@article{pandit_smith_birnbaum_brudno_2024, title={A biomaterial platform for T cell-specific gene delivery}, volume={177}, ISSN={["1878-7568"]}, DOI={10.1016/j.actbio.2024.02.013}, abstractNote={Efficient T cell engineering is central to the success of CAR T cell therapy but involves multiple time-consuming manipulations, including T cell isolation, activation, and transduction. These steps add complexity and delay CAR T cell manufacturing, which takes a mean time of 4 weeks. To streamline T cell engineering, we strategically combine two critical engineering solutions - T cell-specific lentiviral vectors and macroporous scaffolds - that enable T cell activation and transduction in a simple, single step. The T cell-specific lentiviral vectors (referred to as STAT virus) target T cells through the display of an anti-CD3 antibody and the CD80 extracellular domain on their surface and provide robust T cell activation. Biocompatible macroporous scaffolds (referred to as Drydux) mediate robust transduction by providing effective interaction between naïve T cells and viral vectors. We show that when unstimulated peripheral blood mononuclear cells (PBMCs) are seeded together with STAT lentivirus on Drydux scaffolds, T cells are activated, selectively transduced, and reprogrammed in a single step. Further, we show that the Drydux platform seeded with PBMCs and STAT lentivirus generates tumor-specific functional CAR T cells. This potent combination of engineered lentivirus and biomaterial scaffold holds promise for an effective, simple, and safe avenue for in vitro and in vivo T cell engineering. Manufacturing T cell therapies involves lengthy and labor-intensive steps, including T cell selection, activation, and transduction. These steps add complexity to current CAR T cell manufacturing protocols and limit widespread patient access to this revolutionary therapy. In this work, we demonstrate the combination of engineered virus and biomaterial platform that, together, enables selective T cell activation and transduction in a single step, eliminating multistep T cell engineering protocols and significantly simplifying the manufacturing process.}, journal={ACTA BIOMATERIALIA}, author={Pandit, Sharda and Smith, Blake E. and Birnbaum, Michael E. and Brudno, Yevgeny}, year={2024}, month={Mar}, pages={157–164} }
 @article{pandit_agarwalla_song_jansson_dotti_brudno_2024, title={Implantable CAR T cell factories enhance solid tumor treatment}, volume={308}, ISSN={["1878-5905"]}, DOI={10.1016/j.biomaterials.2024.122580}, abstractNote={Chimeric Antigen Receptor (CAR) T cell therapy has produced revolutionary success in hematological cancers such as leukemia and lymphoma. Nonetheless, its translation to solid tumors faces challenges due to manufacturing complexities, short-lived in vivo persistence, and transient therapeutic impact. We introduce 'Drydux' - an innovative macroporous biomaterial scaffold designed for rapid, efficient in-situ generation of tumor-specific CAR T cells. Drydux expedites CAR T cell preparation with a mere three-day turnaround from patient blood collection, presenting a cost-effective, streamlined alternative to conventional methodologies. Notably, Drydux-enabled CAR T cells provide prolonged in vivo release, functionality, and enhanced persistence exceeding 150 days, with cells transitioning to memory phenotypes. Unlike conventional CAR T cell therapy, which offered only temporary tumor control, equivalent Drydux cell doses induced lasting tumor remission in various animal tumor models, including systemic lymphoma, peritoneal ovarian cancer, metastatic lung cancer, and orthotopic pancreatic cancer. Drydux's approach holds promise in revolutionizing solid tumor CAR T cell therapy by delivering durable, rapid, and cost-effective treatments and broadening patient accessibility to this groundbreaking therapy.}, journal={BIOMATERIALS}, author={Pandit, Sharda and Agarwalla, Pritha and Song, Feifei and Jansson, Anton and Dotti, Gianpietro and Brudno, Yevgeny}, year={2024}, month={Jul} }
 @article{vanblunk_srikanth_pandit_kuznetsov_brudno_2023, title={Absorption rate governs cell transduction in dry macroporous scaffolds}, volume={1}, ISSN={["2047-4849"]}, DOI={10.1039/d2bm01753a}, abstractNote={Dry, macroporous scaffolds efficiently transduce T cells, but the mechanism for this transduction has not been studied previously. We report that liquid volume and resultant differences in liquid absorption rates governs cell transduction efficiency.}, journal={BIOMATERIALS SCIENCE}, author={VanBlunk, Madelyn and Srikanth, Vishal and Pandit, Sharda S. and Kuznetsov, Andrey V. and Brudno, Yevgeny}, year={2023}, month={Jan} }
 @article{moody_durham_dayton_brudno_2023, title={Loading Intracranial Drug-Eluting Reservoirs Across the Blood-Brain Barrier With Focused Ultrasound}, volume={49}, ISSN={["1879-291X"]}, DOI={10.1016/j.ultrasmedbio.2023.03.012}, abstractNote={Objective Efficient, sustained and long-term delivery of therapeutics to the brain remains an important challenge to treatment of diseases such as brain cancer, stroke and neurodegenerative disease. Focused ultrasound can assist movement of drugs into the brain, but frequent and long-term use has remained impractical. Single-use intracranial drug-eluting depots show promise but are limited for the treatment of chronic diseases as they cannot be refilled non-invasively. Refillable drug-eluting depots could serve as a long-term solution, but refilling is hindered by the blood–brain barrier (BBB), which prevents drug refills from accessing the brain. In this article, we describe how focused ultrasound enables non-invasive loading of intracranial drug depots in mice. Methods Female CD-1 mice (n = 6) were intracranially injected with click-reactive and fluorescent molecules that are capable of anchoring in the brain. After healing, animals were treated with high-intensity focused ultrasound and microbubbles to temporarily increase the permeability of the blood–brain barrier and deliver dibenzocyclooctyne (DBCO)–Cy7. The mice were perfused, and the brains were imaged via ex vivo fluorescence imaging. Results Fluorescence imaging indicated small molecule refills are captured by intracranial depots as long as 4 wk after administration and are retained for up to 4 wk based on fluorescence imaging. Efficient loading was dependent on both focused ultrasound and the presence of refillable depots in the brain as absence of