@article{lin_volkel_cao_hook_polak_clark_san miguel_timp_tuck_velev_et al._2024, title={A primordial DNA store and compute engine}, volume={8}, ISSN={1748-3387 1748-3395}, url={http://dx.doi.org/10.1038/s41565-024-01771-6}, DOI={10.1038/s41565-024-01771-6}, journal={Nature Nanotechnology}, publisher={Springer Science and Business Media LLC}, author={Lin, Kevin N. and Volkel, Kevin and Cao, Cyrus and Hook, Paul W. and Polak, Rachel E. and Clark, Andrew S. and San Miguel, Adriana and Timp, Winston and Tuck, James M. and Velev, Orlin D. and et al.}, year={2024}, month={Aug} }
 @inbook{yagci_kelkar_johnson_sen_keung_2024, title={Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities}, ISBN={9781071640500 9781071640517}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-0716-4051-7_2}, DOI={10.1007/978-1-0716-4051-7_2}, abstractNote={The advent of locus-specific protein recruitment technologies has enabled a new class of studies in chromatin biology. Epigenome editors (EEs) enable biochemical modifications of chromatin at almost any specific endogenous locus. Their locus-specificity unlocks unique information including the functional roles of distinct modifications at specific genomic loci. Given the growing interest in using these tools for biological and translational studies, there are many specific design considerations depending on the scientific question or clinical need. Here, we present and discuss important design considerations and challenges regarding the biochemical and locus specificities of epigenome editors. These include how to: account for the complex biochemical diversity of chromatin; control for potential interdependency of epigenome editors and their resultant modifications; avoid sequestration effects; quantify the locus specificity of epigenome editors; and improve locus-specificity by considering concentration, affinity, avidity, and sequestration effects.}, booktitle={Methods in Molecular Biology}, publisher={Springer US}, author={Yagci, Z. Begum and Kelkar, Gautami R. and Johnson, Tyler J. and Sen, Dilara and Keung, Albert J.}, year={2024}, pages={23–55} }
 @article{rudibaugh_tam_estridge_stuppy_keung_2024, title={Single-Cell Assessment of Human Stem Cell-Derived Mesolimbic Models and Their Responses to Substances of Abuse}, volume={3}, ISSN={2674-1172}, url={http://dx.doi.org/10.3390/organoids3020009}, DOI={10.3390/organoids3020009}, abstractNote={The mesolimbic pathway connects ventral tegmental area dopaminergic neurons and striatal medium spiny neurons, playing a critical role in reward and stress behaviors. Exposure to substances of abuse during development and adulthood has been linked to adverse outcomes and molecular changes. The rise of human cell repositories and whole-genome sequences enables human functional genomics ‘in a dish’, offering insights into human-specific responses to substances of abuse. Continued development of new models is needed, and the characterization of in vitro models is also necessary to ensure appropriate experimental designs and the accurate interpretation of results. This study introduces new culture conditions for generating medium spiny neurons and dopaminergic neurons with an early common media, allowing for coculture and assembloid generation. It then provides a comprehensive characterization of these and prior models and their responses to substances of abuse. Single-cell analysis reveals cell-type-specific transcriptomic responses to dopamine, cocaine, and morphine, including compound and cell-type-specific transcriptomic signatures related to neuroinflammation and alterations in signaling pathways. These findings offer a resource for future genomics studies leveraging human stem cell-derived models.}, number={2}, journal={Organoids}, publisher={MDPI AG}, author={Rudibaugh, Thomas P. and Tam, Ryan W. and Estridge, R. Chris and Stuppy, Samantha R. and Keung, Albert J.}, year={2024}, month={Jun}, pages={126–147} }
 @inbook{fadri_lee_keung_2024, title={Summary of ChIP-Seq Methods and Description of an Optimized ChIP-Seq Protocol}, ISBN={9781071640500 9781071640517}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-0716-4051-7_22}, DOI={10.1007/978-1-0716-4051-7_22}, abstractNote={Chromatin immunoprecipitation (ChIP) is an invaluable method to characterize interactions between proteins and genomic DNA, such as the genomic localization of transcription factors and post-translational modification of histones. DNA and proteins are reversibly and covalently crosslinked using formaldehyde. Then the cells are lysed to release the chromatin. The chromatin is fragmented into smaller sizes either by micrococcal nuclease (MN) or sonication and then purified from other cellular components. The protein-DNA complexes are enriched by immunoprecipitation (IP) with antibodies that target the epitope of interest. The DNA is released from the proteins by heat and protease treatment, followed by degradation of contaminating RNAs with RNase. The resulting DNA is analyzed using various methods, including polymerase chain reaction (PCR), quantitative PCR (qPCR), or sequencing. This protocol outlines each of these steps for both yeast and human cells. This chapter includes a contextual discussion of the combination of ChIP with DNA analysis methods such as ChIP-on-Chip, ChIP-qPCR, and ChIP-Seq, recent updates on ChIP-Seq data analysis pipelines, complementary methods for identification of binding sites of DNA binding proteins, and additional protocol information about ChIP-qPCR and ChIP-Seq.}, booktitle={Methods in Molecular Biology}, publisher={Springer US}, author={Fadri, Maria Theresa M. and Lee, Jessica B. and Keung, Albert J.}, year={2024}, pages={419–447} }
 @misc{polak_keung_2023, title={A molecular assessment of the practical potential of DNA-based computation}, volume={81}, ISSN={["1879-0429"]}, DOI={10.1016/j.copbio.2023.102940}, abstractNote={The immense information density of DNA and its potential for massively parallelized computations, paired with rapidly expanding data production and storage needs, have fueled a renewed interest in DNA-based computation. Since the construction of the first DNA computing systems in the 1990s, the field has grown to encompass a diverse array of configurations. Simple enzymatic and hybridization reactions to solve small combinatorial problems transitioned to synthetic circuits mimicking gene regulatory networks and DNA-only logic circuits based on strand displacement cascades. These have formed the foundations of neural networks and diagnostic tools that aim to bring molecular computation to practical scales and applications. Considering these great leaps in system complexity as well as in the tools and technologies enabling them, a reassessment of the potential of such DNA computing systems is warranted.}, journal={CURRENT OPINION IN BIOTECHNOLOGY}, author={Polak, Rachel E. and Keung, Albert J.}, year={2023}, month={Jun} }
 @article{han_lee_indermaur_keung_2023, title={Chaetocin disrupts the SUV39H1-HP1 interaction independent of SUV39H1 methyltransferase activity}, volume={480}, ISSN={["1470-8728"]}, url={https://doi.org/10.1042/BCJ20220528}, DOI={10.1042/BCJ20220528}, abstractNote={Chemical tools to control the activities and interactions of chromatin components have broad impact on our understanding of cellular and disease processes. It is important to accurately identify their molecular effects to inform clinical efforts and interpretations of scientific studies. Chaetocin is a widely used chemical that decreases H3K9 methylation in cells. It is frequently attributed as a specific inhibitor of the histone methyltransferase activities of SUV39H1/SU(VAR)3–9, although prior observations showed chaetocin likely inhibits methyltransferase activity through covalent mechanisms involving its epipolythiodixopiperazine disulfide ‘warhead’ functionality. The continued use of chaetocin in scientific studies may derive from the net effect of reduced H3K9 methylation, irrespective of a direct or indirect mechanism. However, there may be other molecular impacts of chaetocin on SUV39H1 besides inhibition of H3K9 methylation levels that could confound the interpretation of past and future experimental studies. Here, we test a new hypothesis that chaetocin may have an additional downstream impact aside from inhibition of methyltransferase activity. Using a combination of truncation mutants, a yeast two-hybrid system, and direct in vitro binding assays, we show that the human SUV39H1 chromodomain (CD) and HP1 chromoshadow domain (CSD) directly interact. Chaetocin inhibits this binding interaction through its disulfide functionality with some specificity by covalently binding with the CD of SUV39H1, whereas the histone H3–HP1 interaction is not inhibited. Given the key role of HP1 dimers in driving a feedback cascade to recruit SUV39H1 and to establish and stabilize constitutive heterochromatin, this additional molecular consequence of chaetocin should be broadly considered.}, number={6}, journal={BIOCHEMICAL JOURNAL}, author={Han, Linna and Lee, Jessica B. and Indermaur, Elaine W. and Keung, Albert J.}, year={2023}, month={Mar}, pages={421–432} }
 @article{volkel_lin_hook_timp_keung_tuck_2023, title={FrameD: framework for DNA-based data storage design, verification, and validation}, volume={39}, ISSN={["1367-4811"]}, url={https://doi.org/10.1093/bioinformatics/btad572}, DOI={10.1093/bioinformatics/btad572}, abstractNote={Abstract
               
                  Motivation
                  DNA-based data storage is a quickly growing field that hopes to harness the massive theoretical information density of DNA molecules to produce a competitive next-generation storage medium suitable for archival data. In recent years, many DNA-based storage system designs have been proposed. Given that no common infrastructure exists for simulating these storage systems, comparing many different designs along with many different error models is increasingly difficult. To address this challenge, we introduce FrameD, a simulation infrastructure for DNA storage systems that leverages the underlying modularity of DNA storage system designs to provide a framework to express different designs while being able to reuse common components.
