Biology subjects

Daniels, A.

Publications and source records attributed to Daniels, A..

9 recordsLinked to original sources

bioRxiv2026

Targeting DNMT1 augments anti-tumor CD8⁺ T cell function

Chronic stimulation of CD8 T cells within the tumor microenvironment (TME) induces a hypofunctional state characterized by diminished cytotoxicity and functionally impaired anti-tumor function, known as exhaustion. Exhaustion is associated with epigenetic changes that remain relatively stable despite interventions like immune checkpoint inhibition (ICI). Although epigenetic changes are potentially reversible, reports of therapeutic strategies to effectively restore function in exhausted CD8 T cells remain limited. Here, we report DNA methyltransferase 1 (DNMT1) inhibition (DNMT1i) in counteracting CD8+ T cell dysfunction during the anti-tumor response. We show that DNMT1i synergizes with ICI to rescue the tumor cell killing activity of chronically stimulated CD8 T cells in a melanoma model. DNMT1i mitigates transcriptional features of exhaustion while inducing a divergent effector program. DNMT1i attenuates the global increase in chromatin accessibility associated with exhaustion and enables epigenetic remodeling of the exhausted chromatin landscape upon restimulation. Finally, DNMT1i enhances the effector function of melanoma patient-derived tumor infiltrating lymphocytes after prolonged ex vivo expansion. These studies establish DNMT1 targeting as a promising strategy to counteract CD8 T cell exhaustion and potentiate ICI efficacy.

immunology↗

bioRxiv2026

AAV-NRF2 protects retinal and choroidal vasculature in a GDF15-dependent manner in an oxidative damage model of AMD

Oxidative stress is proposed to be a driver of age-related diseases. Age-related macular degeneration is one such disease, where the retinal pigment epithelium (RPE) is affected early in the disease. Vasculature damage also occurs, sometimes preceding RPE damage. To model some aspects of dry AMD, we used the NaIO3 mouse model of oxidative damage. Disruption of the deep retinal vascular plexus, disorganization and death of capillaries within the choriocapillaris, and marked electroretinographic decline were observed. AAV overexpressing the transcription factor, NRF2, which induces anti-oxidation enzymes and represses inflammation, was tested for protection of damage. The BEST1 promoter limited expression to the RPE. The RPE, photoreceptors, and vascular architecture in both retinal and choroidal compartments were protected. Conditioned medium from RPE-choroid explants, infected by AAV8/BEST1-NRF2, was sufficient to transfer partial protection in vivo, indicating that NRF2 induces a protective secreted factor(s). Analysis of RNA-seq data identified growth differentiation factor 15 (GDF15) as a candidate downstream mediator. Injection of recombinant GDF15 reproduced key protective phenotypes in vivo, whereas Gdf15-deficiency attenuated NRF2-mediated rescue. Pharmacologic inhibition of TGF-{beta} receptor signaling diminished NRF2 associated protection, supporting involvement of this signaling pathway. In a laser-induced choroidal neovascularization model, intravitreal GDF15 injection reduced fluorescein leakage and lesion size. These findings support a model in which NRF2 activation in the RPE induces expression of GDF15, which is capable of protecting the RPE, photoreceptors, and the retinal and choroidal vasculature. NRF2 and GDF15 have therapeutic potential for ocular diseases, as well as for other diseases with vascular pathology.

cell biology↗

bioRxiv2025

AAV NRF2 Gene Therapy Preserves Retinal Structure and Function in Rodent Models of Oxidative Damage

Dry age-related macular degeneration is the most frequent cause of visual impairment in individuals over age 50 in developed countries. It is characterized by subretinal deposits of oxidized proteins and lipids and results in progressive loss of high acuity vision. One major risk factor is smoking, which causes oxidative stress in many tissues, including the eye. We previously showed that an adeno-associated viral vector expressing human NRF2 (AAV8/Best1-Nrf2), a transcription factor that regulates responses to oxidative damage, slowed degeneration in mouse models of another blinding disorder, retinitis pigmentosa, which also includes oxidative stress. Here, our AAV8/Best1-Nrf2 vector was tested in a model of oxidative stress wherein sodium iodate was injected systemically, as this is often used to model dry age-related macular degeneration. Sodium iodate causes acute oxidative damage to supporting cells of the retina, the retinal pigment epithelial cells, and ultimately leads to photoreceptor death. Subretinal injection of AAV8/Best1-Nrf2 led to protection of the retinal pigment epithelium and photoreceptors, as well as preservation of visual function, in rat and mouse sodium iodate models. AAV8/Best1-Nrf2 may serve as an effective gene-agnostic therapy for diseases with oxidative stress, including dry age-related macular degeneration. ETOCOxidative stress occurs in diseases that lead to vision loss, including age-related macular degeneration (AMD). AMD is commonly modeled by injection of sodium iodate (S.I.), an oxidizing agent. AAV-mediated delivery of Nrf2, a transcription factor that regulates oxidative stress, protects the eye from S.I. damage in rat and mouse models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/663952v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@357e57org.highwire.dtl.DTLVardef@1f9986dorg.highwire.dtl.DTLVardef@186537corg.highwire.dtl.DTLVardef@1d31aad_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

