-depletion-induced increases in Hi-C map disorder, deterioration of chromosome territories, and cell-to-cell conformational heterogeneity are largely reversible when WT-like LAD-nuclear-envelope interactions are restored. The original and recovered conformational states of chromatin are nearly indistinguishable by bulk Hi-C contact matrix; the corresponding Pearson correlation coefficient is 0.999902. The direct experimentally testable prediction is that restoration of functional LAD-lamina interactions will promote recovery of young/WT-like 3D chromatin architecture after lamina-dependent architectural disruption.
Internal tandem duplication mutations in FLT3 ( FLT3 ITD ) occur in approximately 30% of patients with acute myeloid leukemia (AML) and are among the most common genetic alterations in this disease. FLT3 ITD is a major driver of AML and is associated with poor clinical outcomes. Although FLT3 inhibitors (FLT3is) have significantly improved outcomes for patients with FLT3 ITD + AML, acquired resistance remains a major barrier to durable clinical benefit. Reactivation of RAS/MAPK signaling, often driven by activating NRAS mutations, is a major mechanism of FLT3i resistance in AML; however, effective strategies to overcome this resistance remain lacking. Here, we identify ribonucleotide reductase (RNR) as a critical therapeutic vulnerability in NRAS -driven FLT3i-resistant FLT3 ITD + AML. Activation of RAS signaling through SPRY3 loss or oncogenic NRAS mutations confers robust resistance to FLT3is, whereas pharmacologic inhibition of RNR with multiple inhibitors, as well as siRNA-mediated RNR suppression, reverses FLT3i resistance and restores FLT3i sensitivity across multiple FLT3 ITD + AML models in vitro . In vivo , clofarabine, an FDA-approved RNR inhibitor (RNRi), significantly overcomes NRAS mutation-driven FLT3i resistance. In combination with FLT3 inhibition, clofarabine markedly suppresses the progression of FLT3i-resistant AML and significantly prolongs survival in cell line-derived xenograft (CDX) models. Importantly, the therapeutic efficacy of the gilteritinib/clofarabine combination was independently validated in two genetically distinct patient-derived xenograft (PDX) models harboring different NRAS mutations, demonstrating robust reduction of leukemia burden and confirming the generalizability of RNR inhibition in primary FLT3i-resistant AML. Together, these findings identify a previously unrecognized therapeutic vulnerability in FLT3i-resistant FLT3 mut + AML and establish RNR inhibition as an effective strategy to overcome FLT3i resistance, providing a strong rationale for the clinical evaluation of RNRis in combination with FLT3is in patients with resistant AML. Although FLT3 inhibitors (FLT3i) are an important therapeutic advance in FLT3 ITD + AML, resistance commonly develops. We identified ribonucleotide reductase (RNR) as a new key vulnerability in NRAS -driven FLT3i-resistant AML and demonstrated that multiple RNRis, including the FDA-approved agent clofarabine, restore FLT3i sensitivity and enhance antileukemic activity, supporting a clinically actionable combination strategy.
Impaired insulin transport across the endothelium contributes to insulin resistance, a poorly understood condition implicated in diabetes and many other chronic diseases. Insulin has been reported to undergo non-receptor-mediated endocytosis resembling fluid-phase uptake through unclear mechanisms. Here we show in mice with diet-induced insulin resistance that endothelial-specific deletion of the depalmitoylase acyl-protein thioesterase 1 (APT1) improved glucose tolerance and insulin sensitivity without affecting chronic inflammation or capillary structure. Endothelial APT1 deficiency increased interstitial insulin levels in mice confirmed by in-situ microneedle-based sampling. In cultured human microvascular cells, APT1 inhibition enhanced cell transport of high-dose insulin independent of the insulin receptor and canonical endocytic machinery. Unexpectedly, live-cell imaging revealed insulin rapidly localizing to mitochondria prior to endolysosomal trafficking, even at physiological insulin concentrations. APT1 inhibition delayed mitochondrial discharge of insulin to lysosomes. Cyclosporin A, an immunosuppressant known to affect mitochondrial function, preserved mitochondrial insulin content and promoted insulin transport in cultured cells, and enhanced interstitial insulin delivery in mice. Proteomic analysis revealed two palmitoylated proteins, PACS1 and YTHDF2, required for APT1-mediated mitochondrial-endolysosomal trafficking of insulin. A mitochondrial insulin shuttle in endothelial cells may participate in the physiological adaptation to hyperinsulinemia and its regulation by palmitoylation suggests a novel approach to insulin resistance.