               
               
                  Results
                  We demonstrate the utility of FrameD and the need for a common simulation platform using a case study. Our case study compares designs that utilize strand copies differently, some that align strand copies using multiple sequence alignment algorithms and others that do not. We found that the choice to include multiple sequence alignment in the pipeline is dependent on the error rate and the type of errors being injected and is not always beneficial. In addition to supporting a wide range of designs, FrameD provides the user with transparent parallelism to deal with a large number of reads from sequencing and the need for many fault injection iterations. We believe that FrameD fills a void in the tools publicly available to the DNA storage community by providing a modular and extensible framework with support for massive parallelism. As a result, it will help accelerate the design process of future DNA-based storage systems.
               
               
                  Availability and implementation
                  The source code for FrameD along with the data generated during the demonstration of FrameD is available in a public Github repository at https://github.com/dna-storage/framed, (https://dx.doi.org/10.5281/zenodo.7757762).
               }, number={10}, journal={BIOINFORMATICS}, author={Volkel, Kevin D. and Lin, Kevin N. and Hook, Paul W. and Timp, Winston and Keung, Albert J. and Tuck, James M.}, editor={Kelso, JanetEditor}, year={2023}, month={Oct} }
 @article{estridge_o’neill_keung_2023, title={Matrigel Tunes H9 Stem Cell-Derived Human Cerebral Organoid Development}, volume={2}, ISSN={2674-1172}, url={http://dx.doi.org/10.3390/organoids2040013}, DOI={10.3390/organoids2040013}, abstractNote={Human cerebral organoids are readily generated from human embryonic stem cells and human induced pluripotent stem cells and are useful in studying human neurodevelopment. Recent work with human cerebral organoids have explored the creation of different brain regions and the impacts of soluble and mechanical cues. Matrigel is a gelatinous, heterogenous mixture of extracellular matrix proteins, morphogens, and growth factors secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. It is a core component of almost all cerebral organoid protocols, generally supporting neuroepithelial budding and tissue polarization; yet, its roles and effects beyond its general requirement in organoid protocols are not well understood, and its mode of delivery is variable, including the embedding of organoids within it or its delivery in soluble form. Given its widespread usage, we asked how H9 stem cell-derived hCO development and composition are affected by Matrigel dosage and delivery method. We found Matrigel exposure influences organoid size, morphology, and cell type composition. We also showed that greater amounts of Matrigel promote an increase in the number of choroid plexus (ChP) cells, and this increase is regulated by the BMP4 pathway. These results illuminate the effects of Matrigel on human cerebral organoid development and the importance of delivery mode and amount on organoid phenotype and composition.}, number={4}, journal={Organoids}, publisher={MDPI AG}, author={Estridge, R. Chris and O’Neill, Jennifer E. and Keung, Albert J.}, year={2023}, month={Oct}, pages={165–176} }
 @article{tam_keung_2023, title={Profiling transcriptomic responses of human stem cell-derived medium spiny neuron-like cells to exogenous phasic and tonic neurotransmitters}, volume={126}, ISSN={["1095-9327"]}, DOI={10.1016/j.mcn.2023.103876}, abstractNote={Transcriptomic responses to neurotransmitters contribute to the complex processes driving memory and addiction. Advances in both measurement methods and experimental models continue to improve our understanding of this regulatory layer. Here we focus on the experimental potential of stem cell derived neurons, currently the only ethical model that can be used in reductionist and experimentally perturbable studies of human cells. Prior work has focused on generating distinct cell types from human stem cells, and has also shown their utility in modeling development and cellular phenotypes related to neurodegeneration. Here we seek an understanding of how stem cell derived neural cultures respond to perturbations experienced during development and disease progression. This work profiles transcriptomic responses of human medium spiny neuron-like cells with three specific goals. We first characterize transcriptomic responses to dopamine and dopamine receptor agonists and antagonists presented in dosing patterns mimicking acute, chronic, and withdrawal regimens. We also assess transcriptomic responses to low and persistent tonic levels of dopamine, acetylcholine, and glutamate to better mimic the in vivo environment. Finally, we identify similar and distinct responses between hMSN-like cells derived from H9 and H1 stem cell lines, providing some context for the extent of variability these types of systems will likely pose for experimentalists. The results here suggest future optimizations of human stem cell derived neurons to increase their in vivo relevance and the biological insights that can be garnered from these models.}, journal={MOLECULAR AND CELLULAR NEUROSCIENCE}, author={Tam, Ryan W. and Keung, Albert J.}, year={2023}, month={Sep} }
 @article{rudibaugh_stuppy_keung_2023, title={Reactive Oxygen Species Mediate Transcriptional Responses to Dopamine and Cocaine in Human Cerebral Organoids}, volume={24}, ISSN={1422-0067}, url={http://dx.doi.org/10.3390/ijms242216474}, DOI={10.3390/ijms242216474}, abstractNote={Dopamine signaling in the adult ventral forebrain regulates behavior, stress response, and memory formation and in neurodevelopment regulates neural differentiation and cell migration. Excessive dopamine levels, including those due to cocaine use in utero and in adults, could lead to long-term adverse consequences. The mechanisms underlying both homeostatic and pathological changes remain unclear, in part due to the diverse cellular responses elicited by dopamine and the reliance on animal models that exhibit species-specific differences in dopamine signaling. In this study, we use the human-derived ventral forebrain organoid model of Xiang-Tanaka and characterize their response to cocaine or dopamine. We explore dosing regimens of dopamine or cocaine to simulate acute or chronic exposure. We then use calcium imaging, cAMP imaging, and bulk RNA-sequencing to measure responses to cocaine or dopamine exposure. We observe an upregulation of inflammatory pathways in addition to indicators of oxidative stress following exposure. Using inhibitors of reactive oxygen species (ROS), we then show ROS to be necessary for multiple transcriptional responses of cocaine exposure. These results highlight novel response pathways and validate the potential of cerebral organoids as in vitro human models for studying complex biological processes in the brain.}, number={22}, journal={International Journal of Molecular Sciences}, publisher={MDPI AG}, author={Rudibaugh, Thomas T. and Stuppy, Samantha R. and Keung, Albert J.}, year={2023}, month={Nov}, pages={16474} }
 @article{lee_tuck_keung_2023, title={Reimaging DNA as the Code of (More Than) Life}, volume={119}, number={11}, journal={Chemical Engineering Progress}, author={Lee, Magdelene and Tuck, James and Keung, Albert}, year={2023}, month={Nov}, pages={34–37} }
 @article{volkel_tomek_keung_tuck_2022, title={DINOS: Data INspired Oligo Synthesis for DNA Data Storage}, volume={18}, ISSN={["1550-4840"]}, url={http://dx.doi.org/10.1145/3510853}, DOI={10.1145/3510853}, abstractNote={As interest in DNA-based information storage grows, the costs of synthesis have been identified as a key bottleneck. A potential direction is to tune synthesis for data. Data strands tend to be composed of a small set of recurring code word sequences, and they contain longer sequences of repeated data. To exploit these properties, we propose a new framework called DINOS. DINOS consists of three key parts: (i) The first is a hierarchical strand assembly algorithm, inspired by gene assembly techniques that can assemble arbitrary data strands from a small set of primitive blocks. (ii) The assembly algorithm relies on our novel formulation for how to construct primitive blocks, spanning a variety of useful configurations from a set of code words and overhangs. Each primitive block is a code word flanked by a pair of overhangs that are created by a cyclic pairing process that keeps the number of primitive blocks small. Using these primitive blocks, any data strand of arbitrary length can be assembled, theoretically. We show a minimal system for a binary code with as few as six primitive blocks, and we generalize our processes to support an arbitrary set of overhangs and code words. (iii) We exploit our hierarchical assembly approach to identify redundant sequences and coalesce the reactions that create them to make assembly more efficient.
          
            We evaluate DINOS and describe its key characteristics. For example, the number of reactions needed to make a strand can be reduced by increasing the number of overhangs or the number of code words, but increasing the number of overhangs offers a small advantage over increasing code words while requiring substantially fewer primitive blocks. However, density is improved more by increasing the number of code words. We also find that a simple redundancy coalescing technique is able to reduce reactions by 90.6% and 41.2% on average for decompressed and compressed data, respectively, even when the smallest data fragments being assembled are 16 bits. With a simple padding heuristic that finds even more redundancy, we can further decrease reactions for the same operating point up to 91.1% and 59% for decompressed and compressed data, respectively, on average. Our approach offers greater density by up to 80% over a prior general purpose gene assembly technique. Finally, in an analysis of synthesis costs in which we make 1 GB volume using
            de novo
            synthesis versus making only the primitive blocks with
            de novo
            synthesis and otherwise assembling using DINOS, we estimate DINOS as 10
            5
            × cheaper than
            de novo
            synthesis.
