BioSciences Publications

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https://hdl.handle.net/1911/78269

BioSciences faculty publications. For works published before Summer 2014, please see the Biochemistry & Cell Biology and Ecology & Evolutionary Biology collections.

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RACER-m leverages structural features for sparse T cell specificity prediction
(AAAS, 2024) Wang, Ailun; Lin, Xingcheng; Chau, Kevin Ng; Onuchic, José N.; Levine, Herbert; George, Jason T.; Center for Theoretical Biological Physics
Reliable prediction of T cell specificity against antigenic signatures is a formidable task, complicated by the immense diversity of T cell receptor and antigen sequence space and the resulting limited availability of training sets for inferential models. Recent modeling efforts have demonstrated the advantage of incorporating structural information to overcome the need for extensive training sequence data, yet disentangling the heterogeneous TCR-antigen interface to accurately predict MHC-allele-restricted TCR-peptide interactions has remained challenging. Here, we present RACER-m, a coarse-grained structural model leveraging key biophysical information from the diversity of publicly available TCR-antigen crystal structures. Explicit inclusion of structural content substantially reduces the required number of training examples and maintains reliable predictions of TCR-recognition specificity and sensitivity across diverse biological contexts. Our model capably identifies biophysically meaningful point-mutant peptides that affect binding affinity, distinguishing its ability in predicting TCR specificity of point-mutants from alternative sequence-based methods. Its application is broadly applicable to studies involving both closely related and structurally diverse TCR-peptide pairs.
CrysFormer: Protein structure determination via Patterson maps, deep learning, and partial structure attention
(AIP Publishing LLC, 2024) Pan, Tom; Dun, Chen; Jin, Shikai; Miller, Mitchell D.; Kyrillidis, Anastasios; Phillips, George N., Jr.
Determining the atomic-level structure of a protein has been a decades-long challenge. However, recent advances in transformers and related neural network architectures have enabled researchers to significantly improve solutions to this problem. These methods use large datasets of sequence information and corresponding known protein template structures, if available. Yet, such methods only focus on sequence information. Other available prior knowledge could also be utilized, such as constructs derived from x-ray crystallography experiments and the known structures of the most common conformations of amino acid residues, which we refer to as partial structures. To the best of our knowledge, we propose the first transformer-based model that directly utilizes experimental protein crystallographic data and partial structure information to calculate electron density maps of proteins. In particular, we use Patterson maps, which can be directly obtained from x-ray crystallography experimental data, thus bypassing the well-known crystallographic phase problem. We demonstrate that our method, CrysFormer, achieves precise predictions on two synthetic datasets of peptide fragments in crystalline forms, one with two residues per unit cell and the other with fifteen. These predictions can then be used to generate accurate atomic models using established crystallographic refinement programs.
Breakdown of Boltzmann-type models for the alignment of self-propelled rods
(Elsevier, 2024) Murphy, Patrick; Perepelitsa, Misha; Timofeyev, Ilya; Lieber-Kotz, Matan; Islas, Brandon; Igoshin, Oleg A.; Center for Theoretical Biological Physics
Studies in the collective motility of organisms use a range of analytical approaches to formulate continuous kinetic models of collective dynamics from rules or equations describing agent interactions. However, the derivation of these kinetic models often relies on Boltzmann’s “molecular chaos” hypothesis, which assumes that correlations between individuals are short-lived. While this assumption is often the simplest way to derive tractable models, it is often not valid in practice due to the high levels of cooperation and self-organization present in biological systems. In this work, we illustrated this point by considering a general Boltzmann-type kinetic model for the alignment of self-propelled rods where rod reorientation occurs upon binary collisions. We examine the accuracy of the kinetic model by comparing numerical solutions of the continuous equations to an agent-based model that implements the underlying rules governing microscopic alignment. Even for the simplest case considered, our comparison demonstrates that the kinetic model fails to replicate the discrete dynamics due to the formation of rod clusters that violate statistical independence. Additionally, we show that introducing noise to limit cluster formation helps improve the agreement between the analytical model and agent simulations but does not restore the agreement completely. These results highlight the need to both develop and disseminate improved moment-closure methods for modeling biological and active matter systems.
Observing one-divalent-metal-ion-dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo
(eLife Sciences Publications Ltd, 2024) Chang, Caleb; Zhou, Grace; Gao, Yang
Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The histidine-metal (His-Me) superfamily nucleases are renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how the single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.
