Biology subjects

Johnson, D. L.

Publications and source records attributed to Johnson, D. L..

13 recordsLinked to original sources

bioRxiv2026

Comparative Effects of Hypoxic vs. Normoxic Mesenchymal Stem Cell-Derived Extracellular Vesicles on Tissue Repair Following Volumetric Muscle Loss (VML)

Volumetric muscle loss (VML) is an irreversible muscle injury that results in chronic functional impairment. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) can facilitate tissue repair through immunomodulatory, angiogenic, and anti-fibrotic effects. However, their low yield and poor on-site retention limit their therapeutic efficacy. Hypoxia can boost MSC metabolism, proliferation, and EV production. Hypoxic (3% O2) preconditioning of MSCs increased the yield of EVs (30-300 nm) by 1.5-fold but decreased the expression of characteristic EV markers (i.e., CD81, ICAM, and FLOT1). Fibrin hydrogels promote skeletal muscle regeneration and can sequester EVs via integrins or electrostatic interactions. We hypothesized that encapsulating EVs in fibrin hydrogels would further enhance regeneration and prolong the retention of EVs at the VML injury site. VML was created by removing [~]20% of the gastrocnemius-soleus muscles mass in mice using a 3 mm biopsy punch. EVs (4.48x1010 particles/mL) derived from MSCs cultured under hypoxic (Hypo-EV) or normoxic (Norm-EV) conditions were encapsulated within fibrin hydrogels and implanted at the VML injury site. Fibrin hydrogels containing PBS (PFG) were used as controls. On day 14 post-injury, Norm-EV treatment resulted in increased muscle mass, angiogenesis, and myofiber regeneration relative to the Hypo-EV group. Both the Norm-EV and Hypo-EV treatment groups reduced macrophage infiltration at the injury site compared to the PFG. These findings highlight that while both Norm-EV and Hypo-EV exhibit immunomodulatory effects, they differ in their regenerative potential. We speculate that hypoxic conditions could have caused MSCs to prioritize survival over repair-promoting activities, thereby producing EVs with less pro-regenerative signals. The increased quantity of EVs in response to hypoxia doesnt compensate for their diminished regenerative potential, highlighting the importance of quality over quantity when considering EVs for therapeutic applications. Graphical AbstractJain et al., Comparative Effects of Hypoxic vs. Normoxic Mesenchymal Stem Cell-Derived Extracellular Vesicles on Tissue Repair Following Volumetric Muscle Loss (VML) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/697216v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@79a1feorg.highwire.dtl.DTLVardef@17a5f41org.highwire.dtl.DTLVardef@103c2e8org.highwire.dtl.DTLVardef@1f13e13_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Chromatin stability safeguards mitochondrial homeostasis and prevents mTORC1 hyperactivation

Cells dynamically regulate chromatin in response to nutrient flux which promotes the transcriptional changes necessary for adaptation. The mechanistic target of rapamycin complex 1 (mTORC1) kinase integrates nutrient signaling with chromatin regulation, yet whether chromatin stability feeds back to mTORC1 activation and stress adaption remains unknown. We previously identified histone H3 at lysine 37 (H3K37) as essential for the response to mTORC1 stress such that mutation of H3K37 to alanine (H3K37A) causes cell death upon mTORC1 inhibition. Herein, we show that H3K37-dependent chromatin stability prevents proteasome-mediated histone degradation, restricts mTORC1 signaling, and safeguards mitochondrial homeostasis during mTORC1 stress. Genetic interaction analyses reveal that H3K37A combined with mutants that destabilize chromatin, including loss of the Set2 H3K36 methyltransferase, Rpd3S histone deacetylase, or multiple histone deposition pathways, causes synthetic lethality when mTORC1 is inhibited. Transcriptome analysis indicates that H3K37A misregulates the mitochondrial transcriptome during mTORC1 stress, which increases mitochondrial reactive oxygen species (ROS) and triggers lethal mitochondrial retrograde signaling. Inactivation of retrograde signaling, or ROS neutralization, rescues viability of H3K37A and chromatin stability mutants during mTORC1 stress. These findings establish chromatin stability as a key safeguard that restrains mTORC1 activity and prevents toxic mitochondrial stress during metabolic adaptation.