          }, number={3}, journal={ACM JOURNAL ON EMERGING TECHNOLOGIES IN COMPUTING SYSTEMS}, publisher={Association for Computing Machinery (ACM)}, author={Volkel, Kevin and Tomek, Kyle J. and Keung, Albert J. and Tuck, James M.}, year={2022}, month={Jul} }
 @article{tam_keung_2022, title={Human Pluripotent Stem Cell-Derived Medium Spiny Neuron-like Cells Exhibit Gene Desensitization}, volume={11}, ISSN={["2073-4409"]}, url={https://www.mdpi.com/2073-4409/11/9/1411}, DOI={10.3390/cells11091411}, abstractNote={Gene desensitization in response to a repeated stimulus is a complex phenotype important across homeostatic and disease processes, including addiction, learning, and memory. These complex phenotypes are being characterized and connected to important physiologically relevant functions in rodent systems but are difficult to capture in human models where even acute responses to important neurotransmitters are understudied. Here through transcriptomic analysis, we map the dynamic responses of human stem cell-derived medium spiny neuron-like cells (hMSN-like cells) to dopamine. Furthermore, we show that these human neurons can reflect and capture cellular desensitization to chronic versus acute administration of dopamine. These human cells are further able to capture complex receptor crosstalk in response to the pharmacological perturbations of distinct dopamine receptor subtypes. This study demonstrates the potential utility and remaining challenges of using human stem cell-derived neurons to capture and study the complex dynamic mechanisms of the brain.}, number={9}, journal={CELLS}, author={Tam, Ryan W. and Keung, Albert J.}, year={2022}, month={May} }
 @article{meanor_keung_rao_2022, title={Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity}, volume={23}, ISSN={["1422-0067"]}, url={https://www.mdpi.com/1422-0067/23/3/1691}, DOI={10.3390/ijms23031691}, abstractNote={Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.}, number={3}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Meanor, Jenna N. and Keung, Albert J. and Rao, Balaji M.}, year={2022}, month={Feb} }
 @misc{meanor_keung_rao_gera_2022, title={Yeast Display Guided Selection of pH-Dependent Binders}, ISBN={9781071622841 9781071622858}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-0716-2285-8_16}, DOI={10.1007/978-1-0716-2285-8_16}, abstractNote={pH-dependent antigen binding has proven useful in engineering next-generation therapeutics specifically via antibody recycling technology. This technology allows for half-lifeHalf-life extension, thereby lowering the amount and frequency of dosing of therapeutics. Cell sorting, coupled with display techniques, has been used extensively for the selection of high-affinity binders. Herein, we describe a cell sorting methodology utilizing yeast surface display for selection of binding proteins with strong binding at physiological pH and weak to no binding at acidic pH. This methodology can be readily applied to engineer proteins and/or antibodies that do not have pH-dependent binding or for selection of de novo pH-dependent bindersPh dependent binder using library-based methods.}, journal={Methods in Molecular Biology}, publisher={Springer US}, author={Meanor, Jenna N. and Keung, Albert J. and Rao, Balaji M. and Gera, Nimish}, year={2022}, pages={293–311} }
 @misc{matange_tuck_keung_2021, title={DNA stability: a central design consideration for DNA data storage systems}, volume={12}, ISSN={["2041-1723"]}, url={https://doi.org/10.1038/s41467-021-21587-5}, DOI={10.1038/s41467-021-21587-5}, abstractNote={AbstractData storage in DNA is a rapidly evolving technology that could be a transformative solution for the rising energy, materials, and space needs of modern information storage. Given that the information medium is DNA itself, its stability under different storage and processing conditions will fundamentally impact and constrain design considerations and data system capabilities. Here we analyze the storage conditions, molecular mechanisms, and stabilization strategies influencing DNA stability and pose specific design configurations and scenarios for future systems that best leverage the considerable advantages of DNA storage.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Matange, Karishma and Tuck, James M. and Keung, Albert J.}, year={2021}, month={Mar} }
 @article{sen_voulgaropoulos_keung_2021, title={Effects of early geometric confinement on the transcriptomic profile of human cerebral organoids}, volume={21}, ISSN={["1472-6750"]}, url={https://doi.org/10.1186/s12896-021-00718-2}, DOI={10.1186/s12896-021-00718-2}, abstractNote={Abstract
                Background
                Human cerebral organoids (hCO) are attractive systems due to their ability to model important brain regions and transcriptomics of early in vivo brain development. To date, they have been used to understand the effects of genetics and soluble factors on neurodevelopment. Interestingly, one of the main advantages of hCOs are that they provide three dimensionality that better mimics the in vivo environment; yet, despite this central feature it remains unclear how spatial and mechanical properties regulate hCO and neurodevelopment. While biophysical factors such as shape and mechanical forces are known to play crucial roles in stem cell differentiation, embryogenesis and neurodevelopment, much of this work investigated two dimensional systems or relied on correlative observations of native developing tissues in three dimensions. Using hCOs to establish links between spatial factors and neurodevelopment will require the use of new approaches and could reveal fundamental principles of brain organogenesis as well as improve hCOs as an experimental model.
              
                Results
                Here, we investigated the effects of early geometric confinements on transcriptomic changes during hCO differentiation. Using a custom and tunable agarose microwell platform we generated embryoid bodies (EB) of diverse shapes mimicking several structures from embryogenesis and neurodevelopment and then further differentiated those EBs to whole brain hCOs. Our results showed that the microwells did not have negative gross impacts on the ability of the hCOs to differentiate towards neural fates, and there were clear shape dependent effects on neural lineage specification. In particular we observed that non-spherical shapes showed signs of altered neurodevelopmental kinetics and favored the development of medial ganglionic eminence-associated brain regions and cell types over cortical regions. Transcriptomic analysis suggests these mechanotransducive effects may be mediated by integrin and Wnt signaling.
              
                Conclusions
                The findings presented here suggest a role for spatial factors in brain region specification during hCO development. Understanding these spatial patterning factors will not only improve understanding of in vivo development and differentiation, but also provide important handles with which to advance and improve control over human model systems for in vitro applications.
              }, number={1}, journal={BMC BIOTECHNOLOGY}, author={Sen, Dilara and Voulgaropoulos, Alexis and Keung, Albert J.}, year={2021}, month={Oct} }
 @article{sen_drobna_keung_2021, title={Evaluation of UBE3A antibodies in mice and human cerebral organoids}, volume={11}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-021-85923-x}, abstractNote={AbstractUBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. While commercially available antibodies have been widely employed to determine UBE3A localization, an extensive analysis and comparison of the performance of different UBE3A antibodies has not been conducted. Here we evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition, we also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models. Our results reveal that among the seven antibodies tested, three antibodies demonstrated substantial nonspecific immunoreactivity. While four antibodies show specific localization patterns in both mouse brain sections and human cerebral organoids, these antibodies varied significantly in background signals and staining patterns in undifferentiated human pluripotent stem cells.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Sen, Dilara and Drobna, Zuzana and Keung, Albert J.}, year={2021}, month={Mar} }
 @article{lee_caywood_lo_levering_keung_2021, title={Mapping the dynamic transfer functions of eukaryotic gene regulation}, volume={12}, ISSN={["2405-4720"]}, DOI={10.1016/j.cels.2021.08.003}, abstractNote={Biological information can be encoded within the dynamics of signaling components, which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions-modulating the pulse frequency, amplitude, and pulse width of light-regulating the binding of an epigenome editor to a fluorescent reporter. This system revealed tunable gene expression and filtering behaviors and provided a quantification of the limit to the amount of information that can be reliably transferred across a single promoter as ∼1.7 bits. Using a library of over 100 orthogonal chromatin regulators, we further determined that chromatin state could be used to tune mutual information and expression levels, as well as completely alter the input-output transfer function of the promoter. This system unlocks the information-rich content of eukaryotic gene regulation.}, number={11}, journal={CELL SYSTEMS}, author={Lee, Jessica B. and Caywood, Leandra M. and Lo, Jennifer Y. and Levering, Nicholas and Keung, Albert J.}, year={2021}, month={Nov}, pages={1079-+} }
 @article{waldman_rao_keung_2021, title={Mapping the residue specificities of epigenome enzymes by yeast surface display}, volume={28}, ISSN={2451-9456}, url={http://dx.doi.org/10.1016/j.chembiol.2021.05.022}, DOI={10.1016/j.chembiol.2021.05.022}, abstractNote={Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here, we describe a versatile and scalable approach which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first use this system to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, a large panel of commercially available anti-acetylation antibodies are screened for their specificities, identifying many suitable and unsuitable reagents. Furthermore, this approach enables efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4 and uncovers residue interdependencies affecting p300 activity. These results show that using yeast surface display to study histone modifications is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications.}, number={12}, journal={Cell Chemical Biology}, publisher={Elsevier BV}, author={Waldman, Alison C. and Rao, Balaji M. and Keung, Albert J.}, year={2021}, month={Dec}, pages={1772–1779.e4} }
