2023
2023 Writing Style
Dec 19
KK
Pre-T cell receptor self-MHC sampling restricts thymocyte dedifferentiation
T-cell receptor (TCR) β-selection is the first major checkpoint in T cell development and is critical for establishing a diverse and robust TCR repertoire. During this step, MHC-I-mediated pre-TCR signaling promotes maturation to the double positive (DP) stage; however, the mechanism and role of thymic stromal MHC in this process are highly controversial. To address this fundamental question, the authors employed an in vitro thymocyte differentiation model with wild-type precursors differentiated on MHC-I positive or negative stroma. ScRNA-seq analysis of sorted T cell precursors showed that cells from MHC- stroma successfully progressed to the DP stage, bypassing the β-selection checkpoint in the absence of MHC. Further analysis identified a subset of DP cells from the stromal MHC- samples which expressed both lymphoid progenitor marker genes as well as myeloid and stem-like genes (e.g., Spi1, Mpo, Cd34), suggesting that MHC-mediated pre-TCR signaling is important for maintaining lineage commitment. Although this study failed to replicate its in vitro findings in mice, this work provides valuable insight into a fundamental stage of development.
AJM
Ageing-associated changes in transcriptional elongation influence longevity
Background: Aging impairs diverse cellular processes. Transcription efficiency is a fundamental regulator to all cellular processes. Although the transcriptomic landscape is known to undergo extensive remodeling in aging, it is not known if the process of transcription itself is altered and how this may impact various cellular processes. Herein, we aim to address the hypothesis: accelerated transcription elongation due to aging-associated loss of histone density hinders transcriptional fidelity and drives increased incidence of aging-associated disease. Through the completion of this project, we will establish an impactful driver of aging-associated transcriptional changes across cellular processes.
Preliminary data: To establish common transcriptional defects in aging across mammals and tissue types, we surveyed transcriptomes from rat, mouse, human, fly, and metazoan samples. We quantified the read coverage across introns to determine the kinetics of transcription, Interesting, we revealed an association of age with increased Pol II transcription speed across all species and tissues tested. These findings were validated through a pulse-chase-like test for incorporation of radiolabeled nucleotides across ages. Given chromatin structure is a determinant of Pol II speed, we investigated histone transcript expression and performed mononucleosome sequencing. Indeed, histone expression and overall nucleosome density correlated with Pol II speed in samples.
Approach:
Aim 1: Demonstrate accelerated transcriptional elongation impacts organismal health and longevity.
To this end, we will use ours and other deep sequencing datasets to determine a preference for Pol II increased speed that may disproportionately impact cellular processes. Further, we will establish a casual relationship of increased Pol II speed with the prevalence of transcriptional errors (i.e. misalignment rate, generation of circular RNAs, splicing changes) known to induce age-related disease. Lastly, we will use fly and metazoan Pol II mutants for life-span and organ function to determine functional outcomes.
Aim 2: Demonstrate a casual relationship of diminished histone density on transcriptional elongation.
To this end, we will utilize metazoan and flies with mutations to regulate histone quantity and evaluate Pol II speed. In parallel, we will incorporate diets and drugs known to extend lifespan and track changes in transcription.
Pitfalls/Alternatives: We are assuming that all primary cells used across ages are comparable, however, aging has been documented to cause an accumulation of longer-lived cells. In order to robustly evaluate the rate of transcription in similar cells, we can incorporate cell that have been subjected to replicative senescence.
Dec 6
KK
Epitranscriptomic Modification of MicroRNA Increases Atherosclerosis Susceptibility
Background/significance: Several major risk factors for atherosclerotic cardiovascular disease (ASCVD) increase redox stress on vascular tissue, which can cause 8OH-G oxidative modifications of guanine, leading to a G-to-U transversion. MicroRNAs, which are critical epitranscriptomic regulators, could have major changes in binding to cognate mRNAs when undergoing this G-to-U transversion, leading us to ask whether this mechanism contributes to endothelial dysfunction in atherosclerosis.
Preliminary data: We performed a screen where we treated HUVECs with oxLDL, isolated 8OH-G RNAs using electrophoresis, and performed CLIP-sequencing to identify oxLDL-enhanced miRs. Of the 6 enriched miRs we identified, we observed that 8OH-G miR-483 targets PCSK9, is enriched in serum of hypercholesteremic humans and mice, and was 3-fold higher in low-flow regions of mouse aortas, leading us to hypothesize that 8OH-G modification of miR-483 contributes to ASCVD pathogenesis.
Innovation/approach: We will first transfect HUVECs with miR-483, modified miR-483, or scrambled control, then perform RNA-seq to identify transcriptomic changes. Based on these changes and predicted miR targets, we will perform luciferase reporter assays to confirm differential mRNA targeting by miR-483 and 8OH-G miR-483. We will evaluate the in vivo role of 8OH-G miR-483 using our AAV-mPCSK9 + PCAL atherosclerosis model, infecting mice with lentiviral vectors overexpressing miR483, modified miR-483, or scramble controls under an endothelial cell promoter gene. We will evaluate atherosclerosis progression by quantifying plaque formation and measuring carotid blood flow.
Pitfalls/alternatives:
Because other miRs could contribute to ASCVD, we can perform additional experiments using the other miRs identified in our screen. We could also use luciferase assays to determine whether 8OH-G modification prevents miR-483 target binding or whether it changes the mRNAs that miR-483 binds to.
Nov 15
KK
iPS-cell-derived microglia promote brain organoid maturation via cholesterol transfer
Background: Although 3D-cultured human organoids provide valuable insight into development and disease conditions, existing brain organoid models fail to capture the essential role of microglia during human neural development.
Significance/innovation: To address this critical knowledge gap, we developed a novel iPSC-derived organoid model that incorporates microglia during development.
Approach: Our model takes advantage of our technique to differentiate primitive macrophages from iPSCs (iMac), which we demonstrated can differentiate into microglia-like cells when co-cultured with neuronal cells. We will first generate cerebral brain organoids and iMacs from the same iPSCs, then add iMacs to the organoid culture at the appropriate developmental stage. We will confirm the ability of iMacs to differentiate into microglia-like states in this model via flow cytometry and scRNA-seq, comparing co-cultured organoids to iMacs or organoids alone. We will also examine the contributions of iMac-derived microglia to neuronal development in this model by examining neuronal and glial states via flow cytometry and scRNA-seq, and we will perform electrophysiological experiments to verify improved neuronal functions in our model. We will also compare findings from our model to published transcriptomic data of human and mouse brains and microglia.