Event-based models (EBMs) are used to infer ordering of biomarker alteration patterns with respect to disease progression. However, EBM approaches rely on computationally expensive permutation-based inference, assumptions of feature independence, and likelihood optimization that can limit scalability and stability in high-dimensional settings. Here, we introduce Fast Event-Based Model (FastEBM), a scalable, uncertainty aware, Markov- chain-based framework that reformulates disease progression inference as a subject-ordering problem on a data-driven diffusion manifold. The progression uncertainty, used to derive positional variance diagrams, is quantified using first-passage-time variability derived directly from the inferred Markov process. Using synthetic experiments varying feature dimensionality, cohort size, noise level, and feature-correlation structure, we compared FastEBM with established methods, including Gaussian mixture model EBM (GMM-EBM), kernel density estimation EBM (KDE-EBM), and discriminative EBM (DEBM). FastEBM achieved the best accuracy and runtime. In low-subject/high-dimensional stress tests, FastEBM retained event-order recovery. FastEBM remained robust in simulations containing correlated and redundant features after decorrelation and feature-group handling. We applied FastEBM to real-world data to characterize biomarker progression in Alzheimer's disease. First, we evaluated a low-dimensional multi- modal dataset from The Alzheimer's Disease Prediction Of Longitudinal Evolution (TAD- POLE) challenge. Second, to demonstrate high-dimensional disease progression mapping, we applied FastEBM to regional cortical tau-PET data from the Alzheimer's Disease Neuroimaging Initiative (ADNI). In both cases, FastEBM recovered progression patterns broadly consistent with the literature, also revealing lateralized progression trends. These results show that diffusion-based Markov geometry provides a scalable and robust alternative to conventional event-based modeling. FastEBM is available at: https://github.com/sjusc07/FastEBM .
Ferroptosis is driven by the accumulation of oxidatively damaged membrane phospholipids, making membrane lipid composition a central determinant of cell death sensitivity. While fatty acid chain length and degree of unsaturation are well-established regulators of ferroptosis, whether fatty acid stereochemistry contributes to ferroptosis susceptibility is mostly unexplored. Here, we systematically screened structurally diverse fatty acids for their ability to modulate ferroptosis and unexpectedly identified trans-unsaturated fatty acids as potent sensitizers. Compared with its cis counterpart linoleic acid, the trans polyunsaturated fatty acid (PUFA) linoelaidic acid more strongly enhanced lipid peroxidation and promoted the accumulation of ferroptosis-susceptible phospholipid species. Unexpectedly, the trans monounsaturated fatty acid petroselaidic acid also sensitized cells to ferroptosis, whereas its cis stereoisomer petroselinic acid suppressed ferroptosis. Mechanistically, petroselaidic acid required stearoyl-CoA desaturase-dependent conversion to a PUFA, directly demonstrating that double-bond geometry can redirect fatty acid metabolic fate through altered recognition by lipid metabolic enzymes. Although linoelaidic acid and petroselaidic acid followed distinct metabolic pathways, both converged on phospholipid remodeling that expanded pools of ferroptosis-susceptible membrane lipids. Together, our findings demonstrate that fatty acid double-bond geometry determines their metabolic fate and the membrane phospholipid composition, establishing lipid stereochemistry as a previously unrecognized structural determinant of ferroptosis sensitivity.
The Caffeinated Coli Educational Module brings inquiry-driven learning to high schools, introducing students to scientific research, synthetic biology, and genetic engineering. The module focuses on the exploration of a genetically engineered strain of E. coli modified to grow exclusively on caffeine and, as such, can be used as a measurement device to determine the amount of caffeine in a liquid or beverage. Students conduct two bioassay experiments using these bacteria. During this process, they learn to create cultures and then measure bacterial growth followed by calculating the caffeine concentrations of unknown samples using their own data. This flexible module contains five weeks of original lectures and student learning materials that follow Next Generation Science Standards (NGSS), allowing the content to be adapted for basic, intermediate, or advanced biology courses. Upon completion of the module, students and teachers expressed that the most memorable aspects of the module include collaboration with peers, hands-on learning of content, and the opportunity to interact with the professor/mentors through office hours. Already implemented in 8 Texas high schools over the past two academic years, our module inspires STEM learning while bringing 21 st century biology research to new audiences.