 @article{tomek_volkel_indermaur_tuck_keung_2021, title={Promiscuous molecules for smarter file operations in DNA-based data storage}, volume={12}, ISSN={["2041-1723"]}, url={https://doi.org/10.1038/s41467-021-23669-w}, DOI={10.1038/s41467-021-23669-w}, abstractNote={AbstractDNA holds significant promise as a data storage medium due to its density, longevity, and resource and energy conservation. These advantages arise from the inherent biomolecular structure of DNA which differentiates it from conventional storage media. The unique molecular architecture of DNA storage also prompts important discussions on how data should be organized, accessed, and manipulated and what practical functionalities may be possible. Here we leverage thermodynamic tuning of biomolecular interactions to implement useful data access and organizational features. Specific sets of environmental conditions including distinct DNA concentrations and temperatures were screened for their ability to switchably access either all DNA strands encoding full image files from a GB-sized background database or subsets of those strands encoding low resolution, File Preview, versions. We demonstrate File Preview with four JPEG images and provide an argument for the substantial and practical economic benefit of this generalizable strategy to organize data.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Tomek, Kyle J. and Volkel, Kevin and Indermaur, Elaine W. and Tuck, James M. and Keung, Albert J.}, year={2021}, month={Jun} }
 @article{annabi_baker_boettiger_chakraborty_chen_corbett_correia_dahlman_oliveira_ertuerk_et al._2021, title={Voices of biotech research}, volume={39}, ISSN={["1546-1696"]}, DOI={10.1038/s41587-021-00847-1}, abstractNote={Nature Biotechnology asks a selection of faculty about the most exciting frontier in their field and the most needed technologies for advancing knowledge and applications.}, number={3}, journal={NATURE BIOTECHNOLOGY}, author={Annabi, Nasim and Baker, Matthew and Boettiger, Alistair and Chakraborty, Debojyoti and Chen, Yvonne and Corbett, Kizzmekia S. and Correia, Bruno and Dahlman, James and Oliveira, Tulio and Ertuerk, Ali and et al.}, year={2021}, month={Mar}, pages={281–286} }
 @misc{sen_keung_2020, title={Capturing complex epigenetic phenomena through human multicellular systems}, volume={16}, ISSN={["2468-4511"]}, DOI={10.1016/j.cobme.2020.05.008}, abstractNote={Epigenetic states inherently define a wide range of complex biological phenotypes and processes in development and disease. Accurate cellular modeling would ideally capture the epigenetic complexity of these processes as well as the accompanying molecular changes in chromatin biochemistry including in DNA and histone modifications. Here we highlight recent work that demonstrate how multicellular systems provide a natural approach to capture complex epigenetic phenomena. They accomplish this through more closely matching the in vivo environment and through the intrinsic nature of multicellular systems being able to generate and model multiple distinct cellular states, all within one system. We also discuss challenges and limitations of such systems, efforts to tune and modulate epigenetics directly in multicellular systems, and how molecular interventional approaches could advance and improve the utility of these models.}, journal={CURRENT OPINION IN BIOMEDICAL ENGINEERING}, author={Sen, Dilara and Keung, Albert J.}, year={2020}, month={Dec}, pages={34–41} }
 @article{lin_volkel_tuck_keung_2020, title={Dynamic and scalable DNA-based information storage}, volume={11}, ISSN={["2041-1723"]}, url={https://doi.org/10.1038/s41467-020-16797-2}, DOI={10.1038/s41467-020-16797-2}, abstractNote={AbstractThe physical architectures of information storage systems often dictate how information is encoded, databases are organized, and files are accessed. Here we show that a simple architecture comprised of a T7 promoter and a single-stranded overhang domain (ss-dsDNA), can unlock dynamic DNA-based information storage with powerful capabilities and advantages. The overhang provides a physical address for accessing specific DNA strands as well as implementing a range of in-storage file operations. It increases theoretical storage densities and capacities by expanding the encodable sequence space and simplifies the computational burden in designing sets of orthogonal file addresses. Meanwhile, the T7 promoter enables repeatable information access by transcribing information from DNA without destroying it. Furthermore, saturation mutagenesis around the T7 promoter and systematic analyses of environmental conditions reveal design criteria that can be used to optimize information access. This simple but powerful ss-dsDNA architecture lays the foundation for information storage with versatile capabilities.}, number={1}, journal={NATURE COMMUNICATIONS}, publisher={Springer Science and Business Media LLC}, author={Lin, Kevin N. and Volkel, Kevin and Tuck, James M. and Keung, Albert J.}, year={2020}, month={Jun} }
 @article{sen_voulgaropoulos_drobna_keung_2020, title={Human Cerebral Organoids Reveal Early Spatiotemporal Dynamics and Pharmacological Responses of UBE3A}, volume={15}, ISSN={["2213-6711"]}, url={https://doi.org/10.1016/j.stemcr.2020.08.006}, DOI={10.1016/j.stemcr.2020.08.006}, abstractNote={Angelman syndrome is a complex neurodevelopmental disorder characterized by delayed development, intellectual disability, speech impairment, and ataxia. It results from the loss of UBE3A protein, an E3 ubiquitin ligase, in neurons of the brain. Despite the dynamic spatiotemporal expression of UBE3A observed in rodents and the potential clinical importance of when and where it is expressed, its expression pattern in humans remains unknown. This reflects a common challenge of studying human neurodevelopment: prenatal periods are hard to access experimentally. In this work, human cerebral organoids reveal a change from weak to strong UBE3A in neuronal nuclei within 3 weeks of culture. Angelman syndrome human induced pluripotent stem cell-derived organoids also exhibit early silencing of paternal UBE3A, with topoisomerase inhibitors partially rescuing UBE3A levels and calcium transient phenotypes. This work establishes human cerebral organoids as an important model for studying UBE3A and motivates their broader use in understanding complex neurodevelopmental disorders.}, number={4}, journal={STEM CELL REPORTS}, author={Sen, Dilara and Voulgaropoulos, Alexis and Drobna, Zuzana and Keung, Albert J.}, year={2020}, month={Oct}, pages={845–854} }
 @article{tomek_volkel_simpson_hass_indermaur_tuck_keung_2019, title={Driving the Scalability of DNA-Based Information Storage Systems}, volume={8}, ISSN={["2161-5063"]}, url={https://doi.org/10.1021/acssynbio.9b00100}, DOI={10.1021/acssynbio.9b00100}, abstractNote={The extreme density of DNA presents a compelling advantage over current storage media; however, to reach practical capacities, new systems for organizing and accessing information are needed. Here, we use chemical handles to selectively extract unique files from a complex database of DNA mimicking 5 TB of data and design and implement a nested file address system that increases the theoretical maximum capacity of DNA storage systems by five orders of magnitude. These advancements enable the development and future scaling of DNA-based data storage systems with modern capacities and file access capabilities.}, number={6}, journal={ACS SYNTHETIC BIOLOGY}, author={Tomek, Kyle J. and Volkel, Kevin and Simpson, Alexander and Hass, Austin G. and Indermaur, Elaine W. and Tuck, James M. and Keung, Albert J.}, year={2019}, month={Jun}, pages={1241–1248} }
 @article{sen_voulgaropoulos_drobna_keung_2019, title={Human cerebral organoids capture the spatiotemporal complexity and disease dynamics of UBE3A}, url={https://doi.org/10.1101/742213}, DOI={10.1101/742213}, abstractNote={SUMMARY Human neurodevelopment and its associated diseases are complex and challenging to study. This has driven recent excitement for human cerebral organoids (hCOs) as research and screening tools. These models are steadily proving their utility; however, it remains unclear what limits they will face in recapitulating the complexities of neurodevelopment and disease. Here we show that their utility extends to key (epi)genetic and disease processes that are complex in space and time. Specifically, hCOs capture UBE3A’s dynamically imprinted expression and subcellular localization patterns. Furthermore, given UBE3A’s direct links to Angelman Syndrome and Autism Spectrum Disorder, we show that hCOs respond to candidate small molecule therapeutics. This work demonstrates that hCOs can provide important insights to focus the scope of mechanistic and therapeutic strategies including revealing difficult to access prenatal developmental time windows and cell types key to disease etiology.}, author={Sen, Dilara and Voulgaropoulos, Alexis and Drobna, Zuzana and Keung, Albert J.}, year={2019}, month={Aug} }
 @article{liao_ttofali_slotkowski_denny_cecil_leenay_keung_beisel_2019, title={Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis}, volume={10}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-019-10747-3}, abstractNote={AbstractCRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.}, journal={NATURE COMMUNICATIONS}, author={Liao, Chunyu and Ttofali, Fani and Slotkowski, Rebecca A. and Denny, Steven R. and Cecil, Taylor D. and Leenay, Ryan T. and Keung, Albert J. and Beisel, Chase L.}, year={2019}, month={Jul} }