Increasing hub disruption parallels dementia severity in autosomal dominant Alzheimer disease

Hub regions in the brain, recognized for their roles in ensuring efficient information transfer, are vulnerable to pathological alterations in neurodegenerative conditions, including Alzheimer Disease (AD). Given their essential role in neural communication, disruptions to these hubs have profound implications for overall brain network integrity and functionality. Hub disruption, or targeted impairment of functional connectivity at the hubs, is recognized in AD patients. Computational models paired with evidence from animal experiments hint at a mechanistic explanation, suggesting that these hubs may be preferentially targeted in neurodegeneration, due to their high neuronal activity levels--a phenomenon termed "activity-dependent degeneration". Yet, two critical issues were unresolved. First, past research hasnt definitively shown whether hub regions face a higher likelihood of impairment (targeted attack) compared to other regions or if impairment likelihood is uniformly distributed (random attack). Second, human studies offering support for activity-dependent explanations remain scarce. We applied a refined hub disruption index to determine the presence of targeted attacks in AD. Furthermore, we explored potential evidence for activity-dependent degeneration by evaluating if hub vulnerability is better explained by global connectivity or connectivity variations across functional systems, as well as comparing its timing relative to amyloid beta deposition in the brain. Our unique cohort of participants with autosomal dominant Alzheimer Disease (ADAD) allowed us to probe into the preclinical stages of AD to determine the hub disruption timeline in relation to expected symptom emergence. Our findings reveal a hub disruption pattern in ADAD aligned with targeted attacks, detectable even in pre-clinical stages. Notably, the disruptions severity amplified alongside symptomatic progression. Moreover, since excessive local neuronal activity has been shown to increase amyloid deposition and high connectivity regions show high level of neuronal activity, our observation that hub disruption was primarily tied to regional differences in global connectivity and sequentially followed changes observed in A{beta} PET cortical markers is consistent with the activity-dependent degeneration model. Intriguingly, these disruptions were discernible 8 years before the expected age of symptom onset. Taken together, our findings not only align with the targeted attack on hubs model but also suggest that activity-dependent degeneration might be the cause of hub vulnerability. This deepened understanding could be instrumental in refining diagnostic techniques and developing targeted therapeutic strategies for AD in the future.

neuroscience↗

bioRxiv2023

Coupling remote sensing and eDNA to monitor environmental impact: A pilot to quantify the environmental benefits of sustainable agriculture in the Brazilian Amazon

Monitoring is essential to ensure that environmental goals are being achieved, including those of sustainable agriculture. Growing interest in environmental monitoring provides an opportunity to improve monitoring practices. Approaches that directly monitor land cover change and biodiversity annually by coupling the wall-to-wall coverage from remote sensing and the site-specific community composition from environmental DNA (eDNA) can provide timely, relevant results for parties interested in the success of sustainable agricultural practices. To ensure that the measured impacts are due to the environmental projects and not exogenous factors, sites where projects have been implemented should be benchmarked against counterfactuals (no project) and control (natural habitat) sites. Results can then be used to calculate diverse sets of indicators customized to monitor different projects. Here, we report on our experience developing and applying one such approach to assess the impact of shaded cocoa projects implemented by the Instituto de Manejo e Certificacao Florestal e Agricola (IMAFLORA) near Sao Felix do Xingu, in Para, Brazil. We used the Continuous Degradation Detection (CODED) and LandTrendr algorithms to create a remote sensing-based assessment of forest disturbance and regeneration, estimate carbon sequestration, and changes in essential habitats. We coupled these remote sensing methods with eDNA analyses using arthropod-targeted primers by collecting soil samples from intervention and counterfactual pasture field sites and a control secondary forest. We used a custom set of indicators from the pilot application of a coupled monitoring framework called TerraBio. Our results suggest that, due to IMAFLORAs shaded cocoa projects, over 400 acres were restored in the intervention area and the community composition of arthropods in shaded cocoa is closer to second-growth forests than that of pastures. In reviewing the coupled approach, we found multiple aspects worked well, and we conclude by presenting multiple lessons learned.

