Biology subjects

Friedman, A.

Publications and source records attributed to Friedman, A..

28 records · Page 2Linked to original sources

bioRxiv2021

Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline

Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita (DC), frequently resulting in spontaneous bone marrow failure. While knockout of telomerase in mice has been instrumental in highlighting the importance of telomere length maintenance at an organismal level, it may not be representative of human telomeropathy mutations in vivo. A DC mutation in the shelterin protein TPP1 (K170{Delta}) that compromises telomerase recruitment to telomeres but leaves other functions of TPP1 and the integrity of the telomerase holoenzyme intact is a physiologically relevant tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mutant mouse knocked in for the equivalent of the TPP1 K170{Delta} mutation (TPP1 K82{Delta}) and investigated both its bone marrow and germline compartments in unprecedented detail. TPP1 K82{Delta} caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82{Delta} caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of either empty or severely disorganized seminiferous tubules, and a decrease in both spermatogonia and spermatocytes. It is intriguing that both TPP1 K82{Delta} mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to a robust infertility phenotype at steady state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice. Telomere length maintenance in the human bone marrow can ensure progression to reproductive age, while that in the mouse germline can help meet the elevated demand for sperm to produce multiple offspring.

cell biology↗

bioRxiv2021

A landscape of synergistic drug combinations in non-small-cell lung cancer

Targeted therapeutics have advanced cancer treatment, but single agent activity remains limited by de novo and acquired resistance. Combining targeted drugs is broadly seen as a way to improve treatment outcome, motivating the ongoing search for efficacious combinations. To identify synergistic targeted therapy combinations and study the impact of tumor heterogeneity on combination outcome, we systematically tested over 5,000 two drug combinations at multiple doses across a collection of 81 non-small cancer cell lines. Both known and novel synergistic combinations were identified. Strikingly, very few combinations yield synergy across the majority of cell line models. Importantly, synergism mainly arises due to sensitization of single agent resistant models, rather than further sensitize already sensitive cell lines, frequently via dual targeting of a single or two highly interconnected pathways. This drug combinations resource, the largest of its kind should help delineate new synergistic regimens by facilitating the understanding of drug synergism in cancer.

cancer biology↗

bioRxiv2021

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

biochemistry↗

bioRxiv2020

Ventral tegmental dopamine neurons control the impulse vector during motivated behavior

The Ventral Tegmental Area (VTA) is a major source of dopamine, especially to the limbic brain regions. Despite decades of research, the function of VTA dopamine neurons remains controversial. Here, using a novel head-fixed behavioral system with five orthogonal force sensors, we show for the first time that distinct populations of VTA dopamine activity precisely represent the impulse vector (force exerted over time) generated by the animal. Optogenetic excitation of VTA dopamine neurons quantitatively determines impulse in the forward direction, and optogenetic inhibition produces impulse in the backward direction. At the same time, these neurons also regulate the initiation and execution of anticipatory licking. Our results indicate that VTA controls the magnitude, direction, and duration of force used to move towards or away from any motivationally relevant stimuli. One Sentence SummaryVTA dopamine bidirectionally controls impulse vector and anticipatory behavior

neuroscience↗

bioRxiv2020

Limited Environmental Serine Confers Sensitivity to PHGDH Inhibition in Brain Metastasis

A hallmark of metastasis is the adaptation of tumor cells to new environments. Although it is well established that the metabolic milieu of the brain is severely deprived of nutrients, particularly the amino acids serine and its catabolite glycine, how brain metastases rewire their metabolism to survive in the nutrient-limited environment of the brain is poorly understood. Here we demonstrate that cell-intrinsic de novo serine synthesis is a major determinant of brain metastasis. Whole proteome comparison of triple-negative breast cancer (TNBC) cells that differ in their capacity to colonize the brain reveals that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is the most significantly upregulated protein in cells that efficiently metastasize to the brain. Genetic silencing or pharmacological inhibition of PHGDH attenuated brain metastasis and improved overall survival in mice, whereas expression of catalytically active PHGDH in a non-brain trophic cell line promoted brain metastasis. Collectively, these findings indicate that nutrient availability determines serine synthesis pathway dependence in brain metastasis, and suggest that PHGDH inhibitors may be useful in the treatment of patients with cancers that have spread to the brain. Statement of SignificanceOur study highlights how limited serine and glycine availability within the brain microenvironment potentiates tumor cell sensitivity to serine synthesis inhibition. This finding underscores the importance of studying cancer metabolism in physiologically-relevant contexts, and provides a rationale for using PHGDH inhibitors to treat brain metastasis.