Pitfalls/alternatives: Our model attempts to mimic the wave of primitive macrophages which arrive to the developing brain and develop into microglia. We could compare our results to other models, which have added mature microglia to organoid cultures. We could additionally compare myelination states in our model to other models.
Oct 18
KK
Transcriptional linkage analysis with in vivo AAV-Perturb-seq
Background/significance: As the number of genetic variants associated with disease increases, so does the need to understand the biological mechanisms through which these variants contribute to disease. Existing methods to perform large-scale genetic screens are limited to in vitro experiments or to tissues and cell types amenable to lentiviral infection, significantly limiting their translatability.
Innovation: To overcome current limitations, we propose AAV-Perturb-seq, an AAV-mediated direct in vivo single-cell CRISPR screening technique, which will allow for broader application of genetic screenings in vivo.
Approach: We developed an AAV-based CRISPR screening library to deliver gRNAs to target tissues in LSL-Cas9 mice. To test the efficacy of our method, we will infect mice with i.v. injection of a brain-specific AAV.PHP.B library, using genes from the complex 22q11.2 deletion syndrome as a disease model. To maximize tissue applicability of our method, we will perform single-nucleus RNA-sequencing, testing both 5' and 3' capture methods. We will then analyze the effects of these mutations on cellular phenotypes by comparing transcriptomes of nuclei with detected gRNA expression to control nuclei from the same cell type.
Pitfalls/alternative approaches: We will optimize viral titers to ensure that only one copy number per cell is inserted. Due to the depth limitations of snRNA-seq, we will test methods, such as insertion of sortable nuclear tags, into our constructs. Finally, our method assumes that any change in cellular phenotype is due to genomic perturbation within the same cell and not due to environmental changes.
AJM
The β1-adrenergic receptor links sympathetic nerves to T cell exhaustion
CD8 T cell exhaustion to chronic stimuli is a double-edged sword that is crucial to immune regulation but can prevent efficient clearance of pathogens and is a leading cause of ineffective immune checkpoint blockade (ICB) cancer therapies. Chronic antigen stimulation leading to T cell exhaustion has been well studied in vitro; however, how the stress response in tissue contributes to T cell exhaustion progression is less understood. Our preliminary work established the upregulation of beta1-adrenergic receptor ADRB1 on terminally exhausted T cells in vivo and ablation limits the T cell exhaustion to chronic stimuli; thus, this project investigates the hypothesis: Sympathetic stress response hormones released due to pathogens and malignant disease promotes CD8 T cell exhaustion. We will establish the importance of sympathetic signals in the tumor microenvironment to the prevalence of exhausted T cells through modulation of ADRB1 expression, local cognate catecholamine levels, and tumor clearence. Further, to establish the usefulness of modulating stress responses in tumor microenviornments, we will investigate the synergy of beta-blockers with ICB in a well-established model of ICB-resistant melanoma. Through this work, we will reveal a potent regulatory mechanism dictating tumor infiltrating T cell exhaustion that can improve ICB therapy.
Oct 4th
KK
Genotoxic effects of base and prime editing in human hematopoietic stem cells
Base editing (BE) and prime editing (PE) are two novel technologies that allow gene editing at single-nucleotide resolution, but their cellular toxicity is unknown. To evaluate their relative efficiency and toxicity compared to Cas9 KO, the authors used two types of BE to target the B2M gene in human hematopoietic stem/progenitor cells (HSPC). The BEs were less toxic than Cas9 and more efficiently knocked out the target gene, but DNA sequencing revealed that BEs still caused large deletions and translocations. The authors transplanted HSPC colonies into immune-deficient mice and found that BE-edited samples repopulated more effectively than Cas9 samples but less effectively than controls. Whole exome sequencing of HSPCs from the in vitro and in vivo experiments also showed an increased off-target mutational burden from a cytidine BE but not an adenine BE. While this study highlighted several limitations of BE technologies, it also demonstrates their potential for future applications.
AJM
Reductive carboxylation epigenetically instructs T cell differentiation
T cell differentiation into effector versus memory cells is characterized by distinct metabolic programs regulated by the enzyme IDH2; However, how metabolic changes drive differentiation fates remains poorly understood. ATAC-Seq of T cells supplemented with IDH2-regulated metabolites revealed changes in metabolite ratios directly regulate downstream target activity to impact chromatin openness at memory-relevant genes. Importantly, use of IDH2 inhibitors (IDH2i) during chimeric antigen receptor (CAR) T cell generation increased proportion of T cells with memory traits and boosted tumor control following re-challenge. These findings demonstrate a regulatory importance of metabolic programs in cell differentiation that can be translated to improve autologous cell transfer therapy efficacy.
Bill
‘Alternative splicing modulation by G-quadruplexes’, https://pubmed.ncbi.nlm.nih.gov/35504902/
While alternative splicing is an important contributor to gene regulation, less is known about the mechanisms regulating alternative splicing itself; G-quadruplex (G4) formation is proposed as one such mechanism. A first analysis reveals that DNA G-quadruplex motifs are enriched 3-fold near splice junctions. Secondly, RNA G4s are enriched at excluded exons (experimentally validated), but also promote inclusion elsewhere. Associating RNA G4s with splicing outcomes is significant because RNA G4s are more stable than DNA G4s, suggesting that RNA G4s may have increased functional impact in the transcriptome compared to the genome. A concern is that the authors report greater fold increase on non-template (coding) DNA strands, but do not acknowledge that such an association is expected, since G4s have been strongly linked with gene promoters in the past. While these findings are suggestive, an experiment is needed to verify that, among many factors influencing gene expression, G4 formation alone can change the outcome of an expression pathway.
09/13/2023
AJM
Intracellular tPA–PAI-1 interaction determines VLDL assembly in hepatocytes
Low levels of tissue plasminogen activator (tPA) is a biomarker for cardiovascular disease, assumed to be from tPA’s well-established thrombolytic activity; however, low tPA levels can directly contribute to increased circulating cholesterol-bound apolipoprotein B (apoB) levels, suggesting a regulatory relationship. Herein, the authors investigated tPA’s influence on very low- and low-density lipoprotein (VLDL, LDL) and apoB loading, revealing tPA knock-down in hepatocytes increased plasma lipid-loaded apoB. Proximity ligation assays revealed direct interactions of tPA to apoB in hepatocytes through tPA’s non-catalytic Kringle 2 (K2) domain to prevent apoB lipid loading. Interestingly, tPA’s K2 domain is inhibited by SERPINE1 (PAI-1) and humans deficient for SERPINE1 have reduced plasma lipid-loaded apoB. These findings expand tPA’s importance in cardiovascular disease and elucidate a novel regulatory mechanism of tPA-SERPINE1 for plasma triglycerides.