 @misc{lee_keung_2018, title={Chromatin Immunoprecipitation in Human and Yeast Cells}, ISBN={9781493977734 9781493977741}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-4939-7774-1_14}, DOI={10.1007/978-1-4939-7774-1_14}, abstractNote={Chromatin immunoprecipitation (ChIP) is an invaluable method to characterize interactions between proteins and genomic DNA, such as the genomic localization of transcription factors and posttranslational modification of histones. DNA and proteins are reversibly and covalently crosslinked using formaldehyde. Then the cells are lysed to release the chromatin. The chromatin is fragmented into smaller sizes either by micrococcal nuclease (MNase) or sonication and then purified from other cellular components. The protein-DNA complexes are enriched by immunoprecipitation (IP) with antibodies that target the epitope of interest. The DNA is released from the proteins by heat and protease treatment, followed by degradation of contaminating RNAs with RNase. The resulting DNA is analyzed using various methods, including PCR, qPCR, or sequencing. This protocol outlines each of these steps for both yeast and human cells.}, journal={Methods in Molecular Biology}, publisher={Springer New York}, author={Lee, Jessica B. and Keung, Albert J.}, year={2018}, pages={257–269} }
 @misc{sen_keung_2018, title={Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities}, ISBN={9781493977734 9781493977741}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-4939-7774-1_3}, DOI={10.1007/978-1-4939-7774-1_3}, abstractNote={The advent of locus-specific protein recruitment technologies has enabled a new class of studies in chromatin biology. Epigenome editors enable biochemical modifications of chromatin at almost any specific endogenous locus. Their locus specificity unlocks unique information including the functional roles of distinct modifications at specific genomic loci. Given the growing interest in using these tools for biological and translational studies, there are many specific design considerations depending on the scientific question or clinical need. Here we present and discuss important design considerations and challenges regarding the biochemical and locus specificities of epigenome editors. These include how to account for the complex biochemical diversity of chromatin; control for potential interdependency of epigenome editors and their resultant modifications; avoid sequestration effects; quantify the locus specificity of epigenome editors; and improve locus specificity by considering concentration, affinity, avidity, and sequestration effects.}, journal={Methods in Molecular Biology}, publisher={Springer New York}, author={Sen, Dilara and Keung, Albert J.}, year={2018}, pages={65–87} }
 @article{park_patel_keung_khalil_2019, title={Engineering Epigenetic Regulation Using Synthetic Read-Write Modules}, volume={176}, ISSN={["1097-4172"]}, DOI={10.1016/j.cell.2018.11.002}, abstractNote={Chemical modifications to DNA and histone proteins are involved in epigenetic programs underlying cellular differentiation and development. Regulatory networks involving molecular writers and readers of chromatin marks are thought to control these programs. Guided by this common principle, we established an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system utilizes synthetic factors that write and read m6A and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we used our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits were able to program epigenetic functions de novo, conceptually validating “read-write” architectures. This work provides a toolkit for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells.}, number={1-2}, journal={CELL}, author={Park, Minhee and Patel, Nikit and Keung, Albert J. and Khalil, Ahmad S.}, year={2019}, month={Jan}, pages={227-+} }
 @article{keung_2018, title={The untapped information content of the epigenome}, volume={114}, number={9}, journal={Chemical Engineering Progress}, author={Keung, Albert J.}, year={2018}, pages={50–54} }
 @article{keung_khalil_2016, title={A unifying model of epigenetic regulation}, volume={351}, ISSN={["1095-9203"]}, DOI={10.1126/science.aaf1647}, abstractNote={
            Single-cell tracking reveals a common “algorithm” of operation used by chromatin regulators
            
              [Also see Report by
              
                Bintu
                et al.
              
              ]
            
          }, number={6274}, journal={SCIENCE}, author={Keung, Albert J. and Khalil, Ahmad S.}, year={2016}, month={Feb}, pages={661–662} }
 @misc{park_keung_khalil_2016, title={The epigenome: the next substrate for engineering}, volume={17}, ISSN={["1474-760X"]}, DOI={10.1186/s13059-016-1046-5}, abstractNote={We are entering an era of epigenome engineering. The precision manipulation of chromatin and epigenetic modifications provides new ways to interrogate their influence on genome and cell function and to harness these changes for applications. We review the design and state of epigenome editing tools, highlighting the unique regulatory properties afforded by these systems.}, journal={GENOME BIOLOGY}, author={Park, Minhee and Keung, Albert J. and Khalil, Ahmad S.}, year={2016}, month={Aug} }
 @article{keung_joung_khalil_collins_2015, title={Chromatin regulation at the frontier of synthetic biology}, volume={16}, ISSN={1471-0056 1471-0064}, url={http://dx.doi.org/10.1038/NRG3900}, DOI={10.1038/NRG3900}, abstractNote={Synthetic biology approaches to characterize gene regulation have largely used transcription factor circuits in bacteria. However, the multilayered regulation of genes by chromatin in eukaryotes provides opportunities for more sophisticated control of gene expression. This Review describes diverse approaches for engineering eukaryotic chromatin states, the insights gained into physiological gene regulation principles, and the broad potential applications throughout biomedical research and industry. As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including 'epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication.}, number={3}, journal={Nature Reviews Genetics}, publisher={Springer Science and Business Media LLC}, author={Keung, Albert J. and Joung, J. Keith and Khalil, Ahmad S. and Collins, James J.}, year={2015}, month={Feb}, pages={159–171} }
 @article{keung_bashor_kiriakov_collins_khalil_2014, title={Using Targeted Chromatin Regulators to Engineer Combinatorial and Spatial Transcriptional Regulation}, volume={158}, ISSN={0092-8674}, url={http://dx.doi.org/10.1016/J.CELL.2014.04.047}, DOI={10.1016/J.CELL.2014.04.047}, abstractNote={The transcription of genomic information in eukaryotes is regulated in large part by chromatin. How a diverse array of chromatin regulator (CR) proteins with different functions and genomic localization patterns coordinates chromatin activity to control transcription remains unclear. Here, we take a synthetic biology approach to decipher the complexity of chromatin regulation by studying emergent transcriptional behaviors from engineered combinatorial, spatial, and temporal patterns of individual CRs. We fuse 223 yeast CRs to programmable zinc finger proteins. Site-specific and combinatorial recruitment of CRs to distinct intralocus locations reveals a range of transcriptional logic and behaviors, including synergistic activation, long-range and spatial regulation, and gene expression memory. Comparing these transcriptional behaviors with annotated CR complex and function terms provides design principles for the engineering of transcriptional regulation. This work presents a bottom-up approach to investigating chromatin-mediated transcriptional regulation and introduces chromatin-based components and systems for synthetic biology and cellular engineering.}, number={1}, journal={Cell}, publisher={Elsevier BV}, author={Keung, Albert J. and Bashor, Caleb J. and Kiriakov, Szilvia and Collins, James J. and Khalil, Ahmad S.}, year={2014}, month={Jul}, pages={110–120} }
 @article{keung_dong_schaffer_kumar_2013, title={Pan-neuronal maturation but not neuronal subtype differentiation of adult neural stem cells is mechanosensitive}, volume={3}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/SREP01817}, DOI={10.1038/SREP01817}, abstractNote={Most past studies of the biophysical regulation of stem cell differentiation have focused on initial lineage commitment or proximal differentiation events. It would be valuable to understand whether biophysical inputs also influence distal endpoints more closely associated with physiological function, such as subtype specification in neuronal differentiation. To explore this question, we cultured adult neural stem cells (NSCs) on variable stiffness ECMs under conditions that promote neuronal fate commitment for extended time periods to allow neuronal subtype differentiation. We find that ECM stiffness does not modulate the expression of NeuroD1 and TrkA/B/C or the percentages of pan-neuronal, GABAergic, or glutamatergic neuronal subtypes. Interestingly, however, an ECM stiffness of 700 Pa maximizes expression of pan-neuronal markers. These results suggest that a wide range of stiffnesses fully permit pan-neuronal NSC differentiation, that an intermediate stiffness optimizes expression of pan-neuronal genes and that stiffness does not impact commitment to particular neuronal subtypes.}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Keung, Albert J. and Dong, Meimei and Schaffer, David V. and Kumar, Sanjay}, year={2013}, month={May} }