cancer biology↗

bioRxiv2020

A geometric basis for surface habitat complexity and biodiversity

Structurally complex habitats tend to contain more species and higher total abundances than simple habitats. This ecological paradigm is grounded in first principles: species richness scales with area, and surface area and niche density increase with three-dimensional complexity. Here we present a geometric basis for surface habitats that unifies ecosystems and spatial scales. The theory is framed by fundamental geometric constraints among three structure descriptors—surface height, rugosity and fractal dimension—and explains 98% of surface variation in a structurally complex test system: coral reefs. We then show how coral biodiversity metrics (species richness, total abundance and probability of interspecific encounter) vary over the theoretical structure descriptor plane, demonstrating the value of the theory for predicting the consequences of natural and human modifications of surface structure.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ecology↗

bioRxiv2020

Chemical constituents involved in e-cigarette, or vaping product use-associated lung injury (EVALI)

BackgroundThe Centers for Disease Control (CDC) declared e-cigarette (e-cig), or vaping product use-associated lung injury (EVALI) a national outbreak due to the high incidence of emergency department admissions and deaths. Investigators have identified vitamin E acetate (VEA) as the plausible cause for EVALI, based on compounds found in bronchoalveolar lavage fluid. ObjectivesWe defined the chemical constituents present in e-cig cartridges associated with EVALI and compared constituents to medical-grade and cannabidiol (CBD) containing cartridges. MethodsWe measured chemicals and elemental metals in e-liquid and vapor phases of e-cig counterfeit cartridges by Gas Chromatography (GC) and Mass Spectrometry (MS), EPA method TO-15 by GCMS, and ICP-MS analysis. ResultsWe have identified chemical constituents in e-cig vaping tetrahydrocannabinol (THC)-containing counterfeit cartridges compared to medical-grade and cannabidiol (CBD) containing cartridges. Apart from VEA and THC, other potential toxicants correlated with EVALI included solvent-derived hydrocarbons, silicon conjugated compounds, various terpenes, pesticides/plasticizers/polycaprolactones, and metals. These chemicals are known to cause symptoms, such as cough, shortness of breath or chest pain, nausea, vomiting or diarrhea, fatigue, fever, or weight loss, all symptoms presenting in patients with EVALI. ConclusionThis study provides insights into understanding the chemical-induced disease mechanism of acute lung injury.

pharmacology and toxicology↗

bioRxiv2019

The ER cargo receptor SURF4 facilitates efficient erythropoietin secretion

Erythropoietin (EPO), a glycoprotein produced by specialized peritubular fibroblasts in the kidney, is the master regulator of erythropoiesis. EPO is secreted into the plasma in response to tissue hypoxia and stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels. Using this cell line, we performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent sgRNAs resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, consistent with a role for SURF4 as an ER cargo receptor of EPO, we found that disruption of SURF4 resulted in accumulation of EPO in the ER compartment, and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO, ruling out an artifact of heterologous overexpression. This work suggests that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoeisis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

cell biology↗

bioRxiv2019

Blood-brain barrier dysfunction in aging induces hyper-activation of TGF-beta signaling and chronic yet reversible neural dysfunction

Aging involves a decline in neural function that contributes to cognitive impairment and disease. However, the mechanisms underlying the transition from a young-and-healthy to aged-and-dysfunctional brain are not well understood. Here, we report breakdown of the vascular blood-brain barrier (BBB) in aging humans and rodents, which begins as early as middle age and progresses to the end of the lifespan. Gain-of-function and loss-of-function manipulations show that this BBB dysfunction triggers hyperactivation of transforming growth factor {beta} (TGF{beta}) signaling in astrocytes, which is necessary and sufficient to cause neural dysfunction and age-related pathology. Specifically, infusion of the serum protein albumin into the young brain (mimicking BBB leakiness) induced astrocytic TGF{beta} signaling and an aged brain phenotype including aberrant electrocorticographic activity, vulnerability to seizures, and cognitive impairment. Furthermore, conditional genetic knockdown of astrocytic TGF{beta} receptors, or pharmacological inhibition of TGF{beta} signaling, reversed these symptomatic outcomes in aged mice. Finally, we found that this same signaling pathway is activated in aging human subjects with BBB dysfunction. Our study identifies dysfunction in the neurovascular unit as one of the earliest triggers of neurological aging, and demonstrates that the aging brain may retain considerable latent capacity which can be revitalized by therapeutic inhibition of TGF{beta} signaling.

neuroscience↗

bioRxiv2018

Genome-wide prediction of synthetic rescue mediators of resistance to targeted and immunotherapy

Most patients with advanced cancer eventually acquire resistance to targeted therapies, spurring extensive efforts to identify molecular events mediating therapy resistance. Many of these events involve synthetic rescue (SR) interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (the rescuer). Here we perform a genome-wide prediction of SR rescuer genes by analyzing tumor transcriptomics and survival data of 10,000 TCGA cancer patients. Predicted SR interactions are validated in new experimental screens. We show that SR interactions can successfully predict cancer patients response and emerging resistance. Inhibiting predicted rescuer genes sensitizes resistant cancer cells to therapies synergistically, providing initial leads for developing combinatorial approaches to overcome resistance proactively. Finally, we show that the SR analysis of melanoma patients successfully identifies known mediators of resistance to immunotherapy and predicts novel rescuers.

cancer biology↗