Machine learning in biological physics: From biomolecular prediction to design
(National Academy of Sciences, 2024) Martin, Jonathan; Lequerica Mateos, Marcos; Onuchic, José N.; Coluzza, Ivan; Morcos, Faruck; Center for Theoretical Biological Physics
Machine learning has been proposed as an alternative to theoretical modeling when dealing with complex problems in biological physics. However, in this perspective, we argue that a more successful approach is a proper combination of these two methodologies. We discuss how ideas coming from physical modeling neuronal processing led to early formulations of computational neural networks, e.g., Hopfield networks. We then show how modern learning approaches like Potts models, Boltzmann machines, and the transformer architecture are related to each other, specifically, through a shared energy representation. We summarize recent efforts to establish these connections and provide examples on how each of these formulations integrating physical modeling and machine learning have been successful in tackling recent problems in biomolecular structure, dynamics, function, evolution, and design. Instances include protein structure prediction; improvement in computational complexity and accuracy of molecular dynamics simulations; better inference of the effects of mutations in proteins leading to improved evolutionary modeling and finally how machine learning is revolutionizing protein engineering and design. Going beyond naturally existing protein sequences, a connection to protein design is discussed where synthetic sequences are able to fold to naturally occurring motifs driven by a model rooted in physical principles. We show that this model is “learnable” and propose its future use in the generation of unique sequences that can fold into a target structure.
Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells
(Optica Publishing Group, 2024) Nelson, Tyler; Vargas-Hernández, Sofía; Freire, Margareth; Cheng, Siyang; Gustavsson, Anna-Karin; Smalley-Curl Institute;Institute of Biosciences & Bioengineering;Center for Nanoscale Imaging Sciences
Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin – a protein located in the focal adhesion complex – and actin in human osteosarcoma cells.
Nucleosomes play a dual role in regulating transcription dynamics
(National Academy of Sciences, 2024) Brahmachari, Sumitabha; Tripathi, Shubham; Onuchic, José N.; Levine, Herbert; Center for Theoretical Biological Physics
Transcription has a mechanical component, as the translocation of the transcription machinery or RNA polymerase (RNAP) on DNA or chromatin is dynamically coupled to the chromatin torsion. This posits chromatin mechanics as a possible regulator of eukaryotic transcription, however, the modes and mechanisms of this regulation are elusive. Here, we first take a statistical mechanics approach to model the torsional response of topology-constrained chromatin. Our model recapitulates the experimentally observed weaker torsional stiffness of chromatin compared to bare DNA and proposes structural transitions of nucleosomes into chirally distinct states as the driver of the contrasting torsional mechanics. Coupling chromatin mechanics with RNAP translocation in stochastic simulations, we reveal a complex interplay of DNA supercoiling and nucleosome dynamics in governing RNAP velocity. Nucleosomes play a dual role in controlling the transcription dynamics. The steric barrier aspect of nucleosomes in the gene body counteracts transcription via hindering RNAP motion, whereas the chiral transitions facilitate RNAP motion via driving a low restoring torque upon twisting the DNA. While nucleosomes with low dissociation rates are typically transcriptionally repressive, highly dynamic nucleosomes offer less of a steric barrier and enhance the transcription elongation dynamics of weakly transcribed genes via buffering DNA twist. We use the model to predict transcription-dependent levels of DNA supercoiling in segments of the budding yeast genome that are in accord with available experimental data. The model unveils a paradigm of DNA supercoiling-mediated interaction between genes and makes testable predictions that will guide experimental design.
Microbial symbionts buffer hosts from the demographic costs of environmental stochasticity
(Wiley, 2024) Fowler, Joshua C.; Ziegler, Shaun; Whitney, Kenneth D.; Rudgers, Jennifer A.; Miller, Tom E. X.
Species' persistence in increasingly variable climates will depend on resilience against the fitness costs of environmental stochasticity. Most organisms host microbiota that shield against stressors. Here, we test the hypothesis that, by limiting exposure to temporally variable stressors, microbial symbionts reduce hosts' demographic variance. We parameterized stochastic population models using data from a 14-year symbiont-removal experiment including seven grass species that host Epichloë fungal endophytes. Results provide novel evidence that symbiotic benefits arise not only through improved mean fitness, but also through dampened inter-annual variance. Hosts with “fast” life-history traits benefited most from symbiont-mediated demographic buffering. Under current climate conditions, contributions of demographic buffering were modest compared to benefits to mean fitness. However, simulations of increased stochasticity amplified benefits of demographic buffering and made it the more important pathway of host–symbiont mutualism. Microbial-mediated variance buffering is likely an important, yet cryptic, mechanism of resilience in an increasingly variable world.