molecular biology↗

bioRxiv2025

Intersecting effects of social circumstances and transcendent thinking on mid-adolescents' longitudinal functional connectome development

Socially moderated variation in mid-adolescents functional brain network (FBN) development is insufficiently studied, especially within low-SES contexts. Alongside demographic factors, evidence suggests adolescents malleable psychological dispositions may contribute, with possible clinical and educational implications. We applied graph theory to a unique 2-year longitudinal fMRI dataset (N=65) with an ecologically valid 2-hour interview revealing an emergent mid-adolescent psychological disposition, "transcendent thinking" (TT)--adolescents tendency to consider systems-level, ethical, personal implications of social information. Overall, FBNs showed increased segregation and entropy but decreased integration and energy. Modularity increased particularly in somatosensory subnetworks. Classifying participants by two key demographic factors, parents education (PE) and community violence exposure (CVE), FBN changes differed by group, and TT moderated changes in high-CVE/low-PE participants, arguably the most vulnerable group. Machine learning revealed TT and CVE, but not IQ, as principal FBN change predictors across groups. Findings suggest social context and psychological dispositions influence low-SES adolescents FBN development.

neuroscience↗

bioRxiv2024

Maf1 Cooperates with Progesterone Receptor to Repress RNA Polymerase III Transcription of Select tRNAs

Progesterone receptors (PR) can regulate transcription by RNA Polymerase III (Pol III), which transcribes small non-coding RNAs, including all transfer RNAs (tRNAs). We have previously demonstrated that PR is associated with the Pol III complex at tRNA genes and that progestins downregulate tRNA transcripts in breast tumor models. To further elucidate the mechanism of PR-mediated regulation of Pol III, we studied the interplay between PR, the Pol III repressor Maf1, and TFIIIB, a core transcription component. ChIP-seq was performed for PR, the Pol III subunit POLR3A, the TFIIIB component Brf1, and Maf1 in breast cancer cells with or without progestin treatment. Upon progestin exposure, PR localized to approximately half of POLR3A-occupied tRNA genes, with Maf1 co-recruited to many of these PR-POLR3A sites. While progestin treatment did not significantly alter the number of tRNA genes occupied by Pol III or Brf1, Brf1 occupancy was stabilized, as indicated by increased peak amplitudes. Analysis of nascent tRNA transcription revealed a specific progestin-induced downregulation of approximately one-third of highly expressed tRNA genes. This repression was significantly reduced by Maf1 knockdown, indicating that Maf1 is necessary for PR-mediated tRNA transcription downregulation. Overall, these findings demonstrate a ligand-dependent PR-mediated repression of tRNA transcription through Maf1.

molecular biology↗

bioRxiv2024

Assessing the Sustainability of Pacific Walrus Harvest in a Changing Environment

Harvest sustainability is a primary goal of wildlife management and conservation, and in a changing world it is increasingly important to consider environmental drivers of population dynamics alongside harvest in cohesive management plans. This is particularly pertinent for harvested species that are acutely experiencing effects of climate change. The Pacific walrus (Odobenus rosmarus divergens), a critical traditional subsistence resource for indigenous communities, is simultaneously subject to rapid habitat loss associated with diminishing sea ice and an increasing anthropogenic footprint in the Arctic. We developed a theta-logistic population modeling-management framework to evaluate various harvest scenarios combined with four potential climate/disturbance scenarios (ranging from optimistic-pessimistic) which simulates Pacific walrus population dynamics to the end of the 21st century. We considered two types of harvest strategies: (1) adaptive harvest scenarios wherein harvest is calculated as a percentage of the population and annual harvests are updated at set intervals as the population is reassessed, and (2) non-adaptive harvest scenarios wherein annual harvest remains constant. All climate/disturbance scenarios indicated declines of varying severity in Pacific walrus abundance to the end of the 21st century, even in the absence of harvest. However, we found that an adaptive annual harvest of 1.23% of the independent-aged female subset of the population (e.g., 1,280 independent-aged females harvested in 2020, representing contemporary harvest levels) met our criterion for sustainability (>70% probability of maintaining population abundance above maximum net productivity level) under all climate/disturbance scenarios, accepting a medium risk tolerance level of 25%. This suggests that the present rate of Pacific walrus harvest is sustainable and will continue to be--provided the harvest adapts to match changes in population dynamics. Our simulations suggest that a sustainable non-adaptive harvest is also possible, but only at low levels if the population declines as expected. Applying a constant annual harvest of 1,280 independent-aged females (equivalent to contemporary harvest levels of 1.23) exceeded our criterion for sustainability and resulted in a >5% chance of quasi-extinction by the end of the 21st century under three of the four climate/disturbance scenarios we evaluated. Our results highlight the importance of adaptive co-management strategies, and we suggest such modeling frameworks are useful for managing for harvest sustainability in a changing climate.