 @article{mackay_keung_kumar_2012, title={A Genetic Strategy for the Dynamic and Graded Control of Cell Mechanics, Motility, and Matrix Remodeling}, volume={102}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2011.12.048}, DOI={10.1016/j.bpj.2011.12.048}, abstractNote={Cellular mechanical properties have emerged as central regulators of many critical cell behaviors, including proliferation, motility, and differentiation. Although investigators have developed numerous techniques to influence these properties indirectly by engineering the extracellular matrix (ECM), relatively few tools are available to directly engineer the cells themselves. Here we present a genetic strategy for obtaining graded, dynamic control over cellular mechanical properties by regulating the expression of mutant mechanotransductive proteins from a single copy of a gene placed under a repressible promoter. With the use of constitutively active mutants of RhoA GTPase and myosin light chain kinase, we show that varying the expression level of either protein produces graded changes in stress fiber assembly, traction force generation, cellular stiffness, and migration speed. Using this approach, we demonstrate that soft ECMs render cells maximally sensitive to changes in RhoA activity, and that by modulating the ability of cells to engage and contract soft ECMs, we can dynamically control cell spreading, migration, and matrix remodeling. Thus, in addition to providing quantitative relationships between mechanotransductive signaling, cellular mechanical properties, and dynamic cell behaviors, this strategy enables us to control the physical interactions between cells and the ECM and thereby dictate how cells respond to matrix properties.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={MacKay, Joanna L. and Keung, Albert J. and Kumar, Sanjay}, year={2012}, month={Feb}, pages={434–442} }
 @article{keung_asuri_kumar_schaffer_2012, title={Soft microenvironments promote the early neurogenic differentiation but not self-renewal of human pluripotent stem cells}, volume={4}, ISSN={1757-9708}, url={http://dx.doi.org/10.1039/c2ib20083j}, DOI={10.1039/c2ib20083j}, abstractNote={Human pluripotent stem cells (hPSCs) are of great interest in biology and medicine due to their ability to self-renew and differentiate into any adult or fetal cell type. Important efforts have identified biochemical factors, signaling pathways, and transcriptional networks that regulate hPSC biology. However, recent work investigating the effect of biophysical cues on mammalian cells and adult stem cells suggests that the mechanical properties of the microenvironment, such as stiffness, may also regulate hPSC behavior. While several studies have explored this mechanoregulation in mouse embryonic stem cells (mESCs), it has been challenging to extrapolate these findings and thereby explore their biomedical implications in hPSCs. For example, it remains unclear whether hPSCs can be driven down a given tissue lineage by providing tissue-mimetic stiffness cues. Here we address this open question by investigating the regulation of hPSC neurogenesis by microenvironmental stiffness. We find that increasing extracellular matrix (ECM) stiffness in vitro increases hPSC cell and colony spread area but does not alter self-renewal, in contrast to past studies with mESCs. However, softer ECMs with stiffnesses similar to that of neural tissue promote the generation of early neural ectoderm. This mechanosensitive increase in neural ectoderm requires only a short 5-day soft stiffness "pulse", which translates into downstream increases in both total neurons as well as therapeutically relevant dopaminergic neurons. These findings further highlight important differences between mESCs and hPSCs and have implications for both the design of future biomaterials as well as our understanding of early embryonic development.}, number={9}, journal={Integrative Biology}, publisher={Oxford University Press (OUP)}, author={Keung, Albert J. and Asuri, Prashanth and Kumar, Sanjay and Schaffer, David V.}, year={2012}, month={Aug}, pages={1049–1058} }
 @article{jang_koerber_kim_asuri_vazin_bartel_keung_kwon_park_schaffer_2011, title={An Evolved Adeno-associated Viral Variant Enhances Gene Delivery and Gene Targeting in Neural Stem Cells}, volume={19}, ISSN={1525-0016}, url={http://dx.doi.org/10.1038/mt.2010.287}, DOI={10.1038/mt.2010.287}, abstractNote={Gene delivery to, and gene targeting in, stem cells would be a highly enabling technology for basic science and biomedical application. Adeno-associated viral (AAV) vectors have demonstrated the capacity for efficient delivery to numerous cells, but their application to stem cells has been limited by low transduction efficiency. Due to their considerable advantages, however, engineering AAV delivery systems to enhance gene delivery to stem cells may have an impact in stem cell biology and therapy. Therefore, using several diverse AAV capsid libraries—including randomly mutagenized, DNA shuffled, and random peptide insertion variants—we applied directed evolution to create a "designer" AAV vector with enhanced delivery efficiency for neural stem cells (NSCs). A novel AAV variant, carrying an insertion of a selected peptide sequence on the surface of the threefold spike within the heparin-binding site, emerged from this evolution. Importantly, this evolved AAV variant mediated efficient gene delivery to rat, mouse, and human NSCs, as well as efficient gene targeting within adult NSCs, and it is thus promising for applications ranging from basic stem cell biology to clinical translation. Gene delivery to, and gene targeting in, stem cells would be a highly enabling technology for basic science and biomedical application. Adeno-associated viral (AAV) vectors have demonstrated the capacity for efficient delivery to numerous cells, but their application to stem cells has been limited by low transduction efficiency. Due to their considerable advantages, however, engineering AAV delivery systems to enhance gene delivery to stem cells may have an impact in stem cell biology and therapy. Therefore, using several diverse AAV capsid libraries—including randomly mutagenized, DNA shuffled, and random peptide insertion variants—we applied directed evolution to create a "designer" AAV vector with enhanced delivery efficiency for neural stem cells (NSCs). A novel AAV variant, carrying an insertion of a selected peptide sequence on the surface of the threefold spike within the heparin-binding site, emerged from this evolution. Importantly, this evolved AAV variant mediated efficient gene delivery to rat, mouse, and human NSCs, as well as efficient gene targeting within adult NSCs, and it is thus promising for applications ranging from basic stem cell biology to clinical translation.}, number={4}, journal={Molecular Therapy}, publisher={Elsevier BV}, author={Jang, Jae-Hyung and Koerber, James T and Kim, Jung-Suk and Asuri, Prashanth and Vazin, Tandis and Bartel, Melissa and Keung, Albert and Kwon, Inchan and Park, Kook In and Schaffer, David V}, year={2011}, month={Apr}, pages={667–675} }
 @article{ashton_keung_peltier_schaffer_2011, title={Progress and Prospects for Stem Cell Engineering}, volume={2}, ISSN={1947-5438 1947-5446}, url={http://dx.doi.org/10.1146/annurev-chembioeng-061010-114105}, DOI={10.1146/annurev-chembioeng-061010-114105}, abstractNote={Stem cells offer tremendous biomedical potential owing to their abilities to self-renew and differentiate into cell types of multiple adult tissues. Researchers and engineers have increasingly developed novel discovery technologies, theoretical approaches, and cell culture systems to investigate microenvironmental cues and cellular signaling events that control stem cell fate. Many of these technologies facilitate high-throughput investigation of microenvironmental signals and the intracellular signaling networks and machinery processing those signals into cell fate decisions. As our aggregate empirical knowledge of stem cell regulation grows, theoretical modeling with systems and computational biology methods has and will continue to be important for developing our ability to analyze and extract important conceptual features of stem cell regulation from complex data. Based on this body of knowledge, stem cell engineers will continue to develop technologies that predictably control stem cell fate with the ultimate goal of being able to accurately and economically scale up these systems for clinical-grade production of stem cell therapeutics.}, number={1}, journal={Annual Review of Chemical and Biomolecular Engineering}, publisher={Annual Reviews}, author={Ashton, Randolph S. and Keung, Albert J. and Peltier, Joseph and Schaffer, David V.}, year={2011}, month={Jul}, pages={479–502} }
 @article{keung_de juan-pardo_schaffer_kumar_2011, title={Rho GTPases Mediate the Mechanosensitive Lineage Commitment of Neural Stem Cells}, volume={29}, ISSN={1066-5099}, url={http://dx.doi.org/10.1002/stem.746}, DOI={10.1002/stem.746}, abstractNote={Abstract
               Adult neural stem cells (NSCs) play important roles in learning and memory and are negatively impacted by neurological disease. It is known that biochemical and genetic factors regulate self-renewal and differentiation, and it has recently been suggested that mechanical and solid-state cues, such as extracellular matrix (ECM) stiffness, can also regulate the functions of NSCs and other stem cell types. However, relatively little is known of the molecular mechanisms through which stem cells transduce mechanical inputs into fate decisions, the extent to which mechanical inputs instruct fate decisions versus select for or against lineage-committed blast populations, or the in vivo relevance of mechanotransductive signaling molecules in native stem cell niches. Here we demonstrate that ECM-derived mechanical signals act through Rho GTPases to activate the cellular contractility machinery in a key early window during differentiation to regulate NSC lineage commitment. Furthermore, culturing NSCs on increasingly stiff ECMs enhances RhoA and Cdc42 activation, increases NSC stiffness, and suppresses neurogenesis. Likewise, inhibiting RhoA and Cdc42 or downstream regulators of cellular contractility rescues NSCs from stiff matrix- and Rho GTPase-induced neurosuppression. Importantly, Rho GTPase expression and ECM stiffness do not alter proliferation or apoptosis rates indicating that an instructive rather than selective mechanism modulates lineage distributions. Finally, in the adult brain, RhoA activation in hippocampal progenitors suppresses neurogenesis, analogous to its effect in vitro. These results establish Rho GTPase-based mechanotransduction and cellular stiffness as biophysical regulators of NSC fate in vitro and RhoA as an important regulatory protein in the hippocampal stem cell niche.}, number={11}, journal={STEM CELLS}, publisher={Wiley}, author={Keung, Albert J. and de Juan-Pardo, Elena M. and Schaffer, David V. and Kumar, Sanjay}, year={2011}, month={Oct}, pages={1886–1897} }