KK
A viral ADP-ribosyltransferase attaches RNA chains to host proteins
Traditionally, protein-RNA interactions have been understood to be transient and almost exclusively non-covalent. In this paper, the authors found that ADP-ribosyltransferases (ARTs) from the T4 bacteriophage covalently attached NAD-capped RNAs to specific host proteins in E. coli. The authors incubated viral ARTs with known target proteins and radiolabeled NAD-RNA and found that the ARTs attached the labeled NAD-RNA to their targets, which they confirmed via mass spectrometry. In vivo experiments using phages with mutant ARTs found a dramatic reduction in viral production, which the authors hypothesized was due to modification of host translational machinery. While this paper did not identify the direct effects on protein function caused by "RNAylation", the mechanism identified here has significant implications for our understanding of RNA-protein interactions.
AUG 23
AJM
Coupling cellular secretome and transcriptome can permit direct analysis of dynamics underlying cell actions. Herein, the authors analyzed IgG-production using secretion with single-cell sequencing (SEC-Seq) to identify transcriptomic markers associated with the quantity of IgG produced. SEC-Seq utilizes 35um nanovials that are studded with anti-IgG capture antibodies; cells were incubated on the nanovials to accumulate IgG, then stained with oligo-tagged anti-IgG antibodies and directly input into the 10X Chromium system. Nanovials did not significantly impact the reading depth, doublet rate, or gene count. As expected, the authors demonstrated mRNA expression of know IgG-expressing plasma cell protein markers, such as CD38, XBP1, MZB1, and IGHGxs were correlated with IgG production. Interestingly, SEC-Seq enabled identification of CD59, ITM2C, HLA-C, and CYBA as additional correlates to IgG production. This technology can be expanded to investigate a range of cell secretions (cytokines, chemokines, etc.) across multi-omic platforms (sc-ATAC, sc-BCR/TCR, etc.).
KK
RNA polymerase II associates with active genes during DNA replication
Maintaining transcriptional states is essential for cells to keep their identities and functions during DNA replication, and current theories assume that transcriptional machinery must wait for restoration of chromatin structure before resuming transcription. In this study, the authors demonstrate that RNA Pol II and other transcriptional machinery re-associate with active genes to re-initiate transcription immediately after passage of the DNA replication fork. Using proximity ligation assays (PLA) and super-resolution microscopy, the authors found that Pol II and generic transcription factors associated with both the leading and lagging strands of nascent DNA within 5 minutes after synthesis. Additional PLAs using labeled nucleotides showed that Pol II does not need to return to the transcription start site but can immediately resume transcription of immature RNAs. This study challenges existing ideas and provides a new potential mechanism for a fundamental process.
PhyL
Organization of the human intestine at single-cell resolution
The first multiplexed imaging of the intestine was developed by mapping out the organization of single cells across the intestinal tract. The mapped data of cell population, regulation, and distribution spans eight sections of the intestine. Single-nucleus RNA-seq(transcriptions) and CODEX(staining) were used to map the cells throughout the intestine sections. This map can be used as a reference to what a healthy intestinal tract should look like and help identify certain cell types present in gastrointestinal diseases. The single-cell map also shows which cell types have broad or specialized functions by showing how spread the cell type is around the intestines. This multiplex imaging will allow more in-depth research about the intestine and diseases that affect it.
Aug 9
AJM
A framework for individualized splice-switching oligonucleotide therapy
Splice-switch antisense oligonucleotides (ASOs) target cis-regulatory elements to alter target exon inclusiveness and present a promising personalized therapeutic strategy for genetic conditions. However, their utilization has been limited given the effort required to identify ASO-amendable pathogenic mutations. Herein, the authors investigate ASO-targetable ataxia-telangiectasia mutations in a 235 individual cohort to establish a framework for future development of ASOs. 220 unique pathogenic variants were predicted using SpliceAI and REVEL on whole-genome sequencing. Importantly, the authors develop a taxonomy to categorize these mutations as probable/possible/unlikely ASO-amendable variants for further development. For one mutation, c.7865C>T, 32 ASOs were evaluated for enriched functional ATM transcripts and ATM-induced radiation response, of which AT008 was selected for clinical development. In an ongoing trial, one individual with c.7865C>T received intrathecal injection of AT008 for the past three years. Overall, the author’s framework to reveal new pathogenic variants and identify ASO-amendable targets in individuals will promote future development for personalized ASO therapy.
KK
Hepatitis C virus RNA is 5′-capped with flavin adenine dinucleotide
Hepatitis C virus (HCV) is an RNA virus which causes chronic liver infection and cirrhosis and can lead to cancer, and the mechanisms it employs to establish infection are poorly understood. In this study, the authors hypothesized that HCV uses a 5' RNA cap as one tool to overcome host defenses. Using CapZyme enzyme-based RNA sequencing to detect caps, they found that 75% of HCV RNAs had a 5' cap made from the metabolite flavin adenine dinucleotide (FAD). In vitro infection of liver cells determined that removal of FAD or its precursors inhibited HCV replication. Further experiments using reporter cells demonstrated that 5' FAD capping prevented detection by RIG-I, allowing HCV to prevent innate immune activation. This study identifies a novel mechanism for viral immune evasion which can be explored in other pathogens.
PhyL
As COVID-19 cases rose, so did diabetes — no one knows why
Type 1 diabetes cases in young people surged during the COVID pandemic, however there is no conclusive cause for it. The research team first theory is that the COVID virus itself was damaging the pancreas. This theory is unlikely though because there was no substantial damage done to pancreatic cells by the virus. Another possible theory is that a COVID infection might have triggered the immune system to start attacking the body while trying to fight off the virus. Even if the COVID virus is not responsible for Type 1 diabetes, researchers can still look for factors and triggers in the environment and lifestyle of people during the pandemic, such as diet and limited exposure to different illnesses.