ecology↗

bioRxiv2023

Assessing the Population Consequences of Disturbance and Climate Change for the Pacific Walrus

Climate change and anthropogenic disturbance are increasingly affecting wildlife at a global scale. Predicting how varying types and degrees of disturbance may interact to influence population dynamics is a key management challenge. Population Consequences of Disturbance (PCoD) models provide a framework to link effects of anthropogenic disturbance on an individuals behavior and physiology to population-level changes. Bioenergetic models often constitute the basis of these frameworks, wherein an individuals daily energy balance is simulated over the course of its lifetime, allowing many individuals to be subjected to different environmental conditions and ultimately simulate population-level vital rates under varying degrees of disturbance. In the present study, we develop a Pacific walrus (Odobenus rosmarus divergens) PCoD model to encompass the population-level effects of both anthropogenic disturbance and climate change. Pacific walruses are an Arctic/subarctic ice-associated pinniped. As the Arctic has become increasingly ice-free with climate change, walruses spend more time on land-based (rather than ice-based) haulouts from which they must expend more energy to reach foraging areas, and where they have a greater risk of predation and disturbance-based mortalities. Concurrently, sea ice loss is increasing the anthropogenic footprint in Arctic regions (e.g., fisheries, shipping, energy exploration) which creates additional disturbance. We developed a bioenergetic Dynamic Energy Budget (DEB) model for the Pacific walrus and applied it to four scenarios (ranging from optimistic-pessimistic) which incorporate different global sea ice model projections along with varying degrees of anthropogenic disturbance. All scenarios indicated a decline in Pacific walrus carrying capacity and population growth rate (and thus overall abundance) to the end of the 21st century, but demonstrated that the intensity of that decline could be mitigated by global efforts to reduce carbon emissions (i.e., lessening the rate of sea ice loss) and local management and conservation efforts to protect sensitive habitat areas. In summary, we introduced a flexible PCoD modelling framework in a novel context which will prove useful to researchers studying walruses and other species similarly threatened by rapid environmental change.

ecology↗

bioRxiv2023

Ccr4-Not ubiquitin ligase signaling regulates ribosomal protein homeostasis and inhibits 40S ribosomal autophagy

The Ccr4-Not complex containing the Not4 ubiquitin ligase regulates gene transcription and mRNA decay, yet it also has poorly defined roles in translation, proteostasis, and endolysosomal-dependent nutrient signaling. To define how Ccr4-Not mediated ubiquitin signaling regulates these additional processes, we performed quantitative proteomics in the yeast Saccharomyces cerevisiae lacking the Not4 ubiquitin ligase, and also in cells overexpressing either wild-type or functionally inactive ligase. Herein, we provide evidence that both increased and decreased Ccr4-Not ubiquitin signaling disrupts ribosomal protein (RP) homeostasis independently of reduced RP mRNA changes or reductions in known Not4 ribosomal substrates. Surprisingly, we also find that both Not4-mediated ubiquitin signaling, and the Ccr4 subunit, actively inhibit 40S ribosomal autophagy. This 40S autophagy is independent of canonical Atg7-dependent macroautophagy, thus indicating microautophagy activation is responsible. Furthermore, the Not4 ligase genetically interacts with endolysosomal pathway effectors to control both RP expression and 40S autophagy efficiency. Overall, we demonstrate that balanced Ccr4-Not ligase activity maintains RP homeostasis, and that Ccr4-Not ubiquitin signaling interacts with the endolysosomal pathway to both regulate RP expression and inhibit 40S ribosomal autophagy.