Interactive effects of soils, local environmental conditions and herbivores on secondary chemicals in tallow tree
(Oxford University Press, 2024) Xiao, Li; Huang, Wei; Carrillo, Juli; Ding, Jianqing; Siemann, Evan
Plants produce secondary chemicals that may vary along with latitude due to changing abiotic and biotic stress gradients and local environmental conditions. Teasing apart the individual and combined effects of these different abiotic, such as soil nutrients, and biotic factors, such as soil biota and herbivores, on secondary chemicals is critical for understanding plant responses to changing environments. We conducted an experiment at different latitudes in China, using tallow tree (Triadica sebifera) seedlings sourced from a population at 31° N. These seedlings were cultivated in gardens located at low, middle and high latitudes, with either local soil or soil from the original seed collection site (origin soil). The seedlings were exposed to natural levels of aboveground herbivores or had them excluded. Plant secondary chemicals (both foliar and root), aboveground herbivores and soil characteristics were measured. Results showed that most leaf and root secondary metabolites depended on the interaction of the experimental site and soil type. Leaf and root phenolic and tannin concentrations were higher at the middle latitude site, especially in the origin soil. Root and foliar flavonoid concentrations increased when aboveground herbivores were excluded. Microbial communities depended strongly on soil treatment. The different responses of tannins versus flavonoids suggest that these two chemical classes differ in their responses to the varying abiotic and biotic factors in these sites along latitudes. Taken together, our results emphasize the importance of considering the interactive effects of local environmental conditions, soil properties and herbivory in regulating plant chemical defenses.
Geographic differences in body size distributions underlie food web connectance of tropical forest mammals
(Springer Nature, 2024) Beaudrot, Lydia; Acevedo, Miguel A.; Gorczynski, Daniel; Harris, Nyeema C.; Program in Ecology and Evolutionary Biology
Understanding variation in food web structure over large spatial scales is an emerging research agenda in food web ecology. The density of predator–prey links in a food web (i.e., connectance) is a key measure of network complexity that describes the mean proportional dietary breadth of species within a food web. Connectance is a critical component of food web robustness to species loss: food webs with lower connectance have been shown to be more susceptible to secondary extinctions. Identifying geographic variation in food web connectance and its drivers may provide insight into community robustness to species loss. We investigated the food web connectance of ground-dwelling tropical forest mammal communities in multiple biogeographic regions to test for differences among regions in food web connectance and to test three potential drivers: primary productivity, contemporary anthropogenic pressure, and variation in mammal body mass distributions reflective of historical extinctions. Mammal communities from fifteen protected forests throughout the Neo-, Afro-, and Asian tropics were identified from systematic camera trap arrays. Predator–prey interaction data were collected from published literature, and we calculated connectance for each community as the number of observed predator–prey links relative to the number of possible predator–prey links. We used generalized linear models to test for differences among regions and to identify the site level characteristics that best predicted connectance. We found that mammal food web connectance varied significantly among continents and that body size range was the only significant predictor. More possible predator–prey links were observed in communities with smaller ranges in body size and therefore sites with smaller body size ranges had higher mean proportional dietary breadth. Specifically, mammal communities in the Neotropics and in Madagascar had significantly higher connectance than mammal communities in Africa. This geographic variation in contemporary mammalian food web structure may be the product of historical extinctions in the Late Quaternary, which led to greater losses of large-bodied species in the Neotropics and Madagascar thus contributing to higher average proportional dietary breadth among the remaining smaller bodied species in these regions.
Inhibition of Cathepsin S in Autoimmune CD25KO Mouse Improves Sjögren Disease–Like Lacrimal Gland Pathology
(ARVO, 2024) Scholand, Kaitlin K.; Galletti, Jeremias; Haap, Wolfgang; Santos-Ferreira, Tiago; Ullmer, Christoph; de Paiva, Cintia S.