either prevented intracranial loading. Conclusion The ability to target and retain small molecules at predetermined intracranial sites with pinpoint accuracy provides opportunities to continuously deliver drugs to the brain over weeks and months without excessive BBB opening and with minimal off-target side effects. Efficient, sustained and long-term delivery of therapeutics to the brain remains an important challenge to treatment of diseases such as brain cancer, stroke and neurodegenerative disease. Focused ultrasound can assist movement of drugs into the brain, but frequent and long-term use has remained impractical. Single-use intracranial drug-eluting depots show promise but are limited for the treatment of chronic diseases as they cannot be refilled non-invasively. Refillable drug-eluting depots could serve as a long-term solution, but refilling is hindered by the blood–brain barrier (BBB), which prevents drug refills from accessing the brain. In this article, we describe how focused ultrasound enables non-invasive loading of intracranial drug depots in mice. Female CD-1 mice (n = 6) were intracranially injected with click-reactive and fluorescent molecules that are capable of anchoring in the brain. After healing, animals were treated with high-intensity focused ultrasound and microbubbles to temporarily increase the permeability of the blood–brain barrier and deliver dibenzocyclooctyne (DBCO)–Cy7. The mice were perfused, and the brains were imaged via ex vivo fluorescence imaging. Fluorescence imaging indicated small molecule refills are captured by intracranial depots as long as 4 wk after administration and are retained for up to 4 wk based on fluorescence imaging. Efficient loading was dependent on both focused ultrasound and the presence of refillable depots in the brain as absence of either prevented intracranial loading. The ability to target and retain small molecules at predetermined intracranial sites with pinpoint accuracy provides opportunities to continuously deliver drugs to the brain over weeks and months without excessive BBB opening and with minimal off-target side effects.}, number={7}, journal={ULTRASOUND IN MEDICINE AND BIOLOGY}, author={Moody, Christopher T. and Durham, Phillip G. and Dayton, Paul A. and Brudno, Yevgeny}, year={2023}, month={Jul}, pages={1679–1685} }
 @article{agarwalla_ogunnaike_ahn_froehlich_jansson_ligler_dotti_brudno_2022, title={Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells}, volume={3}, ISSN={["1546-1696"]}, DOI={10.1038/s41587-022-01245-x}, abstractNote={Despite their clinical success, chimeric antigen receptor (CAR)-T cell therapies for B cell malignancies are limited by lengthy, costly and labor-intensive ex vivo manufacturing procedures that might lead to cell products with heterogeneous composition. Here we describe an implantable Multifunctional Alginate Scaffold for T Cell Engineering and Release (MASTER) that streamlines in vivo CAR-T cell manufacturing and reduces processing time to a single day. When seeded with human peripheral blood mononuclear cells and CD19-encoding retroviral particles, MASTER provides the appropriate interface for viral vector-mediated gene transfer and, after subcutaneous implantation, mediates the release of functional CAR-T cells in mice. We further demonstrate that in vivo-generated CAR-T cells enter the bloodstream and control distal tumor growth in a mouse xenograft model of lymphoma, showing greater persistence than conventional CAR-T cells. MASTER promises to transform CAR-T cell therapy by fast-tracking manufacture and potentially reducing the complexity and resources needed for provision of this type of therapy. Implantable scaffolds rapidly generate and release anti-tumor CAR-T cells in mice.}, journal={NATURE BIOTECHNOLOGY}, author={Agarwalla, Pritha and Ogunnaike, Edikan A. and Ahn, Sarah and Froehlich, Kristen A. and Jansson, Anton and Ligler, Frances S. and Dotti, Gianpietro and Brudno, Yevgeny}, year={2022}, month={Mar} }
 @article{vanblunk_agarwalla_pandit_brudno_2022, title={Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells}, volume={9}, ISSN={["1940-087X"]}, DOI={10.3791/64036}, abstractNote={Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by viral gene transfer into T cells. The current gold standard of viral gene transfer involves spinoculation of retronectin-coated plates, which is expensive and time-consuming. There is a significant need for efficient and cost-effective methods to generate CAR-T cells. Described here is a method for fabricating inexpensive, dry macroporous alginate scaffolds, known as Drydux scaffolds, that efficiently promote viral transduction of activated T cells. The scaffolds are designed to be used in place of gold standard spinoculation of retronectin-coated plates seeded with virus and simplify the process for transducing cells. Alginate is cross-linked with calcium-D-gluconate and frozen overnight to create the scaffolds. The frozen scaffolds are freeze-dried in a lyophilizer for 72 h to complete the formation of the dry macroporous scaffolds. The scaffolds mediate viral gene transfer when virus and activated T cells are seeded together on top of the scaffold to produce genetically modified cells. The scaffolds produce >85% primary T cell transduction, which is comparable to the transduction efficiency of spinoculation on retronectin-coated plates. These results demonstrate that dry macroporous alginate scaffolds serve as a cheaper and more convenient alternative to the conventional transduction method.}, number={187}, journal={JOVE-JOURNAL OF VISUALIZED EXPERIMENTS}, author={VanBlunk, Madelyn and Agarwalla, Pritha and Pandit, Sharda and Brudno, Yevgeny}, year={2022}, month={Sep} }
