Biology subjects

Kang, E.

Publications and source records attributed to Kang, E..

17 recordsLinked to original sources

bioRxiv2026

Genotoxic and metabolic stress drive divergent senescence programs in human microglia

Microglial dysfunction is a hallmark of brain ageing linked to the accumulation of senescent microglial phenotypes that promote chronic neuroinflammation. Both genotoxic and metabolic stress have been implicated in microglial senescence; yet, whether distinct stressors shape senescence programs remain unclear. Here, we investigated the impact of chronic genotoxic and metabolic stress on senescence-associated phenotypes in the human microglia cell line HMC3. Cells were exposed to doxorubicin to induce sustained DNA damage or to chronic high-glucose conditions to model metabolic stress. Both stress paradigms induced characteristic senescence features including cellular and nuclear hypertrophy, increased senescence-associated {beta} galactosidase activity and reduced metabolic viability without significant cell loss. Both conditions activated the p53-p21 pathway and sustained DNA damage signalling, whereas metabolic stress additionally induced p16 expression and peripheral nuclear localisation of p21, suggesting divergence in senescence regulatory pathways. Mitochondrial alterations were evident under both conditions, Dox-induced stress was associated with downregulation of NRF2-TFAM signalling, whereas HG-induced stress induced NRF2-TFAM activation alongside increased KEAP1 expression, suggesting a constrained antioxidant response. This was accompanied by activation of mitochondrial and antioxidant stress responses that did not restore mitochondrial content. Furthermore, both stressors induced robust inflammatory activation, with genotoxic stress promoting a chemokine-rich senescence-associated secretory phenotype, while metabolic stress induced an interferon-associated inflammatory signature. Collectively, these findings demonstrated that chronic genotoxic and metabolic stress drive distinct yet overlapping senescence programs characterised by morphological changes, mitochondrial remodelling and persistent inflammatory activation. These stress-specific responses may differentially contribute to neurodegenerative processes and disease susceptibility.

neuroscience↗

bioRxiv2026

DNA damage-induced senescence reshapes transcriptomic and functional landscape of human neural progenitor cells

Ageing-related decline in hippocampal neurogenesis has been associated with cognitive impairment and neurodegenerative disease, yet experimentally tractable human models to study the underlying cellular and molecular mechanisms remain limited. Cellular senescence has emerged as a candidate driver of age-related tissue dysfunction, but its induction and consequences in human NPCs have not been well characterized. Here, we established a human in vitro model of NPC senescence using induced pluripotent stem cell-derived NPCs exposed to transient low-dose doxorubicin to activate the DNA damage response (DDR) while minimizing acute cytotoxicity. Doxorubicin-treated NPCs developed a stable senescent phenotype characterized by increased senescence-associated {beta}-galactosidase activity, reduced proliferation, persistent DNA damage, and sustained induction of p21 and p16. Transcriptomic profiling revealed widespread senescence-associated remodeling, including activation of p53 and inflammatory programs and repression of cell cycle and DNA repair pathways. Senescent NPCs exhibited apoptosis resistance despite transcriptional priming of apoptotic pathways and underwent mitochondrial remodeling with a shift towards oxidative metabolism. In parallel, they acquired a senescence-associated secretory phenotype enriched in inflammatory, TGF{beta}-related and pro-angiogenic factors, and conditioned media from these cells promoted angiogenesis in vascular organoids. Importantly, key senescence-associated features were recapitulated in human hippocampal organoids, confirming the robustness of this paradigm in a three-dimensional neural context. Together, these findings establish a tractable human model of DDR-driven NPC senescence and identify senescence as a mechanism linking genotoxic stress to impaired progenitor function, metabolic rewiring, and paracrine niche remodeling relevant to hippocampal ageing and neurodegeneration.

neuroscience↗

bioRxiv2026

Circadian phase and sex shape swimming exercise responses and stereotyped behaviors in mice