Intronic GGGGCC repeat expansions in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This expansion supports a non-canonical form of translational initiation known as repeat-associated non-AUG (RAN) translation to produce toxic dipeptide repeat proteins that contribute to neurodegeneration. Here, we find that the efficiency of RAN translation and its dependency on the 5' 7-methylguanosine mRNA cap are variable across cell types, with both rodent neurons and human iNeurons favoring cap-independent RAN translation from two distinct repeats (CGG and GGGGCC) across multiple reading frames. Treatment with an eIF4E inhibitor that blocks global cap-dependent translation enhances RAN translation specifically in neurons. Intriguingly, cap-independent RAN translation exhibits less reliance on near-cognate codons for initiation than cap-dependent RAN translation. This finding led us to identify a surprising global increase in start codon stringency in neurons as a contributor to the relatively higher cap-independent RAN translation in this cell type. This effect correlates with a cytoplasmic redistribution of eIF1 in neurons and is reversed with neuronal overexpression of the eukaryotic initiation factor eIF5, which relaxes start codon stringency and selectively enhances cap-dependent RAN translation. Taken together, these findings reveal several neuron-specific features of translational regulation that favor cap-independent RAN translation with implications for nucleotide repeat expansion disorder pathogenesis and neuronal translational regulation.
Thermal proteome profiling (TPP) and proteome integral solubility alteration (PISA) assays measure drug-target interactions by monitoring protein thermal stability across the proteome. While detergents are routinely used in lysate-based thermal profiling, the field lacks consensus on whether detergents should be present during the melting step or only added afterward as an extraction buffer, and whether detergent identity matters for this choice. Here, we evaluate how commonly used detergents and the timing of their use in thermal stability workflows affect proteome-wide thermal stability and PISA hit calling in TF-1 lysates. We find that NP-40 and DDM produce highly correlated melting profiles when used exclusively as post-melt extraction buffers, but diverge substantially when present during the melting step. DDM in particular prevents the thermally-induced loss in solubility of large classes of proteins, such as cell surface proteins, and these effects propagate directly into PISA hit calling. Performing the PISA melt in DDM versus NP-40 results in the gain and loss of distinct drug-target interactions for both the PAK4 inhibitor PF-3758309 and the PLK1 inhibitor volasertib. Notably, DDM enables detection of a volasertib-TMEM97 interaction that was previously not detected in NP-40. However, we also find that the stabilization effects of DDM mask the identification of some known PISA hits for these drugs. We further introduce a four-parameter logistic model of protein melting to aid in modeling of these findings and a linear regression framework for PISA hit calling that outperforms pairwise t-tests in low-replicate settings. Together, these results establish detergent selection as a tunable experimental variable in thermal profiling and suggest that some drug-target engagements previously attributed exclusively to intact-cell context may be recoverable in lysates with appropriate buffer conditions.
Current γδ T-cell expansion protocols often sacrifice functionality for yield and largely ignore the context of activation. Here we utilize a tunable alginate microgel system functionalized with anti-CD3 and co-stimulatory antibodies (αCD28 or αCD2) to investigate the impact of biochemical signaling and substrate mechanics on γδ T-cell activation. Microgel-mediated expansion was compared to conventional soluble antibodies and TransAct beads. The microgels enhanced γδ T-cell expansion compared to soluble antibodies, allowed for controlled tuning of differentiation state, and promoted higher NKG2D, IFN-γ and TNF-α expression levels. Functionally, microgel-expanded γδ T-cells exhibited superior cytotoxicity against both solid and liquid tumor targets. This system also allowed elucidation of the differences in stimulation requirements for various donors, based on the starting phenotype. These findings establish a tunable platform for engineering γδ T-cells with improved therapeutic potential. γδ T-cells have shown promising therapeutic effects when used for T cell-based immunotherapy to treat solid tumor. However, achieving rapid expansion of γδ T-cells while maintaining their functionality remains a major challenge, especially given the heterogeneous responses from donors. We demonstrate that a tunable microgel system with flexible presentation of stimulatory cues improves γδ T-cell expansion while preserving cytotoxic function and reveal how starting phenotypes influence responses to activation. These understandings will provide design rationale to enable patient-specific treatment for optimal therapeutic outcomes.