 @article{pandit_palvai_massaro_pierce_brudno_2022, title={Tissue-reactive drugs enable materials-free local depots}, volume={343}, ISSN={["1873-4995"]}, DOI={10.1016/j.jconrel.2022.01.023}, abstractNote={Local, sustained drug delivery of potent therapeutics holds promise for the treatment of a myriad of localized diseases while eliminating systemic side effects. However, introduction of drug delivery depots such as viscous hydrogels or polymer-based implants is highly limited in stiff tissues such as desmoplastic tumors. Here, we present a method to create materials-free intratumoral drug depots through Tissue-Reactive Anchoring Pharmaceuticals (TRAPs). TRAPs diffuse into tissue and attach locally for sustained drug release. In TRAPs, potent drugs are modified with ECM-reactive groups and then locally injected to quickly react with accessible amines within the ECM, creating local drug depots. We demonstrate that locally injected TRAPs create dispersed, stable intratumoral depots deep within mouse and human pancreatic tumor tissues. TRAPs depots based on ECM-reactive paclitaxel (TRAP paclitaxel) had better solubility than free paclitaxel and enabled sustained in vitro and in vivo drug release. TRAP paclitaxel induced higher tumoral apoptosis and sustained better antitumor efficacy than the free drug. By providing continuous drug access to tumor cells, this material-free approach to sustained drug delivery of potent therapeutics has the potential in a wide variety of diseases where current injectable depots fall short.}, journal={JOURNAL OF CONTROLLED RELEASE}, author={Pandit, Sharda and Palvai, Sandeep and Massaro, Nicholas P. and Pierce, Joshua G. and Brudno, Yevgeny}, year={2022}, month={Mar}, pages={142–151} }
 @article{palvai_moody_pandit_brudno_2021, title={On-Demand Drug Release from Click-Refillable Drug Depots}, volume={18}, ISSN={["1543-8392"]}, DOI={10.1021/acs.molpharmaceut.1c00535}, abstractNote={Stimuli-responsive, on-demand release of drugs from drug-eluting depots could transform the treatment of many local diseases, providing intricate control over local dosing. However, conventional on-demand drug release approaches rely on locally implanted drug depots, which become spent over time and cannot be refilled or reused without invasive procedures. New strategies to noninvasively refill drug-eluting depots followed by on-demand release could transform clinical therapy. Here we report an on-demand drug delivery paradigm that combines bioorthogonal click chemistry to locally enrich protodrugs at a prelabeled site and light-triggered drug release at the target tissue. This approach begins with introduction of the targetable depot through local injection of chemically reactive azide groups that anchor to the extracellular matrix. The anchored azide groups then capture blood-circulating protodrugs through bioorthogonal click chemistry. After local capture and retention, active drugs can be released through external light irradiation. In this report, a photoresponsive protodrug was constructed consisting of the chemotherapeutic doxorubicin (Dox), conjugated to dibenzocyclooctyne (DBCO) through a photocleavable ortho-nitrobenzyl linker. The protodrug exhibited excellent on-demand light-triggered Dox release properties and light-mediated in vitro cytotoxicity in U87 glioblastoma cell lines. Furthermore, in a live animal setting, azide depots formed in mice through intradermal injection of activated azide-NHS esters. After i.v. administration, the protodrug was captured by the azide depots with intricate local specificity, which could be increased with multiple refills. Finally, doxorubicin could be released from the depot upon light irradiation. Multiple rounds of depot refilling and light-mediated release of active drug were accomplished, indicating that this system has the potential for multiple rounds of treatment. Taken together, these in vitro and in vivo proof of concept studies establish a novel method for in vivo targeting and on-demand delivery of cytotoxic drugs at target tissues.}, number={10}, journal={MOLECULAR PHARMACEUTICS}, author={Palvai, Sandeep and Moody, Christopher T. and Pandit, Sharda and Brudno, Yevgeny}, year={2021}, month={Oct}, pages={3920–3925} }
 @article{moody_brown_massaro_patel_agarwalla_simpson_brown_zheng_pierce_brudno_2022, title={Restoring Carboxylates on Highly Modified Alginates Improves Gelation, Tissue Retention and Systemic Capture}, volume={138}, ISSN={1742-7061}, url={http://dx.doi.org/10.1016/j.actbio.2021.10.046}, DOI={10.1016/j.actbio.2021.10.046}, abstractNote={Alginate hydrogels are gaining traction for use in drug delivery, regenerative medicine, and as tissue engineered scaffolds due to their physiological gelation conditions, high tissue biocompatibility, and wide chemical versatility. Traditionally, alginate is decorated at the carboxyl group to carry drug payloads, peptides, or proteins. While low degrees of substitution do not cause noticeable mechanical changes, high degrees of substitution can cause significant losses to alginate properties including complete loss of calcium cross-linking. While most modifications used to decorate alginate deplete the carboxyl groups, we propose that alginate modifications that replenish the carboxyl groups could overcome the loss in gel integrity and mechanics. In this report, we demonstrate that restoring carboxyl groups during functionalization maintains calcium cross-links as well as hydrogel shear-thinning and self-healing properties. In addition, we demonstrate that alginate hydrogels modified to a high degree with azide modifications that restore the carboxyl groups have improved tissue retention at intramuscular injection sites and capture blood-circulating cyclooctynes better than alginate hydrogels modified with azide modifications that deplete the carboxyl groups. Taken together, alginate modifications that restore carboxyl groups could significantly improve alginate hydrogel mechanics for clinical applications. Chemical modification of hydrogels provides a powerful tool to regulate cellular adhesion, immune response, and biocompatibility with local tissues. Alginate, due to its biocompatibility and easy chemical modification, is being explored for tissue engineering and drug delivery. Unfortunately, modifying alginate to a high degree of substitution consumes carboxyl group, which are necessary for ionic gelation, leading to poor hydrogel crosslinking. We introduce alginate modifications that restore the alginate's carboxyl groups. We demonstrate that modifications that reintroduce carboxyl groups restore gelation and improve gel mechanics and tissue retention. In addition to contributing to a basic science understanding of hydrogel properties, we anticipate our approach will be useful to create tissue engineered scaffolds and drug delivery platforms.}, journal={Acta Biomaterialia}, publisher={Elsevier BV}, author={Moody, CT and Brown, AE and Massaro, NP and Patel, AS and Agarwalla, PA and Simpson, AM and Brown, AC and Zheng, H and Pierce, JG and Brudno, Y}, year={2022}, month={Jan}, pages={208–217} }