Exercise provides broad health benefits, including improved emotional well-being and cognitive function. Emerging evidence suggests that exercising at different times during the day can have differential effects. However, how circadian phase and sex influence behavioral and physiological responses to exercise remains unclear. To address this question, we examined male and female wild-type mice maintained in either regular (REG, lights on/off at 7AM/7PM) or inverted (INV, lights off/on at 10AM/10PM) light cycles. Mice were then subjected to daily 20-min group swimming exercise sessions at ZT2-3 for 3 weeks. Exercised and sedentary controls mice were then subjected to an open field test (OFT) and blood corticosterone (CORT) measurements 24 hours post-exercise. We quantified several behaviors during swimming: escape attempts, floating, climbing and collisions. We also identified a novel swimming behavior: floating with only nostrils-above-water events (NAWEs). We found that expression of these behaviors was differentially modulated by sex, light-cycle and their interaction. Notably, behavioral differences were more pronounced in REG mice (rest phase). REG mice also lost weight after exercise and had elevated CORT levels compared to mice kept in INV conditions (active phase). Interestingly, OFT behaviors showed significant differences primarily in INV mice, particularly females, when comparing exercised vs sedentary groups. Our novel findings reveal that circadian rhythms and sex significantly interact to shape swimming exercise and stereotyped behaviors in mice. This emphasizes the need to consider the animals circadian phase when designing preclinical studies to match intended behavioral and physiological outcomes. HIGHLIGHTSCircadian phase and sex jointly shape swimming behavior patterns. Newly identified swimming behavior is more prevalent during rest-phase Restphase exercise produced stronger behavioral and physiological effects. Rest-phase exercise resulted in weight loss and elevated stress markers. Active-phase exercised females showed the strongest open field behavioral differences.

physiology↗

bioRxiv2026

Dynamic control of Raf-ERK signaling modulates neuronal activity across biological scales

Neuronal activity robustly engages the extracellular signal-regulated kinase (ERK) signaling pathway through Ca2+-dependent mechanisms; however, whether ERK can acutely and causally modulates ongoing neuronal activity remains unsolved due to complex upstream regulation and diverse subcellular functions. Here, we directly address this question using an optogenetic ERK activator, opto-miniRaf, that enables selective, rapid, graded, and reversible control of ERK signaling. Combining this AAV-compatible system with calcium imaging and electrophysiology, we interrogate ERK functions across biological scales, from cultured neurons, acute brain slices, and the intact brain. Acute optogenetic activation of ERK enhances synchronized network burst activity in cultured rat cortical neurons and increases calcium activity of cortical pyramidal neurons in awake and moving mice following non-invasive light stimulation. Together, these results establish ERK signaling as an acute modulator of neuronal and network activity, positioning opto-miniRaf as a generalizable platform for precise spatiotemporal control of intracellular kinase signaling in complex biological systems.

neuroscience↗

bioRxiv2025

A Human Hippocampal Organoid Model with Sustained Neural Stem Cells Reveals State Shifts Under Glucocorticoid Stress

The human hippocampus is a critical brain region for learning, memory, and stress regulation, distinguished by its ability to sustain neurogenesis after birth. This plasticity is driven by hippocampal neural stem cells (NSCs), which generate new neurons and maintain circuit integrity, but are highly sensitive to environmental and pathological influences. Mechanistic insight into human hippocampal development and neurogenesis remains limited by the absence of physiologically relevant models. Here, we establish an optimized protocol to generate human induced pluripotent stem cell-derived hippocampal organoids that recapitulate key features of hippocampal development. These organoids maintain organized NSC niches, support ongoing neurogenesis, and generate hippocampus-specific cell types. Cellular, transcriptomic, and electrophysiological analyses confirm progressive neuronal maturation, synapse formation, and functional activity, highlighting the physiological relevance of the system. Using this model, we modeled excess prenatal glucocorticoid exposure with dexamethasone, which perturbed NSC dynamics by reducing proliferation and inducing a precocious quiescent-like state. RNA sequencing revealed downregulation of NSC activation genes and upregulation of quiescence- and autophagy-associated programs, suggesting that glucocorticoid signaling enforces an early transition toward quiescence. These findings reveal a mechanism by which excessive glucocorticoid exposure may impair hippocampal growth. Together, this study introduces a robust human hippocampal organoid platform for dissecting the regulation of hippocampal development and for modeling the impact of environmental stressors on human neurogenesis.

neuroscience↗

bioRxiv2025

Harvesting more reads from single-cell combinatorial barcoding data with scarecrow