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July 27
KK
Loss of CDK4/6 activity in S/G2 phase leads to cell cycle reversal
Mitosis, a fundamental cellular process, has long been understood to become irreversible at the "restriction point" of the cell cycle, defined by CDK2 inactivation of Rb. This study challenges the existing model and proposes a new model where the decision to proliferate is dependent on competition between "molecular clocks" for mitosis and cell cycle exit. The authors performed high-throughput single-cell imaging and tracking to follow cell fates after inhibition of various mitogen signaling molecules, and they found that CDK2 activity and Rb phosphorylation are reversible in all cells. They performed further experiments manipulating nutrient availability in addition to mitogen signaling to shift the balance between the proposed "molecular clocks" and were able to successfully direct cells toward specific fates. This study provides a new model for a key cellular switch underlying an essential cellular process.
AJM
Alternative CDC20 translational isoforms tune mitotic arrest duration
Errors in spindle assembly initiate mitotic arrest through inhibiting CDC20 interactions with anaphase-promoting complex (APC/C); however, how cells regulate the duration of mitotic arrest to prevent premature mitotic exit is unclear. Herein, the authors found three co-occurring CDC20 isoforms, Met1, Met43, and Met88, that result from leaky ribosome scanning due to alternative start codons within the same mRNA, known as proteoforms. Differential proteoform enrichment through CRISPR-Cas9 editing revealed Met1 and Met43 can both independently activate APC/C-mediated mitotic progression, while Met43 is not inhibited by spindle-assembly checkpoint proteins MAD2 and CDK. In normal cell cycling, the differential turnover rate of Met1 and Met43 proteins leads to decreased Met1:Met43 ratio, allowing Met43 to activate APC/C and cause mitotic progression, a mechanism that is commonly exploited across cancers to escape mitotic checkpoints and resist anti-mitotic drugs. The authors characterized a novel mechanism of mitotic control with the potential to bolster anti-mitotic drug efficacy.
Phyd
Plastic pollution on the world’s coral reefs
Ivy
Structural basis of human separase regulation by securin and CDK1–cyclin B1
Chromosome segregation is triggered by separase that cleaves the cohesion subunit SCC1 and activated by degradation of securin and cyclin B; however, the regulation of separase is unclear. Therefore, the authors used cryogenic electron microscopy to examine the structures of human separase. In result they found that CDK1-cyclin B1 binds and inhibits separase which provides the molecular basis for control of chromosome separase; CDK1-cyclin B1 inhibits separase by deploying pseudo substrate motifs and loops. From this we see how it allows for future prevention of aneuploidy and tumorigenesis.
July 13
AJM
Reprogramming tumour-associated macrophages to outcompete cancer cells
While the genetic alterations underlying cancers are unique across individuals, malignant tumors all arise from highly competitive transformed cell that similarly exploit microenvironmental dynamics. Herein, the authors utilized hyperactive MYC (MYCHI)-derived mammary cancer to investigate cell-to-cell competition driving tumor growth. Single-cell RNA-Seq revealed MYCHI cell competitiveness relies on increased mTORC1-dependent protein metabolism. Under low-protein diets, MYCHI tumors are enriched in mTORC1-activated macrophages. Receptor knock-out and fluorescent tracing studies demonstrated that as professional scavengers, macrophages are more fit in engulfing and utilizing cellular debris in low-protein environments, reducing tumor burden by starving MYCHI tumors without impairing macrophage activation. Although this paper focuses on MYC-driven mammary tumors, understanding fundamental environmental cues that control cell-to-cell competition is crucial in cancer biology.
Ivy
GDF15 promotes weight loss by enhancing energy expenditure in muscle
Recombinant GDF15 (growth differentiation factor 15) is a better alternative for weight loss compared to a caloric restriction. The authors compared mice that were fed a high fat diet and treated with GDF15 versus a caloric restriction; the ones treated with GDF15 were given either one or five nmol per kg. In result they found that GDF15 exponentially decreased body fat in mice to around 17.0% while the ones on caloric restrictions only lost around 5%; this is because GDF15 signaling through the GFRAL (GDNF family receptor alpha-like) suppresses the appetite while not exerting energy expenditure in the muscles. From this weight loss will now have an effective strategy.
KK
Self-patterning of human stem cells into post-implantation lineages
In vitro cultures of human pluripotent stem cells (hPSCs) have failed to replicate embryonic structures, limiting the ability to model human embryonic development. In this study, the authors report a new culture approach using hPSCs maintained under conditions supporting intermediate pluripotency states, causing the cells to form 3D aggregates which eventually resemble human embryos 9 days post-fertilization. The authors confirmed the similarity of these cultures to known embryonic states using single-cell RNA sequencing and fluorescent microscopy. While these "human Extra-Embryoids" have some major differences to human embryos, this model will allow scientists to study embryonic development in new ways.
PhyL
A mitotic glue for shattered chromosomes
Two types of proteins, CIP2A and TOPBP1, are responsible for aiding in chromothripsis, which is incorrectly rearranging shattered chromosome fragments back together. Chromosomes that come from chromothripsis are frequently found in cancer tumor cells. Cells lacking CIP2A and TOPBP1 cannot produce chromothriptic chromosomes and will trigger an immune response. Tethering back broken chromosomes is crucial for the viability of a cell. This new information can potentially help to develop future cancer treatments and understand how cancer cells are made.
June 28
AJM
A pan-influenza antibody inhibiting neuraminidase via receptor mimicry
Current vaccine strategies to seasonal influenza rely on predicting variants, necessitating yearly vaccination with fluctuating efficacy. Herein, the authors screened 200 healthy individuals for antibody reactivity to influenza neuraminidase (NA) in search of a potential prophylactic monoclonal antibody. One participant harbored diverse NA-reactive antibodies derived from a single clonal family; three of these antibodies displayed broad neutralization capacity to 15 seasonal and animal circulating strains and one, denoted FNI9, retained reactivity to recent evolutionary viral escape variants. Prophylactic administration of FNI9 was able to prevent severe disease in mouse models challenged with current circulating strains of influenza. While this work provides a basis for a new prophylactic for current and potential zoonotic influenza strains, it does not necessarily negate the need for yearly vaccines and monitoring of variants.