 @article{moody_palvai_brudno_2020, title={Click cross-linking improves retention and targeting of refillable alginate depots}, volume={112}, url={https://doi.org/10.1016/j.actbio.2020.05.033}, DOI={10.1016/j.actbio.2020.05.033}, abstractNote={Injectable alginate hydrogels have demonstrated utility in tissue engineering and drug delivery applications due in part to their mild gelation conditions, low host responses and chemical versatility. Recently, the potential of these gels has expanded with the introduction of refillable hydrogel depots - alginate gels chemically decorated with click chemistry groups to efficiently capture prodrug refills from the blood. Unfortunately, high degrees of click group substitution on alginate lead to poor viscoelastic properties and loss of ionic cross-linking. In this work, we introduce tetrabicyclononyne (tBCN) agents that covalently cross-link azide-modified alginate hydrogels for tissue engineering and drug delivery application in vivo. Adjusting cross-linker concentration allowed tuning the hydrogel mechanical properties for tissue-specific mechanical strength. The bioorthogonal and specific click reaction creates stable hydrogels with improved in vivo properties, including improved retention at injected sites. Azide-alginate hydrogels cross-linked with tBCN elicited minimal inflammation and maintained structural integrity over several months and efficiently captured therapeutics drug surrogates from the circulation. Taken together, azide-alginate hydrogels cross-linked with tBCN convey the benefits of alginate hydrogels for use in tissue engineering and drug delivery applications of refillable drug delivery depots. Ionically cross-linked, injectable alginate biomaterials hold promise in many different clinical settings. However, adding new chemical functionality to alginate can disrupt their ionic cross-linking, limiting their utility. We have developed a “click” cross-linking strategy to improve the mechanical properties and tissue function of modified alginate biomaterials and enable them to capture small molecule drugs from the blood. We show that click cross-linked materials remain in place better than ionically cross-linked materials and efficiently capture payloads from the blood. Development of click cross-linking for refillable depots represents a crucial step toward clinical application of this promising drug delivery platform.}, journal={Acta Biomaterialia}, publisher={Elsevier BV}, author={Moody, Christopher T. and Palvai, Sandeep and Brudno, Yevgeny}, year={2020}, month={Aug}, pages={112–121} }
 @article{palvai_bhangu_akgun_moody_hall_brudno_2020, title={In Vivo Targeting Using Arylboronate/Nopoldiol Click Conjugation}, volume={31}, ISSN={["1520-4812"]}, url={https://doi.org/10.1021/acs.bioconjchem.0c00453}, DOI={10.1021/acs.bioconjchem.0c00453}, abstractNote={Bioorthogonal click reactions yielding stable and irreversible adducts are in high demand for in vivo applications, including in biomolecular labelling, diagnostic imaging and drug delivery. Previously, we reported a novel bioorthogonal "click" reaction based on the coupling of ortho-acetyl arylboronates and thiosemicarbazide-functionalized nopoldiol. We now report that a detailed structural analysis of the arylboronate/nopoldiol adduct by X-ray crystallography and 11B NMR reveals that the bioorthogonal reactants form, unexpectedly, a tetracyclic adduct through the cyclization of the distal nitrogen into the semithiocarbazone leading to a strong B-N dative bond and two new 5-membered rings. The cyclization adduct, which protects the boronate unit against hydrolytic breakdown, sheds light on the irreversible nature of this polycondensation. The potential of this reaction to work in a live animal setting was studied through in vivo capture of fluorescently labelled molecules in vivo. Arylboronates were introduced into tissues through intradermal injection of their activated NHS esters, which react with amines in the extracellular matrix. Fluorescently labelled nopoldiol molecules were administered systemically and were efficiently captured by the arylboronic acids in a location-specific manner. Taken together, these in vivo proof of concept studies establish arylboronate/nopoldiol bioorthogonal chemistry as a candidate for wide array of applications in chemical biology and drug delivery.}, number={10}, journal={BIOCONJUGATE CHEMISTRY}, publisher={American Chemical Society (ACS)}, author={Palvai, Sandeep and Bhangu, Jasmine and Akgun, Burcin and Moody, Christopher T. and Hall, Dennis G. and Brudno, Yevgeny}, year={2020}, month={Oct}, pages={2288–2292} }
 @article{mcnamara_brudno_miller_ham_aizenberg_chaikof_mooney_2020, title={Regenerating Antithrombotic Surfaces through Nucleic Acid Displacement}, volume={6}, ISSN={["2373-9878"]}, DOI={10.1021/acsbiomaterials.0c00038}, abstractNote={Blood-contacting devices are commonly coated with antithrombotic agents to prevent clot formation and to extend the lifespan of the device. However, in vivo degradation of these bioactive surface agents ultimately limits device efficacy and longevity. Here, a regenerative antithrombotic catheter surface treatment is developed using oligodeoxynucleotide (ODN) toehold exchange. ODN strands modified to carry antithrombotic payloads can inhibit the thrombin enzyme when bound to a surface and exchange with rapid kinetics over multiple cycles, even while carrying large payloads. The surface-bound ODNs inhibit thrombin activity to significantly reduce fibrinogen cleavage and fibrin formation, and this effect is sustained after ODN exchange of the surface-bound strands with a fresh antithrombotic payload. This study presents a unique strategy for achieving a continuous antithrombotic state for blood-contacting devices using an ODN-based regeneration method.}, number={4}, journal={ACS BIOMATERIALS SCIENCE & ENGINEERING}, author={McNamara, Stephanie L. and Brudno, Yevgeny and Miller, Alex B. and Ham, Hyun Oki and Aizenberg, Michael and Chaikof, Elliot L. and Mooney, David J.}, year={2020}, month={Apr}, pages={2159–2166} }