CD25KO mice are a model of Sjögren disease (SjD) driven by autoreactive T cells. Cathepsin S (CTSS) is a protease crucial for major histocompatibility complex class II presentation that primes T cells. We investigated if a diet containing CTSS inhibitor would improve autoimmune signs in CD25KO mice. Four-week female CD25KO mice were randomly chosen to receive chow containing a CTSS inhibitor (R05461111, 262.5 mg/kg chow) or standard chow for 4 weeks. Cornea sensitivity was measured. Inflammatory score was assessed in lacrimal gland (LG) histologic sections. Flow cytometry of LG and ocular draining lymph nodes (dLNs) investigated expression of Th1 and Th17 cells. Expression of inflammatory, T- and B-cell, and apoptotic markers in the LG were assessed with quantitative PCR. The life span of mice receiving CTSS inhibitor or standard chow was compared. CD4+ T cells from both groups were isolated from spleens and adoptively transferred into RAG1KO female recipients. Mice receiving CTSS inhibitor had better cornea sensitivity and improved LG inflammatory scores. There was a significant decrease in the frequency of CD4+ immune cells and a significant increase in the frequency of CD8+ immune cells in the dLNs of CTSS inhibitor mice. There was a significant decrease in Th1 and Th17 cells in CTSS inhibitor mice in both LGs and dLNs. Ifng, Ciita, and Casp8 mRNA in CTSS inhibitor mice decreased. Mice that received the CTSS inhibitor lived 30% longer. Adoptive transfer recipients with CTSS inhibitor-treated CD4+ T cells had improved cornea sensitivity and lower inflammation scores. Inhibiting CTSS could be a potential venue for the treatment of SjD in the eye and LG.
Insect–microbe interactions and their influence on organisms and ecosystems
(Wiley, 2024) Holt, Jocelyn R.; Cavichiolli de Oliveira, Nathalia; Medina, Raul F.; Malacrinò, Antonino; Lindsey, Amelia R. I.
Microorganisms are important associates of insect and arthropod species. Insect-associated microbes, including bacteria, fungi, and viruses, can drastically impact host physiology, ecology, and fitness, while many microbes still have no known role. Over the past decade, we have increased our knowledge of the taxonomic composition and functional roles of insect-associated microbiomes and viromes. There has been a more recent shift toward examining the complexity of microbial communities, including how they vary in response to different factors (e.g., host genome, microbial strain, environment, and time), and the consequences of this variation for the host and the wider ecological community. We provide an overview of insect–microbe interactions, the variety of associated microbial functions, and the evolutionary ecology of these relationships. We explore the influence of the environment and the interactive effects of insects and their microbiomes across trophic levels. Additionally, we discuss the potential for subsequent synergistic and reciprocal impacts on the associated microbiomes, ecological interactions, and communities. Lastly, we discuss some potential avenues for the future of insect-microbe interactions that include the modification of existing microbial symbionts as well as the construction of synthetic microbial communities.
Electrothermal mineralization of per- and polyfluoroalkyl substances for soil remediation
(Springer Nature, 2024) Cheng, Yi; Deng, Bing; Scotland, Phelecia; Eddy, Lucas; Hassan, Arman; Wang, Bo; Silva, Karla J.; Li, Bowen; Wyss, Kevin M.; Ucak-Astarlioglu, Mine G.; Chen, Jinhang; Liu, Qiming; Si, Tengda; Xu, Shichen; Gao, Xiaodong; JeBailey, Khalil; Jana, Debadrita; Torres, Mark Albert; Wong, Michael S.; Yakobson, Boris I.; Griggs, Christopher; McCary, Matthew A.; Zhao, Yufeng; Tour, James M.
Per- and polyfluoroalkyl substances (PFAS) are persistent and bioaccumulative pollutants that can easily accumulate in soil, posing a threat to environment and human health. Current PFAS degradation processes often suffer from low efficiency, high energy and water consumption, or lack of generality. Here, we develop a rapid electrothermal mineralization (REM) process to remediate PFAS-contaminated soil. With environmentally compatible biochar as the conductive additive, the soil temperature increases to >1000 °C within seconds by current pulse input, converting PFAS to calcium fluoride with inherent calcium compounds in soil. This process is applicable for remediating various PFAS contaminants in soil, with high removal efficiencies ( >99%) and mineralization ratios ( >90%). While retaining soil particle size, composition, water infiltration rate, and cation exchange capacity, REM facilitates an increase of exchangeable nutrient supply and arthropod survival in soil, rendering it superior to the time-consuming calcination approach that severely degrades soil properties. REM is scaled up to remediate soil at two kilograms per batch and promising for large-scale, on-site soil remediation. Life-cycle assessment and techno-economic analysis demonstrate REM as an environmentally friendly and economic process, with a significant reduction of energy consumption, greenhouse gas emission, water consumption, and operation cost, when compared to existing soil remediation practices.