SummaryCombinatorial barcoding technologies for single-cell nucleotide sequencing, such as split-pool ligation protocols, involve sequential rounds of cell barcoding to uniquely tag individual cells. The rapid adoption of combinatorial barcoding in recent years is due in part to its scalability across cells and samples. However, small shifts in barcode positions within sequencing reads caused by technical artifacts, e.g. during barcode incorporation or synthesis, can impact the accurate assignment of reads to cell barcodes. Existing processing tools typically assume barcodes contain fixed-length nucleotide sequences located at fixed positions within reads, overlooking any positional variability. Consequently, reads containing truncated or mispositioned barcodes are discarded during initial data processing steps leading to significant data loss. To solve this limitation and maximise the retention of sequencing reads from single-cell combinatorial barcoding experiments, we introduce scarecrow. Our tool screens a subsample of reads to generate position-specific barcode profiles, which are then used to flexibly identify barcode sequences in each read whilst accounting for positional errors, a phenomenon we refer to as jitter. Barcode matches are then prioritised to minimise nucleotide mismatches and the degree of jitter. These initial profiles are subsequently used to extract and error correct barcode combinations in high throughput sequencing libraries. By incorporating jitter into barcode error correction, scarecrow enables greater data recovery and improved downstream single-cell analyses. Scarecrow is fully open access, implemented in Python, and generates output files using standardised sequence file formats for maximal interoperability. A detailed explanation of the scarecrow workflow can be found in the supplementary materials.

bioinformatics↗

bioRxiv2025

Inflammatory reprogramming of human brain endothelial cells compromises blood-brain barrier integrity in Alzheimer's disease

Blood-brain barrier (BBB) dysfunction is an early feature of Alzheimers disease (AD), yet the endothelial gene-regulatory programs involved remain incompletely understood. We integrate postmortem human single-nucleus transcriptomics with iPSC-based BBB models to define a conserved, inflammation-driven pathway that compromises barrier integrity. We identify an NF-{kappa}B-associated endothelial gene module endoM2 that is elevated in AD, inversely correlated with cognition, and enriched for inflammation and endothelial-to-mesenchymal transition signatures. Cytokine stimulation of iPSC-derived brain endothelial cells induces morphological remodeling, lipid accumulation, junctional disruption, and transcriptomic shifts that mirror endoM2. A targeted drug screen identifies the NF-{kappa}B inhibitor BAY11-7082 as protective against cytokine-induced changes. In our perfusable iPSC-derived BBB-Chip that recapitulates human BBB signatures, single-cell profiling reveals inflammatory endothelial state-specific programs reflecting those in AD brains and demonstrates that BAY11-7082 suppresses cytokine-triggered dysfunction and reverses inflammation-associated gene activation. Together, these findings position cerebrovascular inflammation as a therapeutic target to preserve BBB integrity in AD.

neuroscience↗

bioRxiv2025

Vascular-Perfusable Human 3D Brain-on-Chip

Development and delivery of treatments for neurological diseases are limited by the tight and selective human blood-brain barrier (BBB). Although animal models have been important research and preclinical tools, the rodent BBB exhibits species differences and fails to capture the complexity of human genetics. Microphysiological systems incorporating human-derived cells hold great potential for modeling disease and therapeutic development, with advantages in screening throughput, real-time monitoring, and tunable genetic backgrounds when combined with induced pluripotent stem cell (iPSC) technology. Existing 3D BBB-on-chip systems have incorporated iPSC-derived endothelial cells but not the other major brain cell types from iPSCs, each of which contributes to brain physiology and disease. Here we developed a 3D Brain-Chip system incorporating endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia from iPSCs. To enable this multicellular 3D co-culture in-chip, we designed a GelChip microfluidic platform using a 3D printing-based approach and dextran-based engineered hydrogel. Leveraging this platform, we co-cultured and characterized iPSC-derived brain-on-chips and modeled the brain microvasculature of APOE4, the strongest known genetic risk factor for sporadic Alzheimers disease. These 3D brain-on-chips provide a versatile system to assess BBB vascular morphology and function, investigate downstream neurological effects in disease, and screen therapeutics to optimize delivery to the brain. Significance StatementThe blood-brain barrier (BBB) is both a contributing factor to neurological disease and a major obstacle to its treatment, yet human-relevant models remain limited. Most existing brain-on-chip systems incorporate only subsets of BBB cell types and cannot capture the full cellular complexity of the human neurovascular unit. Here, we establish a vascular-perfusable 3D Brain-Chip using human induced pluripotent stem cell-derived brain cells including endothelial cells, pericytes, astrocytes, neurons, microglia, and oligodendroglia. This system enables systematic analysis of human genetic risk factors, such as APOE4 in Alzheimers disease, and provides a powerful platform to investigate BBB function and dysfunction and accelerate the development of more effective neurological therapies.