ecology↗

bioRxiv2023

Setdb1-loss induces type-I interferons and immune clearance of melanoma

Despite recent advances in the treatment of melanoma, many patients with metastatic disease still succumb to their disease. To identify tumor-intrinsic modulators of immunity to melanoma, we performed a whole-genome CRISPR screen in melanoma and identified multiple components of the HUSH complex, including Setdb1, as hits. We found that loss of Setdb1 leads to increased immunogenicity and complete tumor clearance in a CD8+ T-cell dependent manner. Mechanistically, loss of Setdb1 causes de-repression of endogenous retroviruses (ERVs) in melanoma cells and triggers tumor-cell intrinsic type-I interferon signaling, upregulation of MHC-I expression, and increased CD8+ T-cell infiltration. Furthermore, spontaneous immune clearance observed in Setdb1-/- tumors results in subsequent protection from other ERV-expressing tumor lines, supporting the functional anti-tumor role of ERV-specific CD8+ T-cells found in the Setdb1-/- microenvironment. Blocking the type-I interferon receptor in mice grafted with Setdb1-/- tumors decreases immunogenicity by decreasing MHC-I expression, leading to decreased T-cell infiltration and increased melanoma growth comparable to Setdb1wt tumors. Together, these results indicate a critical role for Setdb1 and type-I interferons in generating an inflamed tumor microenvironment, and potentiating tumor-cell intrinsic immunogenicity in melanoma. This study further emphasizes regulators of ERV expression and type-I interferon expression as potential therapeutic targets for augmenting anti-cancer immune responses.

cancer biology↗

bioRxiv2022

One for all - Human kidney Caki-1 cells are highly susceptible to infection with corona- and other respiratory viruses

In vitro investigations of host-pathogen interactions of viruses are reliant on suitable cell and tissue culture models. Results are only as good as the model they have been generated in. However, choosing cell models for in vitro work often depends on what is available in labs or what has previously been used. Despite the vast increase in coronavirus research activity over the past few years, researchers are still heavily reliant on: non-human cells, for example Vero E6, highly heterogeneous or not fully differentiated cells, such as Calu-3, or naturally unsusceptible cells requiring overexpression of receptors and other accessory factors, such as ACE2 and TMPRSS2. Complex cell models, such as primary cell-derived air-liquid interface epithelial models are highly representative of human tissues but are expensive and time-consuming to develop and maintain. They have limited suitability for large-scale and high-throughput analysis. Using tissue-specific expression pattern as a selection criteria, we identified human kidney cells as an ideal target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and broader coronavirus infection. We show the use of the highly characterized human kidney cell line Caki-1 for infection with three human coronaviruses: Betacoronaviruses SARS-CoV-2 and Middle Eastern respiratory syndrome coronavirus (MERS-CoV) and Alphacoronavirus human coronavirus 229E (hCoV-229E). Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cell lines for the cultivation of the respective virus. Antibody staining against SARS-CoV-2 N protein shows comparable replication rates. Using a panel of 21 antibodies in infected Caki-1 cells using immunocytochemistry shows the location of viral proteins during replication. In addition, Caki-1 cells were found to be susceptible to two other human respiratory viruses, influenza A virus and respiratory syncytial virus, making them an ideal model for cross-comparison of not only a broad range of coronaviruses but respiratory viruses in general. Author SummaryInvestigating how viruses interact with their host relies models used for laboratory research. The closer a model matches the host, the more conclusive results are. Complex cell systems based on primary epithelial or stem cells are the gold standard of in vitro research. However, they are expensive, time consuming, and laborious to establish. Therefore, cell lines remain the backbone of virus research. Despite vastly increased research into human coronaviruses following the COVID-19 pandemic, researchers continue to rely on suboptimal cell line models, for example ells of non-human origin like the VeroE6 African Green Monkey cell line. Using known expression patterns of the entry factors of the COVID-19 causative agent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) we identified the Caki-1 cell line as a target for SARS-CoV-2. This cell line could be shown to be infectable with a wide range of coronaviruses including common cold virus hCoV-229E, epidemic virus MERS-CoV, and SARS-CoV-2 as well as other important respiratory viruses influenza A virus and respiratory syncytial virus. We could show that SARS-CoV-2 proteins can be stained for and localized in Caki-1 cells and the cells are competent of forming a cellular immune response. Together, this makes Caki-1 cells a unique tool for cross-virus comparison in one cell line.

microbiology↗

bioRxiv2021

Direct lysis RT-qPCR of SARS-CoV-2 in cell culture supernatant allows for fast and accurate quantification of virus, opening a vast array of applications

An enormous global effort is being made to study SARS-CoV-2 and develop safe and effective treatments. Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify host factors and treatments to combat the infection. However, quantification of released virus often requires lengthy procedures, such as endpoint dilution assays or reinfection with engineered reporter viruses. Quantification of viral RNA in cell supernatant is faster and can be performed on clinical isolates. However, viral RNA purification is expensive in time and resources and often unsuitable for high-throughput screening. Here, we show a direct lysis RT-qPCR method allowing sensitive, accurate, fast, and cheap quantification of SARS-CoV-2 in culture supernatant. During lysis, the virus is completely inactivated, allowing further processing in low containment areas. This protocol facilitates a wide array of high- and low-throughput applications from basic quantification to studying the biology of SARS-CoV-2 and to identify novel antiviral treatments in vitro.

microbiology↗

bioRxiv2020

Lack of evidence of ACE2 expression and replicative infection by SARS-CoV-2 in human endothelial cells

A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro. Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.

pathology↗