 @article{mackay_keung_kumar_2010, title={A Genetic Strategy for Graded and Dynamic Control of Cell-Matrix Mechanobiology}, volume={98}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2009.12.4004}, DOI={10.1016/j.bpj.2009.12.4004}, abstractNote={Mechanical interactions between cells and the surrounding extracellular matrix, such as adhesion, contraction, and force transduction, play a central role in many fundamental cell behaviors, including proliferation, cell death, and motility. The ability to precisely manipulate the intracellular machinery that regulates these interactions could therefore provide a powerful tool for controlling the mechanical properties of living cells and could also allow us to re-engineer how cells sense and respond to mechanical stimuli in their microenvironment, which would be particularly useful for tissue engineering and cellular technologies where cells are interfaced with synthetic microenvironments. Towards this goal, we have genetically engineered stable cell lines in which we can precisely and dynamically alter the mechanobiological behavior of living cells by varying the activity of signal transduction proteins, such as RhoA GTPase, using constitutively active and dominant negative mutants under the control of a tetracycline-repressible promoter. Through a variety of imaging and biophysical techniques, including atomic force microscopy and traction force microscopy, we have demonstrated graded and dynamic control over cytoskeletal architecture, cell shape and spreading, contractility, and cellular stiffness. In addition, using glioblastoma multiforme as a model system, we show how these cell lines can be used to study the effects of altered cellular mechanical properties on cancer cell motility and invasion.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={MacKay, Joanna L. and Keung, Albert and Kumar, Sanjay}, year={2010}, month={Jan}, pages={730a} }
 @article{peltier_conway_keung_schaffer_2011, title={Akt Increases Sox2 Expression in Adult Hippocampal Neural Progenitor Cells, but Increased Sox2 Does Not Promote Proliferation}, volume={20}, ISSN={1547-3287 1557-8534}, url={http://dx.doi.org/10.1089/scd.2010.0130}, DOI={10.1089/scd.2010.0130}, abstractNote={Multiple extracellular factors have been shown to modulate adult hippocampal neural progenitor cell (NPC) proliferation and self-renewal, and we have previously shown that Akt is an important mediator of the effects of these extracellular factors on NPC proliferation and differentiation. However, very little work has investigated how and whether Akt is involved in maintaining the multipotency of these cells. Here we demonstrate that Akt promotes expression of Sox2, a core transcription factor important for the self-renewal of NPCs. Retroviral-mediated overexpression of wild-type Akt increased Sox2 protein expression, particularly under conditions that promote cell differentiation, whereas Akt inhibition decreased Sox2. Similarly, quantitative reverse transcription (RT)–PCR in differentiating cultures indicated that Akt rescued Sox2 mRNA to levels present under conditions that promote cell proliferation. Additionally, pharmacological inhibition of Akt did not affect Sox2 protein levels in cells constitutively expressing Sox2 from a retroviral vector, indicating that Akt does not affect Sox2 protein stability. Further, in contrast to Akt overexpression, Sox2 overexpression does not increase NPC viable cell number or proliferation yet does inhibit differentiation. Collectively, these results indicate that Akt promotes cell proliferation and maintenance of a multipotent state via two downstream paths.}, number={7}, journal={Stem Cells and Development}, publisher={Mary Ann Liebert Inc}, author={Peltier, Joseph and Conway, Anthony and Keung, Albert J. and Schaffer, David V.}, year={2011}, month={Jul}, pages={1153–1161} }
 @article{lam_cao_umesh_keung_sen_kumar_2010, title={Extracellular matrix rigidity modulates neuroblastoma cell differentiation and N-myc expression}, volume={9}, ISSN={1476-4598}, url={http://dx.doi.org/10.1186/1476-4598-9-35}, DOI={10.1186/1476-4598-9-35}, abstractNote={Neuroblastoma is a pediatric malignancy characterized by tremendous clinical heterogeneity, in which some tumors are extremely aggressive while others spontaneously differentiate into benign forms. Because the degree of differentiation correlates with prognosis, and because differentiating agents such as retinoic acid (RA) have proven to decrease mortality, much effort has been devoted to identifying critical regulators of neuroblastoma differentiation in the cellular microenvironment, including cues encoded in the extracellular matrix (ECM). While signaling between tumor cells and the ECM is classically regarded to be based purely on biochemical recognition of ECM ligands by specific cellular receptors, a number of recent studies have made it increasingly clear that the biophysical properties of the ECM may also play an important role in this cross-talk. Given that RA-mediated neuroblastoma differentiation is accompanied by profound changes in cell morphology and neurite extension, both of which presumably rely upon mechanotransductive signaling systems, it occurred to us that mechanical cues from the ECM might also influence RA-mediated differentiation, which in turn might regulate clinically-relevant aspects of neuroblastoma biology. In this study, we tested this hypothesis by subjecting a series of neuroblastoma culture models to ECM microenvironments of varying mechanical stiffness and examined the regulatory role of ECM stiffness in proliferation, differentiation, and expression of tumor markers. We find that increasing ECM stiffness enhances neuritogenesis and suppresses cell proliferation. Remarkably, increasing ECM stiffness also reduces expression of N-Myc, a transcription factor involved in multiple aspects of oncogenic proliferation that is used for evaluating prognosis and clinical grading of neuroblastoma. Furthermore, the addition of RA enhances all of these effects for all ECM stiffnesses tested. Together, our data strongly support the notion that the mechanical signals from the cellular microenvironment influence neuroblastoma differentiation and do so synergistically with RA. These observations support further investigation of the role of microenvironmental mechanical signals in neuroblastoma proliferation and differentiation and suggest that pharmacological agents that modulate the underlying mechanotransductive signaling pathways may have a role in neuroblastoma therapy.}, number={1}, journal={Molecular Cancer}, publisher={Springer Science and Business Media LLC}, author={Lam, Wilbur A and Cao, Lizhi and Umesh, Vaibhavi and Keung, Albert J and Sen, Shamik and Kumar, Sanjay}, year={2010}, month={Feb} }
 @article{keung_de juan-pardo_schaffer_kumar_2010, title={Mechanotransductive Engineering of Neural Stem Cell Behavior}, volume={98}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2009.12.2181}, DOI={10.1016/j.bpj.2009.12.2181}, abstractNote={Neural stem cells (NSCs) play important roles in learning and memory in the adult mammalian brain and may also serve as a source of cells in cell replacement therapies to treat neurodegenerative diseases. Therefore, investigating how NSC behavior is regulated is crucial to understanding the fundamental biology of the brain as well as in engineering biomedical therapies. Towards these ends, an increasing wealth of knowledge in the NSC field describes a complex picture of biochemical and genetic regulation of NSC self-renewal and differentiation. However, little is known about the biophysical control of NSC behavior by the extracellular matrix (ECM). Here we demonstrate that ECM-derived mechanical signals can act with Rho GTPases to regulate NSC stiffness and differentiation. Culturing NSCs on increasingly stiff ECMs suppresses neurogenesis and enhances gliogenesis, even in the absence of exogenous differentiating agents. This shift is accompanied by enhanced RhoA and Cdc42 activation and increased cellular stiffness. Direct manipulation of RhoA and Cdc42 activity disrupts the ability of NSCs to sense ECM stiffness and tips the balance between neurogenesis and gliogenesis in the presence and absence of exogenous differentiation cues. Inhibitors of a downstream effector of RhoA, Rho kinase, as well as inhibition of myosin II contractility rescues neuronal differentiation of NSCs cultured on stiff substrates as well as for NSCs expressing CA RhoA and CA Cdc42, suggesting that NSC stiffness/contractility regulates NSC differentiation. These results establish Rho GTPase-based mechanotransduction and cellular stiffness as novel regulators of NSC behavior.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Keung, Albert J. and de Juan-Pardo, Elena M. and Schaffer, David V. and Kumar, Sanjay}, year={2010}, month={Jan}, pages={404a–405a} }
 @article{keung_kumar_schaffer_2010, title={Presentation Counts: Microenvironmental Regulation of Stem Cells by Biophysical and Material Cues}, volume={26}, ISSN={1081-0706 1530-8995}, url={http://dx.doi.org/10.1146/annurev-cellbio-100109-104042}, DOI={10.1146/annurev-cellbio-100109-104042}, abstractNote={Stem cells reside in adult and embryonic tissues in a broad spectrum of developmental stages and lineages, and they are thus naturally exposed to diverse microenvironments or niches that modulate their hallmark behaviors of self-renewal and differentiation into one or more mature lineages. Within each such microenvironment, stem cells sense and process multiple biochemical and biophysical cues, which can exert redundant, competing, or orthogonal influences to collectively regulate cell fate and function. The proper presentation of these myriad regulatory signals is required for tissue development and homeostasis, and their improper appearance can potentially lead to disease. Whereas these complex regulatory cues can be challenging to dissect using traditional cell culture paradigms, recently developed engineered material systems offer advantages for investigating biochemical and biophysical cues, both static and dynamic, in a controlled, modular, and quantitative fashion. Advances in the development and use of such systems have helped elucidate novel regulatory mechanisms controlling stem cell behavior, particularly the importance of solid-phase mechanical and immobilized biochemical microenvironmental signals, with implications for basic stem cell biology, disease, and therapeutics.}, number={1}, journal={Annual Review of Cell and Developmental Biology}, publisher={Annual Reviews}, author={Keung, Albert J. and Kumar, Sanjay and Schaffer, David V.}, year={2010}, month={Nov}, pages={533–556} }