neuroscience↗

bioRxiv2025

Germline-targeting HIV Envelope SOSIP immunization more frequently elicits broadly-neutralizing antibody precursor responses in infant compared to juvenile rhesus macaques

A vaccine capable of inducing broadly neutralizing antibodies (bnAbs) is essential for effective prevention against HIV in children and adolescents. Germline-targeting vaccine strategies aim to stimulate bnAb precursor B cells through carefully designed immunogens, such as the stabilized SOSIP trimers, which mimic native HIV envelope (Env) proteins while presenting key neutralizing epitopes to germline B cell receptors. Given the ability of children living with HIV to develop bnAbs earlier and at a higher frequency than adults, we compared the immunogenicity of a CD4 binding site (CD4bs) bnAb germline-targeting SOSIP trimer immunization strategy in infant (n = 5) and juvenile (n = 4) rhesus macaques (RMs). Animals received 3 doses of the germline-targeting BG505 GT1.1 immunogen, followed by 3 boosts of wild-type BG505 SOSIP, each adjuvanted with the TLR7/8 agonist, 3M-052-SE. After 1.5 years, the RMs were further boosted with a mixed clade B Env trimer nanoparticle to enhance heterologous virus neutralization responses. This germline-targeting strategy induced equivalent titers of neutralizing antibodies in both groups of RMs, yet the infants exhibited a higher magnitude of vaccine-specific IgG binding. Notably, after 3 doses of BG505 GT1.1 SOSIP, infants had higher BG505 GT1.1-specific IgD- B cells. Upon completion of the vaccine regimen, 4 of 5 infants developed a CD4bs bnAb precursor response detectable in serum compared to only 1 of 4 juveniles. Finally, administration of the mixed clade B nanoparticle was able to increase the breadth of antibody responses in 3 of 5 infants and 2 of 4 juveniles. These results suggest that immunization in early-life may enhance bnAb induction and highlight the potential for future pediatric HIV-1 vaccine strategies. Author SummaryVaccines that can generate antibody responses that have breadth and potency in neutralization can protect children and adolescents from acquiring multi-strain viruses like HIV. Increasingly, vaccine strategies have become better tailored to elicit these broadly neutralizing antibodies (bnAbs), such as the germline-targeting HIV envelope antigens, which have shown great promise. To determine if we could elicit protective antibody responses against HIV in early life, we immunized infant and juvenile monkeys using the same vaccine strategy. In this study, we found that while both infants and juveniles developed similar levels of antibodies that could neutralize HIV, the infants showed stronger antibody responses in several key areas. Moreso, infants developed the type of early antibody response that researchers believe is needed to eventually evolve into broadly neutralizing antibodies. These findings suggest that initiating HIV vaccination earlier in life may offer a better chance at generating the bnAbs needed to protect against various HIV strains. Our work highlights the potential benefits of initiating an HIV vaccine regimen in childhood.

immunology↗

bioRxiv2025

Deciphering Cell-Type and Temporal-Specific Matrisome Expression Signatures in Human Cortical Development and Neurodevelopmental Disorders via scRNA-Seq Meta-Analysis

Human cortical development is a complex process involving the proliferation, differentiation, and migration of progenitor cells, all coordinated within a dynamic extracellular matrix (ECM). ECM plays a crucial role in guiding these processes, yet its specific contributions and the implications of its dysregulation in neurodevelopmental disorders (NDDs) remain underexplored. In this study, we conducted a meta-analysis of single-cell RNA sequencing (scRNA-seq) data from 37 donors, gestational weeks (GWs) 8 to 26 across six independent studies to elucidate cell type-specific matrisome gene expression signatures and their dynamics in the developing human cortex. Our analysis identified distinct matrisome gene signatures across various cell types, with significant temporal changes during cortical development. Notably, a substantial proportion of matrisome genes are associated with NDDs, exhibiting cell type, temporal and disease specificity. These findings highlight the critical role of cell type-specific matrisome regulation in cortical development and its potential involvement in NDD pathogenesis. This study provides a comprehensive map of cell type-specific matrisome signatures in the developing human cortex and highlights the importance of ECM in both normal development and the pathogenesis of NDDs.