KK
Transfer learning enables predictions in network biology
In order to model high-dimensional transcriptomic regulatory networks, large amounts of data are needed, hampering efforts to characterize networks in rare diseases and tissues. In this study, the authors took advantage of transfer learning to create model called Geneformer to predict transcriptomic networks. Geneformer was pretrained on a set of 30M single-cell transcriptomes to learn gene relationships in a generalized context, and model fine-tuning was performed on smaller context-specific data sets. After fine-tuning, Geneformer successfully predicted transcriptomic network changes in multiple contexts including heart disease, in silico gene perturbation, and Notch signaling during development. While the authors of this study could have more clearly explained the rationale for their methodology and testing, this model is an exciting application of transfer learning that can power network characterization in novel contexts.
Ivy
SC -β (stem cell beta) cells have the potential to provide a cure for patients with type 1 diabetes. SC-β cells have similar features to human β cells such as their ability to secrete insulin in response to glucose and to reverse severe diabetes. Even with these similarities there still seems to be inefficiencies in these derived cells. This is shown when the authors utilized single cell RNA sequencing to characterize the transcriptional environment of SC-islets to demonstrate that SC-β cells express many, but not all-important genes found in primary β cells. Therefore, the authors began modulating the chromatin state of SC-β cells to drive the cells closer to its vivo counterpart. In result, soon β cells will be able to be created from stem cells providing patients with diabetes a new life.
PhyL
Stem cell–derived heart cells injected into first patient
The clinical trial that uses iPSC to regenerate smooth heart muscle cells was a success. The cells are cultured by reverting skin and blood into an embryonic state while also eliminating any pluripotent stem cells to avoid teratomas. Once injected, patients will have to go onto an immunosuppressant to protect the injected cells. Recent studies from the group show risks of the treatment such as partial engraftment as well as scar tissue formation. Ten more patients are scheduled to be enrolled before 2024 to continue developing the treatment.
June 14
KK
Myelin dysfunction drives amyloid-β deposition in models of Alzheimer’s disease
Alzheimer's disease (AD) is a devastating neurodegenerative disease characterized by harmful amyloid-β (Aβ) plaque deposits in the brain, and the cause of plaque formation is unknown. The authors of this study observed myelin damage in brain samples from patients with AD and hypothesized that loss of myelin integrity could be an upstream risk factor for Aβ. To test this hypothesis, they bred AD-prone 5xFAD mice with mice containing myelin defects and found accelerated plaque deposition and neurological changes. Importantly, similar phenotypes persisted using acute demyelination models in 5xFAD mice. The authors performed bulk and snRNA sequencing on microglia and found that AD microglia acquired either "plaque-corralling" or "disease-associated" phenotypes, leading the authors to propose that myelin damage "distracts" microglia from plaque mitigation functions. While this study did not functionally validate the findings of the microglia sequencing, it may have identified a novel mechanism driving AD pathology.
Ivy
Deterministic evolution and stringent selection during preneoplasia
Experimental evolutions have yielded fundamental insights into clonal dynamics in microorganisms and the characteristics of mutant clones; despite these new insights the clonal expansions for malignant cells established in cancers are obscured. Therefore, to identify the expansion of such cells the authors conducted a two-year study on non-malignant HGOs (human gastric organoids) as the basis to studying preneoplasia induced by TP53 deficiency, which in result found that it elicited aneuploidy. Resulting from this, hallmarks of gastro-esophageal tumorigenesis were found in correlation to clonal interference. Overall, the findings implied the predictability in the earliest stages of tumorigenesis and the malleability of HGOs. In the future the authors could research preneoplasia in other cancers.
AJM
Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum
The endoplasmic reticulum (ER) undergoes continual remodeling through autophagy (termed ER-phagy), mediated in part by the ER receptor FAM134B; however, regulatory mechanisms of FAM134B are unknown. Herein, the authors interrogate the role of ubiquitination in FAM134B-regulated ER-phagy via structural modeling and high-resolution imaging. Having established FAM134B is ubiquitinated following induction of ER-phagy, the authors employed Coarse-grained Molecular Dynamics simulations to predict intra- and inter-molecular dynamics, suggesting ubiquitination promotes dense, multimer FAM134B clustering. In vitro membrane models and high-resolution imaging in cells confirmed FAM134B clustering is bolstered by ubiquitination and prompts ER blebbing that colocalized to autophagosome markers. Further work found E3 ligase AMFR is responsible for FAM134B ubiquitination, overall providing a full depiction of a crucial regulatory mechanism of FAM134B-mediated ER-phagy to permit future research in ER remodeling in disease.
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May 31
KK
Peroxisome biogenesis initiated by protein phase separation
Peroxisomes are organelles that are essential to cellular function, but the mechanism through which proteins enter the peroxisome is unknown. In this study, the authors used a yeast model to test the hypothesis that transmembrane proteins form a large pore for molecular transport. The authors expressed an mCherry construct fused to a peroxisome targeting sequence to measure protein import, and they found that mutations in the intrinsically disordered regions (IDR) of peroxisomal proteins Pex5 and Pex13 disrupted peroxisomal transport. The authors then hypothesized that Pex5 and Pex13 IDRs partition cargo via liquid-liquid phase separation (LLPS), and they observed formation of Pex5-Pex13-mCherry condensates in phase-separation buffer consistent with LLPS formation. This work establishes a mechanism for an essential biological process, and future work can characterize the roles of peroxisomal LLPS in human disease.
AJM
Interplay between calcium and sarcomeres directs cardiomyocyte maturation during regeneration
Studying how certain animal species can regenerate their hearts following cardiac injury could provide novel therapeutic strategies. Although cardiomyocyte dedifferentiation has been well studied, mechanisms driving redifferentiation are less appreciated and vital to resume heart function. Herein, trajectory analysis of scRNA-Seq revealed Lrrc10 is correlated with maturation in both embryonic and regenerating cardiomyocyte. Structural analysis of the cardiac dyad in knock-out fish demonstrated Lrrc10 drives the assembly of the cardiac dyad following dedifferentiation, permitting maturation. Future work into Lrrc10’s protein interaction will be necessary to understand its function in this context.
Ivy
Formin-mediated nuclear actin at androgen receptors promotes transcription
The signal-regulated nuclear actin DAAM2 directly promotes actin polymerization which is vital for prostate specific antigen expression in cancer cells; however, it doesn’t explain the clear diagnosis for PAIS (partial androgen insensitivity syndrome) which is characterized by under virilized genital phenotypes. Although CAIS (complete androgen insensitivity syndrome) can be diagnosed, patients with PAIS are left without a clear diagnosis; therefore, to identify the AR activity the authors performed an assay to find the mutation groups responsible for AIS and PAIS. The authors discovered that AR activity was significantly lower in an AR mutation group of AIS compared to the male control group GSF (genital skin fibroblasts), named AIS type II; the AIS type II showed heterozygous variants in the DAAM2 gene in individuals with hypospadias, lack of Mullerian structures, and normal plasma testosterone. Overall, the findings allow us to highlight DAAM2 as a pharmacological target for AR related diseases. In the future they could research how DAAM2 is involved in other disease conditions.