 @article{agarwalla_ogunnaike_ahn_ligler_dotti_brudno_2020, title={Scaffold-Mediated Static Transduction of T Cells for CAR-T Cell Therapy}, volume={9}, ISSN={["2192-2659"]}, url={https://doi.org/10.1002/adhm.202000275}, DOI={10.1002/adhm.202000275}, abstractNote={AbstractChimeric antigen receptor T (CAR‐T) cell therapy has produced impressive clinical responses in patients with B‐cell malignancies. Critical to the success of CAR‐T cell therapies is the achievement of robust gene transfer into T cells mediated by viral vectors such as gamma‐retroviral vectors. However, current methodologies of retroviral gene transfer rely on spinoculation and the use of retronectin, which may limit the implementation of cost‐effective CAR‐T cell therapies. Herein, a low‐cost, tunable, macroporous, alginate scaffold that transduces T cells with retroviral vectors under static condition is described. CAR‐T cells produced by macroporous scaffold‐mediated viral transduction exhibit >60% CAR expression, retain effector phenotype, expand to clinically relevant cell numbers, and eradicate CD19+ lymphoma in vivo. Efficient transduction is dependent on scaffold macroporosity. Taken together, the data show that macroporous alginate scaffolds serve as an attractive alternative to current transduction protocols and have high potential for clinical translation to genetically modify T cells for adoptive cellular therapy.}, number={14}, journal={ADVANCED HEALTHCARE MATERIALS}, publisher={Wiley}, author={Agarwalla, Pritha and Ogunnaike, Edikan A. and Ahn, Sarah and Ligler, Frances S. and Dotti, Gianpietro and Brudno, Yevgeny}, year={2020}, month={Jul} }
 @article{su_huang_ma_liang_dinh_chen_shen_allen_qiao_li_et al._2019, title={Biomimetics: Platelet-Inspired Nanocells for Targeted Heart Repair After Ischemia/Reperfusion Injury (Adv. Funct. Mater. 4/2019)}, volume={29}, ISSN={1616-301X}, url={http://dx.doi.org/10.1002/ADFM.201970019}, DOI={10.1002/ADFM.201970019}, abstractNote={In article number 1803567, Ke Cheng and co-workers introduce platelet-inspired nanocells that target the heart after injury and that incorporates both a prostaglandin E2-modified platelet membrane and cardiac stromal cell-secreted factors. This approach represents a promising therapeutic delivery platform for treating myocardial ischemia/reperfusion injury.}, number={4}, journal={Advanced Functional Materials}, publisher={Wiley}, author={Su, Teng and Huang, Ke and Ma, Hong and Liang, Hongxia and Dinh, Phuong-Uyen and Chen, Justin and Shen, Deliang and Allen, Tyler A. and Qiao, Li and Li, Zhenhua and et al.}, year={2019}, month={Jan}, pages={1970019} }
 @article{wang_sobral_snyder_brudno_gorantla_mooney_2020, title={Clickable, acid labile immunosuppressive prodrugs for in vivo targeting}, volume={8}, url={https://doi.org/10.1039/C9BM01487J}, DOI={10.1039/C9BM01487J}, abstractNote={Clickable immunosuppressive prodrugs enablein vivoreplenishment of drugs in biomaterial depots to maintain long-term immunosuppression in tissue/organ transplantation.}, number={1}, journal={Biomaterials Science}, publisher={Royal Society of Chemistry (RSC)}, author={Wang, Hua and Sobral, Miguel C. and Snyder, Tracy and Brudno, Yevgeny and Gorantla, Vijay S. and Mooney, David J.}, year={2020}, pages={266–277} }
 @article{adams_moody_sollinger_brudno_2019, title={Extracellular-Matrix-Anchored Click Motifs for Specific Tissue Targeting}, volume={17}, ISSN={1543-8384 1543-8392}, url={http://dx.doi.org/10.1021/acs.molpharmaceut.9b00589}, DOI={10.1021/acs.molpharmaceut.9b00589}, abstractNote={Local presentation of cancer drugs by injectable drug eluting depots reduces systemic side effects and improves efficacy. However, local depots deplete their drug stores and are difficult to introduce into stiff tissues, or organs, such as the brain, that can not accommodate increased pressure. We present a method for introducing targetable depots through injection of activated ester molecules into target tissues that react with and anchor themselves to local extracellular matrix (ECM) and subsequently capture systemically-administered small molecules through bioorthogonal click chemistry. A computational model of tissue anchoring depot formation and distribution was verified by histological analysis and confocal imaging of cleared tissues. ECM-anchored click groups do not elicit any noticeable local or systemic toxicity or immune response and specifically capture systemically-circulating molecules at intradermal, intratumoral, and intracranial sites for multiple months. Taken together, ECM-anchoring of click chemistry motifs is a promising approach to specific targeting of both small and large therapeutics, enabling repeated local presentation for cancer therapy and other diseases.}, number={2}, journal={Molecular Pharmaceutics}, publisher={American Chemical Society (ACS)}, author={Adams, Mary R. and Moody, Christopher T. and Sollinger, Jennifer L. and Brudno, Yevgeny}, year={2019}, month={Dec}, pages={392–403} }
 @article{su_huang_ma_liang_dinh_chen_shen_allen_qiao_li_et al._2019, title={Platelet-Inspired Nanocells for Targeted Heart Repair After Ischemia/Reperfusion Injury}, volume={29}, ISSN={["1616-3028"]}, DOI={10.1002/adfm.201803567}, abstractNote={AbstractCardiovascular disease is the leading cause of mortality worldwide. While reperfusion therapy is vital for patient survival post‐heart attack, it also causes further tissue injury, known as myocardial ischemia/reperfusion (I/R) injury in clinical practice. Exploring ways to attenuate I/R injury is of clinical interest for improving post‐ischemic recovery. A platelet‐inspired nanocell (PINC) that incorporates both prostaglandin E2 (PGE2)‐modified platelet membrane and cardiac stromal cell‐secreted factors to target the heart after I/R injury is introduced. By taking advantage of the natural infarct‐homing ability of platelet membrane and the overexpression of PGE2 receptors (EPs) in the pathological cardiac microenvironment after I/R injury, the PINCs can achieve targeted delivery of therapeutic payload to the injured heart. Furthermore, a synergistic treatment efficacy can be achieved by PINC, which combines the paracrine mechanism of cell therapy with the PGE2/EP receptor signaling that is involved in the repair and regeneration of multiple tissues. In a mouse model of myocardial I/R injury, intravenous injection of PINCs results in augmented cardiac function and mitigated heart remodeling, which is accompanied by the increase in cycling cardiomyocytes, activation of endogenous stem/progenitor cells, and promotion of angiogenesis. This approach represents a promising therapeutic delivery platform for treating I/R injury.}, number={4}, journal={ADVANCED FUNCTIONAL MATERIALS}, author={Su, Teng and Huang, Ke and Ma, Hong and Liang, Hongxia and Dinh, Phuong-Uyen and Chen, Justin and Shen, Deliang and Allen, Tyler A. and Qiao, Li and Li, Zhenhua and et al.}, year={2019}, month={Jan} }