neuroscience↗

bioRxiv2024

Behavioral and metabolic effects of extended hippocampal overexpression of the myokine Irisin in an Alzheimers Disease mouse model

Lifestyle interventions such as exercise are promising strategies to reduce the detrimental effects faced by aging populations. In particular, the exercise-induced myokine Irisin could act as a mediator of the positive effects of exercise, even as a potential therapeutic option for Alzheimers disease (AD). However, the fundamental roles of Irisin in counteracting AD neuropathology and other effects have not been fully determined. In addition, whether centrally produced Irisin has any effects is unclear. Here we examined the neurobehavioral effects of overexpressing Irisin directly in the hippocampus of an AD transgenic mouse line (TgCRND8), using adult male mice stereotaxically injected with Irisin-AAV vector. To further mimic chronic exercise, spatial memory and exploratory behaviors were assessed using the novel object recognition (NOR)/open field test (OFT) and Y-Maze tests after 8 weeks. Our results showed that Irisin expression led to several behavioral changes, including increased grooming behavior and slightly improved spatial memory performance in short interval NOR test in Tg mice. In the Y-Maze test however, we observed an increase in time spent in familiar spaces, which could indicate heightened anxiety-like behaviors in mice injected with Irisin-AAV. In addition, we observed significant differences in body and liver size, and in circulating metabolites such as ketone bodies, that were differentially regulated in NTg or Tg mice. Overall, our findings show important neurobehavioral effects of long-lasting overexpression of the exerkine Irisin in the hippocampus. Our results also raise questions about potential detrimental effects and thus warrant further studies to fully dissect how this exercise mediator affects the brain.

neuroscience↗

bioRxiv2024

IL-17A Alters Human Cortical Development in a 3D Ex Vivo Model of Maternal Immune Activation

Human brain development depends on the coordinated interaction of diverse cell types and extracellular matrix (ECM) components, which are essential for proper neurogenesis and cortical organization. Epidemiological and animal studies have demonstrated that maternal immune activation (MIA) disrupts brain development, leading to impaired neurogenesis and increased risk of neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD) and schizophrenia. However, the cellular and molecular mechanisms by which MIA impacts human cortical development remain poorly understood. Here we introduce a 3D ex vivo culture system, termed cerebroids, derived from dorsolateral prefrontal cortex of human fetal brain tissue, which faithfully preserves key developmental processes, along with critical cellular diversity and structural integrity of the developing human cortex. Using this platform, we show that IL-17A, a cytokine strongly implicated in NDDs, induces premature cortical folding, increases cortical thickness, and accelerates neurogenesis and neuronal maturation. Transcriptomic and proteomic analyses reveal significant dysregulation of ECM-related pathways, including the upregulation of proteoglycans such as brevican and versican. Notably, treatment with the anti-inflammatory agent parthenolide, an inhibitor of NF-{kappa}B and HDAC1 pathways, reverses IL-17A-induced cortical abnormalities, restoring normal cortical thickness, folding, and neurogenesis. These findings provide valuable insights into how IL-17A disrupts human cortical development during MIA, advancing our understanding of NDD-associated structural cortical alterations.

neuroscience↗

bioRxiv2023

Algorithm for selecting potential SARS-CoV-2 dominant variants based on POS-NT frequency

COVID-19, currently prevalent worldwide, is caused by a novel coronavirus, SARS-CoV-2. Similar to other RNA viruses, SARS-CoV-2 continues to evolve through random mutations, creating numerous variants, such as Alpha, Beta, and Delta. It is, therefore, necessary to predict the mutations constituting the dominant variant before they are generated. This can be achieved by continuously monitoring the mutation trends and patterns. Hence, in the current study, we sought to design a dominant variant candidate (DVC) selection algorithm. To this end, we obtained COVID-19 sequence data from GISAID and extracted position-nucleotide (POS-NT) frequency ratio data by country and date through data preprocessing. We then defined the dominant dates for each variant in the USA and developed a frequency ratio prediction model for each POS-NT. Based on this model, we applied DVC criteria to develop the selection algorithm, verified for Delta and Omicron. Using Condition 3 as the DVC criterion, 69 and 102 DVC POS-NTs were identified for Delta and Omicron an average of 47 and 82 days before the dominant dates, respectively. Moreover, 13 and 44 Delta- and Omicron-defining POS-NTs were recognized 18 and 25 days before the dominant dates, respectively. We identified all DVC POS-NTs before the dominant dates, including soaring and gently increasing POS-NTs. Considering that we successfully defined all POS-NT mutations for Delta and Omicron, the DVC algorithm may represent a valuable tool for providing early predictions regarding future variants, helping improve global health. Author Summary