May 17
LQ
A druggable copper-signalling pathway that drives inflammation
The mechanism underlying Cd44-regulated inflammation is not well understood. The authors discovered that Cd44 can increase cellular uptake of copper, which can further promote the reduction of NADH to maintain the level of NAD+ to favor cells toward inflammatory state. This discovery was also confirmed in mice with SARS-cov2 infection, endotoxemia, or cecal ligation and puncture.
KK
Genomics of perivascular space burden unravels early mechanisms of cerebral small vessel disease
Perivascular space burden (PVS) is a magnetic resonance imaging marker of cerebral small vessel disease (cSVD), a leading cause of stroke, but the biology of PVS is poorly understood. To identify genetic factors associated with PVS burden, the authors performed GWAS analysis of CHARGE and UK Biobank cohorts (n > 40,000). The authors discovered that most of the identified loci did not correlate with known cSVD risk factors, leading them to conclude that these genes represent novel disease mechanisms. The authors validated these loci using TWAS and eQTL analysis in GTEx and other cohorts, and they used pathway enrichment tools to describe biological functions associated with the identified genes. Though this study used data dominated by patients with European ancestry and the authors did not convincingly explain the rationale behind some of their strategies, this study thoroughly assessed traits associated with an emerging risk factor for cerebrovascular disease and identified novel targets for biological discovery.
AJM
DNMT3A is a well established de novo DNA methyltransferase and crucial to hematopoietic stem cell (HSC) differentiation; However, its importance for rapid cellular changes during acute HSC activation cannot be explained by DNA methylation alone. Herein, the authors reveal DNMT3A is necessary for crucial splicing changes in acute HSC activation that are independent of its methylation function. Instead, co-immunoprecipitation and proximity ligation assays demonstrate DNMT3A recruits core early-spliceosome proteins to active RNA polymerase II. Insufficient DNMT3A led to destabilizing intron retention in thousands of genes necessary for cell cycling and other key cellular processes, leading to reduced global mRNA and protein levels and halting HSC differentiation. Overall this research revealed a novel function of DNMT3A to explain its importance to acute HSC activation. Future work should explore how cancer-related mutations in DNMT3A confer stem-like transformations.
May 3rd
KK
Tracking chromatin state changes using nanoscale photo-proximity labelling
Understanding protein-protein interactions is key to biological discovery, and determining the nature of these transient and multivalent interactions without disrupting them is a major challenge. In this paper, the authors present a nanoscale proximity-labeling technique called µMap to capture close-range interactions between nuclear proteins. µMap incorporates intein-bound Ir-photocatalysts into nuclear proteins via protein trans-splicing, and photoactivation catalyzes the insertion of reactive carbene labels into neighboring proteins. The authors validated this method on H2A in cells expressing a common oncogenic H2A mutation and in cells treated with a selective methyltransferase inhibitor. Though the specificity of the labeling in this technique could have been more effectively demonstrated, this technique represents an advancement with exciting potential.
LQ
Spatial epigenome–transcriptome co-profiling of mammalian tissues
To conquer the technology obstacle of capturing multi-layer information from the same sample, the authors utilized spatial ATAC–RNA-seq and spatial CUT& Tag–RNA-seq on the section of mouse brain and the hippocampus of human brain to get their epigenome and transcription information and investigate how the former influences the latter as well as cell dynamics in the tissue. Many similarities in tissue characteristics were identified by both spatial epigenomic and spatial transcriptomic analyses; notable differences in the tissue features were also noticed indicating the unique function of the two methods in determining cellular states. It becomes possible to uncover new messages buried in tissues through connecting epigenome to transcriptome. This study means biotechnology has reached a new level.
AJM
Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase
Histone modification is tightly regulated and accomplished by an array of protein; yet, how modifications are directed is unique to each protein. In this paper, the authors investigate the histone specificity of Polycomb repressive deubiquitinase (PR-DUB) complex through cryo-electron microscopy and targeted mutagenesis. Three crucial findings are (A) the active component of PR-DUB, BAP1, is responsible for nucleosome binding through its finger-like domain; (B) the conformation of the BAP1’s finger domain is dependent on binding to ASXL; (C) these domains indirectly impact PR-DUB deubiquitinase activity. By elucidating PR-DUB domains’ functions, the authors contextualize several mutations to PR-DUB that are present in various cancers.
April 19th
KK
Programmable protein delivery with a bacterial contractile injection system
One major challenge in developing targeted therapeutics is the ability to deliver protein payloads to specific cells in vivo. In this study, the authors engineered a tool by modifying extracellular contractile injection systems (eCISs), which are bacteria-derived syringe-like complexes that deliver proteins into eukaryotic cells. The authors first used AlphaFold to design modified eCISs that could bind to human membrane receptors, which they validated by delivering GFP and other payloads to several cell lines. The authors demonstrated this technology in vivo using intracranial injections of a Cre-loaded eCIS into loxP-tdTomato mice, assessing brain sections via fluorescent microscopy and flow cytometry. Although this study did not assess target specificity in vivo or convincingly demonstrate a lack of immunogenicity, this technology represents an exciting advance with both basic and translational applications.
LQ
Decrypting drug actions and protein modifications by dose- and time-resolved proteomics
The full-view of the molecular action of a drug used for cancer treatment has not been well developed. The authors adopted LC-MS/MS to measure proteins or peptides collected from cancer cell lines treated with different drugs with different dosages and time points. With the protein datasets, the authors exhibited the function of DecryptM in predicting the molecular mechanism, target, pathway engagement, and potential drug combination for 6 different types of cancer drugs. To truly show the function of DecryptM in the future, the study could also incorporate specimens obtained from cancer patients undergoing treatment.