 @article{keung_healy_kumar_schaffer_2010, title={Biophysics and dynamics of natural and engineered stem cell microenvironments}, volume={2}, ISSN={1939-5094 1939-005X}, url={http://dx.doi.org/10.1002/wsbm.46}, DOI={10.1002/wsbm.46}, abstractNote={AbstractStem cells are defined by their ability to self‐renew and to differentiate into one or more mature lineages, and they reside within natural niches in many types of adult and embryonic tissues that present them with complex signals to regulate these two hallmark properties. The diverse nature of these in vivo microenvironments raises important questions about the microenvironmental cues regulating stem cell plasticity, and the stem cell field has built a strong foundation of knowledge on the biochemical identities and regulatory effects of the soluble, cellular, and extracellular matrix factors surrounding stem cells through the isolation and culture of stem cells in vitro within microenvironments that, in effect, emulate the properties of the natural niche. Recent work, however, has expanded the field's perspective to include biophysical and dynamic characteristics of the microenvironment. These include biomechanical characteristics such as elastic modulus, shear force, and cyclic strain; architectural properties such as geometry, topography, and dimensionality; and dynamic structures and ligand profiles. We will review how these microenvironmental characteristics have been shown to regulate stem cell fate and discuss future research directions that may help expand our current understanding of stem cell biology and aid its application to regenerative medicine. Copyright © 2009 John Wiley & Sons, Inc.This article is categorized under:

Developmental Biology > Stem Cell Biology and Regeneration
}, number={1}, journal={Wiley Interdisciplinary Reviews: Systems Biology and Medicine}, publisher={Wiley}, author={Keung, Albert J. and Healy, Kevin E. and Kumar, Sanjay and Schaffer, David V.}, year={2010}, month={Jan}, pages={49–64} }
 @article{saha_keung_irwin_li_little_schaffer_healy_2008, title={Substrate Modulus Directs Neural Stem Cell Behavior}, volume={95}, ISSN={0006-3495}, url={http://dx.doi.org/10.1529/biophysj.108.132217}, DOI={10.1529/biophysj.108.132217}, abstractNote={Although biochemical signals that modulate stem cell self-renewal and differentiation were extensively studied, only recently were the mechanical properties of a stem cell's microenvironment shown to regulate its behavior. It would be desirable to have independent control over biochemical and mechanical cues, to analyze their relative and combined effects on stem-cell function. We developed a synthetic, interfacial hydrogel culture system, termed variable moduli interpenetrating polymer networks (vmIPNs), to assess the effects of soluble signals, adhesion ligand presentation, and material moduli from 10–10,000 Pa on adult neural stem-cell (aNSC) behavior. The aNSCs proliferated when cultured in serum-free growth media on peptide-modified vmIPNs with moduli of ≥100 Pa. In serum-free neuronal differentiation media, a peak level of the neuronal marker, β-tubulin III, was observed on vmIPNs of 500 Pa, near the physiological stiffness of brain tissue. Furthermore, under mixed differentiation conditions with serum, softer gels (∼100–500 Pa) greatly favored neurons, whereas harder gels (∼1,000–10,000 Pa) promoted glial cultures. In contrast, cell spreading, self-renewal, and differentiation were inhibited on substrata with moduli of ∼10 Pa. This work demonstrates that the mechanical and biochemical properties of an aNSC microenvironment can be tuned to regulate the self-renewal and differentiation of aNSCs.}, number={9}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Saha, Krishanu and Keung, Albert J. and Irwin, Elizabeth F. and Li, Yang and Little, Lauren and Schaffer, David V. and Healy, Kevin E.}, year={2008}, month={Nov}, pages={4426–4438} }
 @article{filler_keung_porter_bent_2006, title={Formation of Surface-Bound Acyl Groups by Reaction of Acyl Halides on Ge(100)−2×1}, volume={110}, ISSN={1520-6106 1520-5207}, url={http://dx.doi.org/10.1021/jp055685}, DOI={10.1021/jp055685+}, abstractNote={We have investigated the reaction of a series of acyl halides, including acetyl chloride, acetyl bromide, acetyl-d3 chloride, benzoyl chloride, and pivaloyl chloride, on Ge(100)-2x1 with multiple internal reflection infrared (MIR-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and density functional theory (DFT). Infrared spectra following saturation exposures of acetyl chloride and acetyl bromide to Ge(100)-2x1 at 310 K are nearly identical, both exhibiting strong nu(C=O) stretching peaks near 1685 cm-1 and no vibrational modes in the nu(Ge-H) region. These data provide strong evidence for the presence of a surface-bound acetyl group on Ge(100)-2x1, which results from a C-X dissociation reaction (where X=Cl, Br). For acetyl chloride, DFT calculations predict that the barrier to C-Cl dissociation is only 1 kcal/mol above a chlorine-bound precursor state and is considerably smaller than barriers leading to the [2+2] C=O cycloaddition and alpha-CH dissociation products. In addition to the C-X dissociation product, both infrared and photoelectron results point to the presence of a second structure for acetyl halides where the oxygen of the surface-bound acetyl group donates charge to a nearby surface atom. This interaction is not observed for benzoyl chloride and pivaloyl chloride.}, number={9}, journal={The Journal of Physical Chemistry B}, publisher={American Chemical Society (ACS)}, author={Filler, Michael A. and Keung, Albert J. and Porter, David W. and Bent, Stacey F.}, year={2006}, month={Feb}, pages={4115–4124} }
 @article{keung_filler_bent_2007, title={Thermal Control of Amide Product Distributions at the Ge(100)-2×1 Surface}, volume={111}, ISSN={1932-7447 1932-7455}, url={http://dx.doi.org/10.1021/jp065278d}, DOI={10.1021/jp065278d}, abstractNote={We have investigated the adsorption of a series of primary, secondary, and tertiary amides including formamide, N-methylformamide, N-methylacetamide, and N,N-dimethylacetamide on the Ge(100)-2×1 surface using multiple internal reflection Fourier transform infrared spectroscopy and density functional theory. At 310 K, primary and secondary amides were observed to form thermodynamically favored N−H dissociation structures with interdimer interactions as well as kinetically favored oxygen dative-bonded structures. The relative surface product distributions could be controlled thermally. Dative-bonded adducts were isolated by exposing the amides to the surface at 240 K, whereas N−H dissociation products were formed by annealing to 450 K. While the acetamides could potentially form “-ene” type products, such products were not observed and instead the acetamides exhibited the same reactivity pattern as the formamides in this study.}, number={1}, journal={The Journal of Physical Chemistry C}, publisher={American Chemical Society (ACS)}, author={Keung, Albert J. and Filler, Michael A. and Bent, Stacey F.}, year={2007}, month={Jan}, pages={411–419} }
 @article{filler_van deventer_keung_bent_2006, title={Carboxylic Acid Chemistry at the Ge(100)-2 × 1 Interface:  Bidentate Bridging Structure Formation on a Semiconductor Surface}, volume={128}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja0549502}, DOI={10.1021/ja0549502}, abstractNote={The reactions of acetic acid, acetic-d3 acid-d, and formic acid with the Ge(100)-2 x 1 surface have been investigated using multiple internal reflection Fourier transform infrared (MIR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and density functional theory (DFT) calculations. The infrared and photoelectron data provide experimental evidence for an O-H dissociation product at 310 K. DFT calculations indicate that the O-H dissociation pathway is significantly favored, both kinetically and thermodynamically, over other potential reaction pathways. All of the carboxylic acids studied exhibit unexpected vibrational modes between 1400 and 1525 cm(-1), which are attributed to the presence of a bidentate bridging structure where both oxygen atoms interact directly with the surface.}, number={3}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Filler, Michael A. and Van Deventer, James A. and Keung, Albert J. and Bent, Stacey F.}, year={2006}, month={Jan}, pages={770–779} }
 @article{keung_filler_porter_bent_2005, title={Tertiary amide chemistry at the Ge(100)-2×1 surface}, volume={599}, ISSN={0039-6028}, url={http://dx.doi.org/10.1016/j.susc.2005.09.035}, DOI={10.1016/j.susc.2005.09.035}, abstractNote={We have investigated the adsorption of several tertiary amides, including N,N-dimethylformamide, N,N-dimethylformamide-d7, 1-methyl-2-pyrrolidinone, and N-methylcaprolactam, on Ge(1 0 0)-2 × 1 using multiple internal reflection Fourier transform infrared spectroscopy and density functional theory. At 310 K, all four tertiary amides were observed to selectively form a dative bond to the germanium surface through the oxygen atom. While previous work has shown that oxygen dative bonds are unstable near room temperature, tertiary amides exhibit delocalization of electron density from nitrogen to oxygen, which appears to increase the stability of the oxygen dative-bonded state. Partial desorption of these surface adducts on the timescale of minutes indicates weakly bound surface adducts with coverage dependent binding energies.}, number={1-3}, journal={Surface Science}, publisher={Elsevier BV}, author={Keung, Albert J. and Filler, Michael A. and Porter, David W. and Bent, Stacey F.}, year={2005}, month={Dec}, pages={41–54} }