bioinformatics↗

bioRxiv2022

ACCELERATING ANTIBODY DESIGN WITH ACTIVE LEARNING

Discovering therapeutic antibody starts by screening antibody library of phage-displayed, transgenic mouse or human B cells. The coverage of those kinds of libraries in the entire antibody sequence space is small; thus, the result highly depends on the quality of the library. Exploring sequence space by mutating a template antibody is also impossible to even with the state-of-the-art screening methods because of the cost. Deep learning helped with its pattern recognition nature to predict target binding, which is only applied to HCDR3 because the number of data deep learning needs increases exponentially. We construct a sequence generation model with transfer learning and active learning to leverage deep learning even in data deficiency. With only six thousands data, the generative model finds nine binding antibody sequences at least per antigen with novel HCDR3.

bioengineering↗

bioRxiv2018

Drosophila ZDHHC8 palmitoylates scribble and Ras64B and controls growth and viability

SummaryPalmitoylation is an important posttranslational modification regulating diverse cellular functions. Consequently, aberrant palmitoylation can lead to diseases such as neuronal disorders or cancer. In humans there are roughly one hundred times more palmitoylated proteins than enzymes catalyzing palmitoylation (palmitoyltransferases). Therefore, it is an important challenge to establish the links between palmitoyltransferases and their targets. From publicly available data, we find that expression of human ZDHHC8 correlates significantly with cancer survival. To elucidate the organismal function of ZDHHC8, we study the Drosophila ortholog of hZDHHC8, CG34449/dZDHHC8. Knockdown of dZDHHC8 causes tissue overgrowth while dZDHHC8 mutants are larval lethal. We provide a list of 159 palmitoylated proteins in Drosophila and present data suggesting that scribble and Ras64B are targets of dZDHHC8.

molecular biology↗

bioRxiv2017

New routes for PTP1B allosteric inhibition by multitemperature crystallography, fragment screening and covalent tethering

Allostery is an inherent feature of proteins and provides alternative routes to regulating function. Small-molecule allosteric inhibitors are often desirable; however, it remains challenging to identify surface sites in proteins which can bind small molecules and modulate function. We identified new allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal \"hidden\" low-occupancy conformational states for protein and ligands. Our results converge on a new allosteric site that is conformationally coupled to the active-site WPD loop, a hotspot for fragment binding, not conserved in the closest homolog, and distinct from other recently reported allosteric sites in PTP1B. Targeting this site with covalently tethered molecules allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure revealed by multitemperature crystallography can be exploited for developing allosteric modulators.

biophysics↗

bioRxiv2017

Extending Chemical Perturbations Of The Ubiquitin Fitness Landscape In A Classroom Setting

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.\n\nBuilds onMavor D, Barlow KA, Thompson S, Barad BA, Bonny AR, Cario CL, Gaskins G, Liu Z, Deming L, Axen SD, Caceres E, Chen W, Cuesta A, Gate R, Green EM, Hulce KR, Ji W, Kenner LR, Mensa B, Morinishi LS, Moss SM, Mravic M, Muir RK, Niekamp S, Nnadi CI, Palovcak E, Poss EM, Ross TD, Salcedo E, See S, Subramaniam M, Wong AW, Li J, Thorn KS, Conchuir SO, Roscoe BP, Chow ED, DeRisi JL, Kortemme T, Bolon DN, Fraser JS. Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting. eLife. 2016.\n\nImpact StatementWe organized a project-based course that used deep mutational scanning in multiple chemical conditions to resolve the inconsistencies between tolerance to mutations in laboratory conditions and sequence conservation over evolutionary timescales.

bioinformatics↗