 @article{brudno_pezone_snyder_uzun_moody_aizenberg_mooney_2018, title={Replenishable drug depot to combat post-resection cancer recurrence}, volume={178}, ISSN={["1878-5905"]}, DOI={10.1016/j.biomaterials.2018.05.005}, abstractNote={Local drug presentation made possible by drug-eluting depots has demonstrated benefits in a vast array of diseases, including in cancer, microbial infection and in wound healing. However, locally-eluting depots are single-use systems that cannot be refilled or reused after implantation at inaccessible sites, limiting their clinical utility. New strategies to noninvasively refill drug-eluting depots could dramatically enhance their clinical use. In this report we present a refillable hydrogel depot system based on bioorthogonal click chemistry. The click-modified hydrogel depots capture prodrug refills from the blood and subsequently release active drugs locally in a sustained manner. Capture of the systemically-administered refills serves as an efficient and non-toxic method to repeatedly refill depots. Refillable depots in combination with prodrug refills achieve sustained release at precancerous tumor sites to improve cancer therapy while eliminating systemic side effects. The ability to target tissues without enhanced permeability could allow the use of refillable depots in cancer and many other medical applications.}, journal={BIOMATERIALS}, author={Brudno, Yevgeny and Pezone, Matthew J. and Snyder, Tracy K. and Uzun, Oktay and Moody, Christopher T. and Aizenberg, Michael and Mooney, David J.}, year={2018}, month={Sep}, pages={373–382} }
 @article{de leon peralta_brudno_kwee_mooney_2016, title={Improving wound healing through the use of tetrazine-modified alginate}, volume={30}, DOI={10.1096/fasebj.30.1_supplement.1177.10}, abstractNote={Ischemic diseases, such as myocardial infarction and peripheral arterial disease, are a leading cause of death globally. There has been increasing interest in treating these diseases with factors that induce angiogenesis, the growth of new blood vessels from pre‐existing blood vessels. We have recently shown that alginate linked to tetrazine helps induce a greater recovery in blood perfusion compared to unmodified alginate in a murine model of hindlimb ischemia. The objective of this project is to understand how this biomaterial induces recovery in blood perfusion. Our hypothesis is that tetrazine acts directly on endothelial cells to induce angiogenesis and also activates immune cells that are proangiogenic. To validate our hypothesis, we have performed in vitro angiogenesis assays to study if tetrazine‐modified alginate induces endothelial cell sprouting. Additionally, we have performed histology and immunohistochemistry on ischemic muscles from our in vivo experiments to see if this biomaterial increases blood vessel density, blood vessel remodeling, and the presence of proangiogenic immune cells. Our findings may lead to a novel method for treating people with ischemic diseases.Support or Funding InformationWyss Institute for Biologically Inspired Engineering, Harvard John A. Paulson School of Engineering and Applied Sciences Research Experiences for Undergraduates (SEAS REU)}, number={Supplement 1}, journal={The FASEB Journal}, author={De Leon Peralta, EJ and Brudno, Y and Kwee, Bj and Mooney, Dj}, year={2016}, month={Apr}, pages={1177.10–1177.10} }
 @article{brudno_desai_kwee_joshi_aizenberg_mooney_2015, title={In Vivo Targeting through Click Chemistry}, volume={10}, ISSN={1860-7179}, url={http://dx.doi.org/10.1002/CMDC.201402527}, DOI={10.1002/CMDC.201402527}, abstractNote={AbstractTargeting small molecules to diseased tissues as therapy or diagnosis is a significant challenge in drug delivery. Drug‐eluting devices implanted during invasive surgery allow the controlled presentation of drugs at the disease site, but cannot be modified once the surgery is complete. We demonstrate that bioorthogonal click chemistry can be used to target circulating small molecules to hydrogels resident intramuscularly in diseased tissues. We also demonstrate that small molecules can be repeatedly targeted to the diseased area over the course of at least one month. Finally, two bioorthogonal reactions were used to segregate two small molecules injected as a mixture to two separate locations in a mouse disease model. These results demonstrate that click chemistry can be used for pharmacological drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug‐eluting vascular grafts and stents.}, number={4}, journal={ChemMedChem}, publisher={Wiley}, author={Brudno, Yevgeny and Desai, Rajiv M. and Kwee, Brian J. and Joshi, Neel S. and Aizenberg, Michael and Mooney, David J.}, year={2015}, month={Feb}, pages={617–620} }
 @article{brudno_mooney_2015, title={On-demand drug delivery from local depots}, volume={219}, ISSN={0168-3659}, url={http://dx.doi.org/10.1016/J.JCONREL.2015.09.011}, DOI={10.1016/J.JCONREL.2015.09.011}, abstractNote={Stimuli-responsive polymeric depots capable of on-demand release of therapeutics promise a substantial improvement in the treatment of many local diseases. These systems have the advantage of controlling local dosing so that payload is released at a time and with a dose chosen by a physician or patient, and the dose can be varied as disease progresses or healing occurs. Macroscale drug depot can be induced to release therapeutics through the action of physical stimuli such as ultrasound, electric and magnetic fields and light as well as through the addition of pharmacological stimuli such as nucleic acids and small molecules. In this review, we highlight recent advances in the development of polymeric systems engineered for releasing therapeutic molecules through physical and pharmacological stimulation.}, journal={Journal of Controlled Release}, publisher={Elsevier BV}, author={Brudno, Yevgeny and Mooney, David J.}, year={2015}, month={Dec}, pages={8–17} }