Chlorophyll fluorescence characteristics and H2O2 contents of Chinese tallow tree are dependent on population origin, nutrients and salinity
(Oxford University Press, 2024) He, Mengyue; Ge, Lihong; Hui, Xue; Li, Wenrao; Ding, Jianqing; Siemann, Evan
Plants from invasive populations often have higher growth rates than conspecifics from native populations due to better environmental adaptability. However, the roles of improved chlorophyll fluorescence or antioxidant defenses in helping them to grow better under adverse situations are insufficient, even though this is a key physiological question for elucidating mechanisms of plant invasion. Here, we conducted experiments with eight native (China) and eight introduced (USA) populations of Chinese tallow tree (Triadica sebifera). We tested how salinity, nutrients (overall amount or N:P in two separate experiments) and their interaction affected T. sebifera aboveground biomass, leaf area, chlorophyll fluorescence and antioxidant defenses. Plants from introduced populations were larger than those from native populations, but salinity and nutrient shortage (low nutrients or high N:P) reduced this advantage, possibly reflecting differences in chlorophyll fluorescence based on their higher PSII maximum photochemical efficiency (Fv/Fm) and PSI maximum photo-oxidizable P700 in higher nutrient conditions. Native population plants had lower Fv/Fm with saline. Except in high nutrients/N:P with salinity, introduced population plants had lower electron transfer rate and photochemical quantum yield. There were no differences in antioxidant defenses between introduced and native populations except accumulation of hydrogen peroxide (H2O2), which was lower for introduced populations. Low nutrients and higher N:P or salinity increased total antioxidant capacity and H2O2. Our results indicate that nutrients and salinity induce differences in H2O2 contents and chlorophyll fluorescence characteristics between introduced and native populations of an invasive plant, illuminating adaptive mechanisms using photosynthetic physiological descriptors in order to predict invasions.
CISD3/MiNT is required for complex I function, mitochondrial integrity, and skeletal muscle maintenance
(National Academy of Sciences, 2024) Nechushtai, Rachel; Rowland, Linda; Karmi, Ola; Marjault, Henri-Baptiste; Nguyen, Thi Thao; Mittal, Shubham; Ahmed, Raheel S.; Grant, DeAna; Manrique-Acevedo, Camila; Morcos, Faruck; Onuchic, José N.; Mittler, Ron; Center for Theoretical Biological Physics
Mitochondria play a central role in muscle metabolism and function. A unique family of iron–sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3–NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions.
Age-Related Differences in the Mouse Corneal Epithelial Transcriptome and Their Impact on Corneal Wound Healing
(ARVO, 2024) Abu-Romman, Anmar; Scholand, Kaitlin K.; Govindarajan, Gowthaman; Yu, Zhiyuan; Pal-Ghosh, Sonali; Stepp, Mary A.; de Paiva, Cintia S.
Aging is a risk factor for dry eye. We sought to identify changes in the aged mouse corneal epithelial transcriptome and determine how age affects corneal sensitivity, re-epithelialization, and barrier reformation after corneal debridement. Corneal epithelium of female C57BL/6J (B6) mice of different ages (2, 12, 18, and 24 months) was collected, RNA extracted, and bulk RNA sequencing performed. Cornea sensitivity was measured with an esthesiometer in 2- to 3-month-old, 12- to 13-month-old, 18- to 19-month-old, and 22- to 25-month-old female and male mice. The 2-month-old and 18-month-old female and male mice underwent unilateral corneal debridement using a blunt blade. Wound size and fluorescein staining were visualized and photographed at different time points, and a re-epithelialization rate curve was calculated. There were 157 differentially expressed genes in aged mice compared with young mice. Several pathways downregulated with age control cell migration, proteoglycan synthesis, and collagen trimerization, assembly, biosynthesis, and degradation. Male mice had decreased corneal sensitivity compared with female mice at 12 and 24 months of age. Aged mice, irrespective of sex, had delayed corneal re-epithelialization in the first 48 hours and worse corneal fluorescein staining intensity at day 14 than young mice. Aged corneal epithelium has an altered transcriptome. Aged mice regardless of sex heal more slowly and displayed more signs of corneal epithelial defects after wounding than young mice. These results indicate that aging significantly alters the corneal epithelium and its ability to coordinate healing.