molecular biology↗

bioRxiv2022

Combination of Volasertib and Rapamycin Inhibits the Regrowth of TSC2-Deficient Tumors

Mutations in TSC1 and TSC2 lead to hyperactivation of mTORC1 and cause Tuberous Sclerosis Complex (TSC) and pulmonary Lymphangioleiomyomatosis (LAM). Rapamycin and rapalogs are potent inhibitors of mTORC1 activity and are approved for the treatment of TSC and LAM. Nevertheless, rapalogs do not cause tumor cell death, and cessation of therapy leads to tumor regrowth. Polo-like kinase 1 (PLK1) interacts with and phosphorylates TSC1, and PLK1 inhibition induces apoptosis and attenuates autophagy in TSC1/TSC2-deficient cells. Here we report that the PLK1 inhibitor volasertib decreases the viability and survival and induces apoptosis in 621-101 cells, a TSC-deficient renal angiomyolipoma cell line from a LAM patient. Combined with rapamycin, volasertib further decreased the survival of 621-101 cells. In vivo, volasertib reduced short-term mouse lung colonization by TSC2-deficient cells and decreased the growth of TSC2-deficient subcutaneous tumors. Mice treated with a combination of volasertib and rapamycin had slower tumor relapse after discontinuation of treatment, compared to rapamycin only. Approximately 35 days after discontinuation of treatment, we observed persistent apoptotic markers and gene expression changes for type I interferon signaling in the regrowth tumors from combination-treated mice, compared to rapamycin only. Notably, combination treatment potently inhibited the growth of subcutaneous tumors derived from the rapamycin-refractory cell line ELT3-245. Taken together, our current work demonstrates that for the management of TSC and LAM disease combination of mTORC1 and PLK1 inhibitors in some cases may be advantageous over currently used rapalog monotherapies.

cancer biology↗

bioRxiv2022

Laboratory evolution and polyploid SCRaMbLE reveal genomic plasticity to synthetic chromosome defects and rearrangements

We have designed, constructed, and debugged a synthetic 753,096 bp version of Saccharomyces cerevisiae chromosome XIV as part of the international Sc2.0 project. We showed that certain synthetic loxPsym recombination sites can interfere with mitochondrial protein localization, that the deletion of one intron (NOG2) reduced fitness, and that a reassigned stop codon can lead to a growth defect. In parallel to these rational debugging modifications, we used Adaptive Laboratory Evolution to generate a general growth defect suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss. The presence of wild-type chromosomes in the hybrid tetraploids increased post-SCRaMbLE viability and heterologous DNA integration, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

synthetic biology↗

bioRxiv2021

Maf1, a repressor of RNA polymerase III-dependent transcription, regulates bone mass

Maf1, a key repressor of RNA polymerase III-mediated transcription, has been shown to promote mesoderm formation in vitro. Here, we show, for the first time, that Maf1 plays a critical role in the regulation of osteoblast differentiation and bone mass. A high bone mass phenotype was noted in mice with global deletion of Maf1 (Maf1-/- mice), as well as paradoxically, in mice that overexpressed MAF1 in cells of the osteoblast lineage (Prx1-Cre;LSL-Maf1 mice). Osteoblasts isolated from Maf1-/- mice showed reduced osteoblastogenesis ex vivo. Prx1-Cre;LSL-MAF1 mice showed enhanced osteoblastogenesis concordant with their high bone mass phenotype. Thus, the high bone mass phenotype in Maf1-/- mice is likely due to the confounding effects of the global absence of Maf1 in Maf1-/- mice. Expectedly, MAF1 overexpression promoted osteoblast differentiation and shRNA-mediated Maf1 downregulation inhibited differentiation of ST2 cells, overall indicating Maf1 enhances osteoblast formation. We also found that, in contrast to MAF1 overexpression, other perturbations that repress RNA pol III transcription, including Brf1 knockdown and the chemical inhibition of RNA pol III by ML-60218, inhibited osteoblast differentiation. All perturbations that decrease RNA pol III transcription, however, enhanced adipogenesis in ST2 cell cultures. RNA-seq was used to determine the basis for these opposing actions on osteoblast differentiation. The modalities used to alter RNA pol III transcription resulted in distinct changes gene expression, indicating that this transcription process is highly sensitive to perturbations and triggers diverse gene expression programs and phenotypic outcomes. Specifically, Maf1 induced genes in ST2 cells known to promote osteoblast differentiation. Furthermore, genes that are induced during osteoblast differentiation displayed codon bias. Together, these results reveal a novel role for Maf1 and RNA pol III-mediated transcription in osteoblast fate determination and differentiation and bone mass regulation.