AJM
BTG1 mutation yields supercompetitive B cells primed for malignant transformation
Diffuse large B cell lymphomas (DLBCL) are typically derived from transformed germinal center B cells (GCB); however, regulatory elements of GCB-dynamics underlying DLBCL pathogenesis have not been characterized. Herein, the authors identified the N-terminus of BTG1 as a common mutation site in DLBCL, but its role in GCB dynamic was unknown. Polysome profiling revealed BTG1 N-terminus mutation (BTGN1MUT) hinders its ability to repress Myc translation, resulting in faster T follicular helper (TFH)-induced Myc-signaling. Utilizing an in vivo system of complementary TFH and BTGN1WT or BTGN1MUT B cells, the authors found rapid Myc-signaling in BTGN1MUT B cells resulted in decreased time between proliferation burst, allowing BTGN1MUT to outcompete BTGN1WT GCBs. Overall, the authors have elucidated a role for a predominate DLBCL oncogene, BTG1, as an important regulator of GCB cell competition as well as established its power to stratify patient outcomes.
April 5th
LQ
The traditional method of calculating BMI lacks sensitivity in accurately identifying the different types of metabolic health conditions. To fill this knowledge gap, the authors developed models using blood multiomics data collected from 1,277 individuals, which include metabolomics-based BMI(MetBMI) , proteomics-based BMI(ProtBMI), clinical labs (chemistries)-based BMI (ChemBMI) and combined omics-based BMI (CombiBMI).The omics-based BMI models more accurately capture the diverse metabolic health states associated with obesity. Notably, the MetBMI model was able to track BMI changes resulting from lifestyle interventions and showed a stronger correlation with the gut microbiome structure.
AJM
Microglia-mediated T cell infiltration drives neurodegeneration in tauopathy
Alzheimer’s disease (AD) is characterized by neurodegeneration sparked by accumulation of amyloid and tau proteins. Inflammation plays a pivotal role in AD neurodegeneration but the impact of adaptive immunity is unresolved. Herein, the authors detail the T cell immune compartment localized to tau-mediated neurodegeneration in mice. Utilizing immunohistochemistry, combined single cell -RNA and TCR-Seq, co-culture systems, and T cell depletion, the authors demonstrate T cells can respond to antigens presented on microglia, expand specifically within the brain tissue, and augment inflammation-driven tau-mediated neurodegeneration. Likewise, they show human AD-patient brain samples demonstrate T cell accumulation in AD plagues as well. Overall, this work demonstrates adaptive immune responses underlying AD pathology and potentiates novel therapeutic discoveries.
KK
A swapped genetic code prevents viral infections and gene transfer
Viral contamination of cell lines and genetically modified organisms (GMO), is a major problem, but previous attempts to encode viral resistance into GMO genomes have had limited success. In this study, the authors further engineered an existing model for virus-resistant E. coli (Syn61Δ3), where all TCG and TCA codons (TCR) and the TAG stop codon were replaced with synonymous alternatives, and the corresponding serine tRNAs and release factor were deleted. To identify factors contributing to successful infection of Syn61Δ3 cells, the authors infected them with phages isolated from the environment, and they found that lytic phages overcame genetic-code-based viral resistance by complementing cellular tRNA with virus-encoded tRNAs which had a high affinity for their cognate codons. The authors then engineered Syn61Δ3 cells for further resistance by re-introducing high-affinity tRNAs encoding for leucine that matched TCR codons, which successfully blocked replication. While this study is limited in the scope of viruses used for testing, it represents a fascinating advancement toward safer and more effective genetic modification.
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March 8th
KK
Many human type I interferons (IFN) signal through JAK-family tyrosine kinases downstream of a common interferon receptor (IFNAR), leading to the question of what determines IFN functional signaling diversity. Because IFNAR activation of the JAK-STAT pathway requires endocytosis, the authors of this study hypothesized that endosomal sorting controls functional diversity in type I IFN signaling. To this aim, they stimulated human retinal epithelial cells (RPE1) with IFNa or IFNb and observed via confocal microscopy that stimulation with IFNa selectively promoted the interaction of IFNAR1 with Hrs (a tyrosine kinase substrate) in the early endosome, and shRNA-mediated depletion confirmed that IFNAR1-Hrs interaction is required for IFNa-induced JAK-STAT signaling. The authors next used co-IP and microscopy to find that the endosomal protein STAM2 constitutively associated with IFNAR1 and the JAK protein TYK2, inhibiting TYK2 activation of STAT1. Blocking endocytosis led to the discovery that Hrs association with STAM2 allowed for STAT1 activation, leading to the conclusion that IFN-IFNAR endocytosis removes STAM blockade of TYK2, allowing JAK-STAT activation. Though the paper could have more precisely defined the mechanisms controlling functional signaling diversity, this work opens fascinating novel avenues of study into cellular signaling.
https://www.nature.com/articles/s41556-022-01085-6/figures/8
LQ
Non-invasive mapping of systemic neutrophil dynamics upon cardiovascular injury
There is currently no non-invasive and background-free approach available for monitoring neutrophil activity changes following a cardiovascular injury. Earlier studies have reported that the neutrophil-specific peptide (NP) has a specific binding affinity to the neutrophil-specific receptor CD177, and that phagocytic immune cells can take up perfluorocarbons (PFCs). The authors modified NP with malPFC (PFC +DSPE-PEG2000maleimide) and injected it into mouse or added it to cell culture medium, later the signals were captured by 1H/19F MRI. During myocardial infarction or LPS stimulation, neutrophils are activated and exhibit an upregulation of CD177, which leads to an increase in the labeling of neutrophils by NPPFC. This method can serve as an indication of inflammation or cardiac damage in human.
AJM
Atherosclerosis progression and severity is impacted by T cell actions within arterial tissues; however, whether T cell actions are in response to systemic changes or driven by plaque-specific autoantigen is a vital, unanswered question. Herein, the authors coupled single-cell RNA and TCR-sequencing to evaluate T cells in plaques, adventitial artery tertiary lymphoid organs (ATLOs), draining lymph nodes (dLNs), and circulation under normal and atherosclerotic conditions. scTCR-seq revealed a significant proportion of plaque-CD8+ T cells are derived from clonal expansion, with clones in ATLOs and dLNs but not present in circulation; thus, it can be inferred that CD8+ T cell actions are driven by plaque-derived antigens. RNA-seq analyses further supports this inference, as plaque-CD8+ T cells across human and mouse datasets are enriched in activation-related markers and devoid of key check-point molecules compared to other locations. This paper provides crucial support to the theory of autoimmune-driven atherosclerosis and the authors call for further controlled in vivo experiments to validate.