 @article{roche_hastings_lewin_shvartsman_brudno_vasilyev_o'brien_walsh_duffy_mooney_et al._2014, title={Comparison of biomaterial delivery vehicles for improving acute retention of stem cells in the infarcted heart}, volume={35}, ISSN={0142-9612}, url={http://dx.doi.org/10.1016/J.BIOMATERIALS.2014.04.114}, DOI={10.1016/J.BIOMATERIALS.2014.04.114}, abstractNote={Cell delivery to the infarcted heart has emerged as a promising therapy, but is limited by very low acute retention and engraftment of cells. The objective of this study was to compare a panel of biomaterials to evaluate if acute retention can be improved with a biomaterial carrier. Cells were quantified post-implantation in a rat myocardial infarct model in five groups (n = 7-8); saline injection (current clinical standard), two injectable hydrogels (alginate, chitosan/β-glycerophosphate (chitosan/ß-GP)) and two epicardial patches (alginate, collagen). Human mesenchymal stem cells (hMSCs) were delivered to the infarct border zone with each biomaterial. At 24 h, retained cells were quantified by fluorescence. All biomaterials produced superior fluorescence to saline control, with approximately 8- and 14-fold increases with alginate and chitosan/β-GP injectables, and 47 and 59-fold increases achieved with collagen and alginate patches, respectively. Immunohistochemical analysis qualitatively confirmed these findings. All four biomaterials retained 50-60% of cells that were present immediately following transplantation, compared to 10% for the saline control. In conclusion, all four biomaterials were demonstrated to more efficiently deliver and retain cells when compared to a saline control. Biomaterial-based delivery approaches show promise for future development of efficient in vivo delivery techniques.}, number={25}, journal={Biomaterials}, publisher={Elsevier BV}, author={Roche, E.T. and Hastings, C.L. and Lewin, S.A. and Shvartsman, D.E. and Brudno, Yevgeny and Vasilyev, N.V. and O'Brien, F.J. and Walsh, C.J. and Duffy, G.P. and Mooney, D.J. and et al.}, year={2014}, month={Aug}, pages={6850–6858} }
 @misc{brudno_aizenberg_mooney_2014, title={Pro-angiogenic factors enhance pericyte function during angiogenesis}, url={http://dx.doi.org/10.1109/nebec.2014.6972741}, DOI={10.1109/nebec.2014.6972741}, abstractNote={Therapeutic stimulation of angiogenesis to re-establish blood flow in ischemic tissues offers great promise as a treatment for patients suffering from cardiovascular disease or trauma. Pericytes, perivascular cells on capillaries play important roles during angiogenesis, but their roles are poorly understood due to a lack of in vitro models to study their behavior. We present two in vitro models to study pericyte detachment and migration from and recruitment to the endothelium. Our results show that pro-angiogenic factors VEGF and Ang2 promote pericyte detachment, while pro-maturation factor PDGF increases pericyte speed and controls pericyte recruitment. Our results demonstrate the importance of temporal control over signaling in promoting vascular growth and vessel maturation and could be useful to promote healing after acute wounds and in chronic conditions such as diabetic ulcers and peripheral artery disease.}, journal={2014 40th Annual Northeast Bioengineering Conference (NEBEC)}, publisher={IEEE}, author={Brudno, Yevgeny and Aizenberg, Michael and Mooney, David}, year={2014}, month={Apr} }
 @article{brudno_silva_kearney_lewin_miller_martinick_aizenberg_mooney_2014, title={Refilling drug delivery depots through the blood}, volume={111}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/PNAS.1413027111}, DOI={10.1073/PNAS.1413027111}, abstractNote={Significance
          Drug delivery depots used in the clinic today are single use, with no ability to refill once exhausted of drug. Our system exploits nucleic acid complementarity to refill drug-delivering depots through the blood. The utility of this approach is demonstrated by its ability to inhibit tumor growth to a greater extent than strategies that rely on enhanced permeability and retention alone. We anticipate our approach will be directly applicable to therapies for many diseases, including cancer, wound healing, and inflammation, and for drug reloading of vascular grafts and stents.}, number={35}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Brudno, Y. and Silva, E. A. and Kearney, C. J. and Lewin, S. A. and Miller, A. and Martinick, K. D. and Aizenberg, M. and Mooney, D. J.}, year={2014}, month={Aug}, pages={12722–12727} }
 @article{shvartsman_storrie-white_lee_kearney_brudno_ho_cezar_mccann_anderson_koullias_et al._2014, title={Sustained Delivery of VEGF Maintains Innervation and Promotes Reperfusion in Ischemic Skeletal Muscles Via NGF/GDNF Signaling}, volume={22}, ISSN={1525-0016}, url={http://dx.doi.org/10.1038/MT.2014.76}, DOI={10.1038/MT.2014.76}, abstractNote={Tissue reinnervation following trauma, disease, or transplantation often presents a significant challenge. Here, we show that the delivery of vascular endothelial growth factor (VEGF) from alginate hydrogels ameliorates loss of skeletal muscle innervation after ischemic injury by promoting both maintenance and regrowth of damaged axons in mice. Nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) mediated VEGF-induced axonal regeneration, and the expression of both is induced by VEGF presentation. Using both <i>in vitro</i> and <i>in vivo</i> modeling approaches, we demonstrate that the activity of NGF and GDNF regulates VEGF-driven angiogenesis, controlling endothelial cell sprouting and blood vessel maturation. Altogether, these studies produce evidence of new mechanisms of VEGF action, further broaden the understanding of the roles of NGF and GDNF in angiogenesis and axonal regeneration, and suggest approaches to improve axonal and ischemic tissue repair therapies.}, number={7}, journal={Molecular Therapy}, publisher={Elsevier BV}, author={Shvartsman, Dmitry and Storrie-White, Hannah and Lee, Kangwon and Kearney, Cathal and Brudno, Yevgeny and Ho, Nhi and Cezar, Christine and McCann, Corey and Anderson, Erin and Koullias, John and et al.}, year={2014}, month={Jul}, pages={1243–1253} }