cell biology↗

bioRxiv2021

HIF-dependent expression of creatine kinase brain isoform (CKB) promotes breast cancer metastasis, and blocking creatine kinase activity with cyclocreatine impairs invasion and improves the efficacy of conventional chemotherapies

The oxygen-responsive Hypoxia Inducible Factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple negative breast cancers (TNBCs) and correlates with poor patient survival. Since inhibiting the HIF transcription factors with small molecules is challenging, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss and gain of function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, formation of invadopodia, and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

cancer biology↗

bioRxiv2020

The mouse cortico-basal ganglia-thalamic network

The cortico-basal ganglia-thalamic loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behavior, and the natural history of many neurological and neuropsychiatric diseases. Classically, the basal ganglia is conceptualized to contain three primary information output channels: motor, limbic, and associative. However, given the roughly 65 cortical areas and two dozen thalamic nuclei that feed into the dorsal striatum, a three-channel view is overly simplistic for explaining the myriad functions of the basal ganglia. Recent works from our lab and others have subdivided the dorsal striatum into numerous functional domains based on convergent and divergent inputs from the cortex and thalamus. To complete this work, we generated a comprehensive data pool of [~]700 injections placed across the striatum, external globus pallidus (GPe), substantia nigra pars reticulata (SNr), thalamic nuclei, and cortex. We identify 14 domains of SNr, 36 in the GPe, and 6 in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify 6 parallel cortico-basal ganglia-thalamic subnetworks that sequentially transduce specific subsets of cortical information with complex patterns of convergence and divergence through every elemental node of the entire cortico-basal ganglia loop. These experiments reveal multiple important novel features of the cortico-basal ganglia network motif. The prototypical sub-network structure is characterized by a highly interconnected nature, with cortical information processing through one or more striatal nodes, which send a convergent output to the SNr and a more parallelized output to the GPe; the GPe output then converges with the SNr. A domain of the thalamus receives the nigral output, and is interconnected with both the striatal domains and the cortical areas that filter into its nigral input source. This study provides conceptual advancement of our understanding of the structural and functional organization of the classic cortico-basal ganglia network.

neuroscience↗

bioRxiv2019

Connectivity characterization of the mouse basolateral amygdalar complex

The basolateral amygdalar complex (BLA) is implicated in behavioral processing ranging from fear acquisition to addiction. Newer methods like optogenetics have enabled the association of circuit-specific functionality to uniquely connected BLA cell types. Thus, a systematic and detailed connectivity profile of BLA projection neurons to inform granular, cell type-specific interrogations is warranted. In this work, we applied computational analysis techniques to the results of our circuit-tracing experiments to create a foundational, comprehensive, multiscale connectivity atlas of the mouse BLA. The analyses identified three domains within the classically defined anterior BLA (BLAa) that house target-specific projection neurons with distinguishable cell body and dendritic morphologies. Further, we identify brain-wide targets of projection neurons located in the three BLAa domains as well as in the posterior BLA (BLAp), ventral BLA (BLAv), lateral (LA), and posterior basomedial (BMAp) nuclei. Projection neurons that provide input to each nucleus are also identifed. Functional characterization of some projection-defined BLA neurons were demonstrated via optogenetic and recording experiments. Hypotheses relating function to connection-defined BLA cell types are proposed.

neuroscience↗