A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system
(Public Library of Science, 2024) Moreno-Campos, Rodrigo; Singleton, Eileen W.; Uribe, Rosa A.; Laboratory of Neural Crest and Enteric Nervous System Development
The vertebrate enteric nervous system (ENS) is a crucial network of enteric neurons and glia resident within the entire gastrointestinal tract (GI). Overseeing essential GI functions such as gut motility and water balance, the ENS serves as a pivotal bidirectional link in the gut-brain axis. During early development, the ENS is primarily derived from enteric neural crest cells (ENCCs). Disruptions to ENCC development, as seen in conditions like Hirschsprung disease (HSCR), lead to the absence of ENS in the GI, particularly in the colon. In this study, using zebrafish, we devised an in vivo F0 CRISPR-based screen employing a robust, rapid pipeline integrating single-cell RNA sequencing, CRISPR reverse genetics, and high-content imaging. Our findings unveil various genes, including those encoding opioid receptors, as possible regulators of ENS establishment. In addition, we present evidence that suggests opioid receptor involvement in the neurochemical coding of the larval ENS. In summary, our work presents a novel, efficient CRISPR screen targeting ENS development, facilitating the discovery of previously unknown genes, and increasing knowledge of nervous system construction.
Beyond microtubules: The cellular environment at the endoplasmic reticulum attracts proteins to the nucleus, enabling nuclear transport
(Elsevier, 2024) Chae, Seok Joo; Kim, Dae Wook; Igoshin, Oleg A.; Lee, Seunggyu; Kim, Jae Kyoung; Center for Theoretical Biological Physics
All proteins are translated in the cytoplasm, yet many, including transcription factors, play vital roles in the nucleus. While previous research has concentrated on molecular motors for the transport of these proteins to the nucleus, recent observations reveal perinuclear accumulation even in the absence of an energy source, hinting at alternative mechanisms. Here, we propose that structural properties of the cellular environment, specifically the endoplasmic reticulum (ER), can promote molecular transport to the perinucleus without requiring additional energy expenditure. Specifically, physical interaction between proteins and the ER impedes their diffusion and leads to their accumulation near the nucleus. This result explains why larger proteins, more frequently interacting with the ER membrane, tend to accumulate at the perinucleus. Interestingly, such diffusion in a heterogeneous environment follows Chapman’s law rather than the popular Fick’s law. Our findings suggest a novel protein transport mechanism arising solely from characteristics of the intracellular environment.
BIGH3 mediates apoptosis and gap junction failure in osteocytes during renal cell carcinoma bone metastasis progression
(Elsevier, 2024) Pan, Tianhong; Liu, Fengshuo; Hao, Xiaoxin; Wang, Shubo; Wasi, Murtaza; Song, Jian H.; Lewis, Valerae O.; Lin, Patrick P.; Moon, Bryan; Bird, Justin E.; Panaretakis, Theocharis; Lin, Sue-Hwa; Wu, Danielle; Farach-Carson, Mary C.; Wang, Liyun; Zhang, Ningyan; An, Zhiqiang; Zhang, Xiang H. -F.; Satcher, Robert L.
Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts.
In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores
(American Society for Microbiology, 2024) Kang, Donghoon; Xu, Qi; Kirienko, Natalia V.
The multidrug-resistant pathogen Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo. Despite its physiological significance, an in vitro, mammalian cell culture model that can be used to characterize the impact and molecular mechanisms of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model to use human bronchial epithelial (16HBE) cellsWe demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipids or genetic disruption of rhamnolipid biosynthesis abrogated the toxicity of the conditioned medium. Furthermore, we also examine the effects of exposure to purified pyoverdine on 16HBE cells. While pyoverdine accumulated within cells, it was largely sequestered within early endosomes, resulting in minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several pro-inflammatory genes. However, pyoverdine potentiated these iron chelators in activating pro-inflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection.

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