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Feb 22, 2023
LQ
Sex-dimorphic and age-dependent organization of 24-hour gene expression rhythms in humans
It hasn't been well studied that how sex and age determine the tissue-specific gene expression rhythms. The authors modeled the circadian rhythm of the genes from GTEx datasets as a sinusoidal function with a fixed period of 24 hours. The study determined clock transcripts showed consistent timing relationships and synchronization across the body. mRNA rhythms varied in tissue-specific functions, such as metabolic pathways and systemic responses. While the network constituted by core genes and other related clock genes was similar between sexes and age groups, gene expression rhythms were highly sex-specific and more stable in females. The authors also observed a general decrease in rhythmic programs with age throughout the body.
AJM
NET formation is independent of gasdermin D and pyroptotic cell death
Neutrophil-release of double-stranded DNA, termed NETosis, has been proposed to be mediated by the pore-forming GSDMD. Herein, the authors employ GSDMD-knockout mouse neutrophils to directly address this hypothesis, finding a lack of GSDMD does not impact the level of dsDNA released. Although an important gap in the field was investigated, the authors do not rigorously evaluate alternatives or provide a novel mechanism to explain their observations.
KK
Low protease activity in B cell follicles promotes retention of intact antigens after immunization
In order to generate effective antibodies to vaccine antigens, antigens must remain intact in the lymph nodes. However, lymph nodes contain high levels of extracellular proteases whose effect on vaccine efficacy in vivo is unknown. In this study, the authors first designed a FRET-based assay with an HIV antigen to determine antigen integrity and found that antigen was protected from protease activity uniquely within follicles. The authors then conjugated the antigen to ferritin to promote selective localization to follicular dendritic cells and performed in vivo vaccination experiments, which led to decreased antigen degradation compared to control. Notably, the authors observed an increase in germinal center B cells which reacted specifically to intact antigen. Though the latter set of experiments did not measure vaccine efficacy using an infection model, this thought-provoking study provides a novel avenue of study for vaccine design.
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Feb 8th, 2023
AJM
Platelet response to influenza vaccination reflects effects of aging
Platelets produce pro-inflammatory mediators and may have a role in age-associated chronic inflammation. In order to address this question, the authors tracked platelet activation in young, and frail or non-frail elderly following influenza vaccination. Marker transcripts and proteins revealed greater basal platelet activation in the elderly, with the frailty enhancing further. Following vaccination, platelet activation was enriched in elderly and remained high in participants with poor antibody response. These findings support platelets are impacted in aging; however, further in vitro and confirmatory experiments are required to assess their importance in age-associated inflammation.
LQ
Loss of epigenetic information as a cause of mammalian aging
The exact cause of aging has not been fully understood. This study adopted inducible changes to the epigenome (ICE) model, which allows for the induction of changes in the epigenome not the transcriptome through breaking non-coding DNA sequences. The authors determined that DNA repair contributes to aging at various levels, such as physiological, cognitive, or molecular, causing epigenetic changes, cellular identity changes, and senescence. All the aging characteristics of the ICE model can be reversed by expressing 4 factors Oct4, Sox2, Klf4, and Myc which can regulate pluripotency in stem cells.
KK
Functional partitioning of transcriptional regulators by patterned charge blocks
While transcriptional regulation is critical to cellular homeostasis and function, relatively little is understood regarding the mechanisms through which cells selectively organize local concentrations of transcriptional machinery. In this study, Lyons et al used the largest intrinsically disordered region (IDR) of the Mediator complex (MED1IDR), a key promoter of transcriptional activation, as a model. Using proteomics and ChIP-seq, the authors found that MED1IDR formed partitions near open chromatin regions in which positive transcriptional regulators were included, and negative transcriptional regulators were excluded. Many of the proteins associated with MED1IDR condensates also contained IDR domains, and further analysis found that these IDR domains contained a high fraction of charged amino acids that were separated into a unique block pattern. Experiments removing this block pattern eliminated association with MED1IDR, and the addition of synthetic charge block regions to proteins which had been excluded from MED1IDR fractions was sufficient to induce inclusion in to MED1IDR fractions. Overall, this study provides critical insight into transcriptional regulatory mechanisms, and future studies could further define how charge block patterning selects for inclusion with transcriptional complexes.
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Jan 11-24, 2023
LQ
Cell-cell metabolite exchange creates a pro-survival metabolic environment that extends lifespan
It is technically challenging to study the physiological impact of metabolite exchange interactions. The authors first reported metabolites generated by yeast cells at the exponential stage could be taken up by the stationary cells, which was proved to be able to extend the lifespan of the cells in the self-establishing metabolically cooperating communities (SeMeCo) model. Also, the SeMeCo cells showed more methionine-consuming compared to cells with wild-type cultures. Glycolytic enzymes upregulation and glycolytic overflow metabolites underlie the longevity of SeMeCo cells, which are induced by increased high-osmolarity glycerol 1, glycerol-3-phosphate phosphatase 1, and 2. In the future, this discovery can be tested if this can slow aging in cells or mice.
KK
Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation
While the roles of various genes and transcription factors during cellular differentiation are well-studied, the role of metabolic molecules such as glucose is unknown. In this study, the authors used proteomics in a model of epidermal differentiation to find that glucose bound a number of RNA-binding proteins, including DDX21, a helicase with ATPase activity. The authors tested binding of glucose and ATP to WT and mutant DDX21 to find that glucose and ATP bind competitively to DDX21, and glucose binding inhibited DDX21 dimerization, leading to DDX21 association with the spliceosome. DDX21 knockdown changed RNA splicing transcripts, identifying an exciting new mechanism regulating differentiation.
AJM
Weight loss can reverse obesity-induced insulin resistance, but whether it eliminates all obesity-associated health risks is unclear. Herein, the authors revealed history of obesity carries higher risk for severe age-related macular degeneration (AMD) due to epigenetic reprogramming in adipose tissue macrophages (ATMs). ATAC-Seq showed persistent accessibility to pro-inflammatory cytokines and angiogenic factors in ATMs due to stearic acid-LPS signaling, despite a return to a healthy weight. Following laser-induced retina injury, ATMs from mice with a history of obesity increased systemic proinflammatory cytokine levels and traveled to the retina to boost pathogenic angiogenesis and inflammation, confirmed by adipose tissue transplant. These results suggest anti-inflammatory interventions are necessary to mitigate obesity-associated health risks even after weight loss.