 @article{maione_brudno_stojadinovic_park_smith_tellechea_leal_kearney_veves_tomic-canic_et al._2015, title={Three-Dimensional Human Tissue Models That Incorporate Diabetic Foot Ulcer-Derived Fibroblasts Mimic <i>In Vivo</i> Features of Chronic Wounds}, volume={21}, ISSN={1937-3384 1937-3392}, url={http://dx.doi.org/10.1089/ten.tec.2014.0414}, DOI={10.1089/ten.tec.2014.0414}, abstractNote={Diabetic foot ulcers (DFU) are a major, debilitating complication of diabetes mellitus. Unfortunately, many DFUs are refractory to existing treatments and frequently lead to amputation. The development of more effective therapies has been hampered by the lack of predictive in vitro methods to investigate the mechanisms underlying impaired healing. To address this need for realistic wound-healing models, we established patient-derived fibroblasts from DFUs and site-matched controls and used them to construct three-dimensional (3D) models of chronic wound healing. Incorporation of DFU-derived fibroblasts into these models accurately recapitulated the following key aspects of chronic ulcers: reduced stimulation of angiogenesis, increased keratinocyte proliferation, decreased re-epithelialization, and impaired extracellular matrix deposition. In addition to reflecting clinical attributes of DFUs, the wound-healing potential of DFU fibroblasts demonstrated in this suite of models correlated with in vivo wound closure in mice. Thus, the reported panel of 3D DFU models provides a more biologically relevant platform for elucidating the cell-cell and cell-matrix-related mechanisms responsible for chronic wound pathogenesis and may improve translation of in vitro findings into efficacious clinical applications.}, number={5}, journal={Tissue Engineering Part C: Methods}, publisher={Mary Ann Liebert Inc}, author={Maione, Anna G. and Brudno, Yevgeny and Stojadinovic, Olivera and Park, Lara K. and Smith, Avi and Tellechea, Ana and Leal, Ermelindo C. and Kearney, Cathal J. and Veves, Aristidis and Tomic-Canic, Marjana and et al.}, year={2015}, month={May}, pages={499–508} }
 @article{brudno_ennett-shepard_chen_aizenberg_mooney_biomaterials_2013, title={Enhancing microvascular formation and vessel maturation through temporal control over multiple pro-angiogenic and pro-maturation factors}, volume={34}, ISSN={0142-9612}, url={http://dx.doi.org/10.1016/J.BIOMATERIALS.2013.08.007}, DOI={10.1016/J.BIOMATERIALS.2013.08.007}, abstractNote={Therapeutic stimulation of angiogenesis to re-establish blood flow in ischemic tissues offers great promise as a treatment for patients suffering from cardiovascular disease or trauma. Since angiogenesis is a complex, multi-step process, different signals may need to be delivered at appropriate times in order to promote a robust and mature vasculature. The effects of temporally regulated presentation of pro-angiogenic and pro-maturation factors were investigated in vitro and in vivo in this study. Pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) cooperatively promoted endothelial sprouting and pericyte detachment in a three-dimensional in vitro EC-pericyte co-culture model. Pro-maturation factors platelet-derived growth factor B (PDGF) and angiopoietin 1 (Ang1) inhibited the early stages of VEGF- and Ang2-mediated angiogenesis if present simultaneously with VEGF and Ang2, but promoted these behaviors if added subsequently to the pro-angiogenesis factors. VEGF and Ang2 were also found to additively enhance microvessel density in a subcutaneous model of blood vessel formation, while simultaneously administered PDGF/Ang1 inhibited microvessel formation. However, a temporally controlled scaffold that released PDGF and Ang1 at a delay relative to VEGF/Ang2 promoted both vessel maturation and vascular remodeling without inhibiting sprouting angiogenesis. Our results demonstrate the importance of temporal control over signaling in promoting vascular growth, vessel maturation and vascular remodeling. Delivering multiple growth factors in combination and sequence could aid in creating tissue engineered constructs and therapies aimed at promoting healing after acute wounds and in chronic conditions such as diabetic ulcers and peripheral artery disease.}, number={36}, journal={Biomaterials}, publisher={Elsevier BV}, author={Brudno, Yevgeny and Ennett-Shepard, A.B. and Chen, R.R. and Aizenberg, M. and Mooney, D.J. and Biomaterials}, year={2013}, month={Dec}, pages={9201–9209} }
 @article{shamis_silva_hewitt_brudno_levenberg_mooney_garlick_2013, title={Fibroblasts Derived from Human Pluripotent Stem Cells Activate Angiogenic Responses In Vitro and In Vivo}, volume={8}, url={http://europepmc.org/abstract/med/24386271}, DOI={10.1371/journal.pone.0083755}, abstractNote={Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.}, number={12}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Shamis, Y and Silva, EA and Hewitt, KJ and Brudno, Y and Levenberg, S and Mooney, DJ and Garlick, JA}, editor={Egles, ChristopheEditorEditor}, year={2013}, pages={e83755} }
 @article{pastor_pape_huang_henderson_lister_ko_mcloughlin_brudno_mahapatra_kapranov_et al._2011, title={Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells}, volume={473}, ISSN={0028-0836 1476-4687}, url={http://dx.doi.org/10.1038/nature10102}, DOI={10.1038/nature10102}, abstractNote={The modified DNA base 5-hydroxymethylcytosine (5hmC), sometimes called the sixth base, is present in the mammalian genome where it is generated by oxidation of 5-methylcytosine (5mC; the fifth base) by enzymes of the Tet family. Four papers in this issue, from the Helin, Zhang, Rao and Reik laboratories, respectively, report on the genome-wide distribution of Tet1 and/or 5hmC in mouse embryonic stem cells using the ChIP-seq technique. Links between Tet1 and transcription regulation — both activation and repression — are revealed. Anjana Rao and colleagues also describe two alternative methods with increased sensitivity for mapping single 5hmC bases. In the associated News & Views, Nathalie Véron and Antoine H. F. M. Peters discuss what these and other recent papers reveal about the role of Tet proteins in regulating DNA methylation and gene expression. 5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals1,2,3,4,5. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC)1,2,4,5,6,7. 5hmC and TET proteins have been implicated in stem cell biology and cancer1,4,5,8,9, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS5. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the ‘poised’ chromatin signature found at developmentally-regulated genes in ES cells.}, number={7347}, journal={Nature}, publisher={Springer Science and Business Media LLC}, author={Pastor, William A. and Pape, Utz J. and Huang, Yun and Henderson, Hope R. and Lister, Ryan and Ko, Myunggon and McLoughlin, Erin M. and Brudno, Yevgeny and Mahapatra, Sahasransu and Kapranov, Philipp and et al.}, year={2011}, month={May}, pages={394–397} }
 @article{brudno_birnbaum_kleiner_liu_2010, title={An in vitro translation, selection and amplification system for peptide nucleic acids}, volume={6}, ISSN={1552-4450 1552-4469}, url={http://dx.doi.org/10.1038/nchembio.280}, DOI={10.1038/nchembio.280}, abstractNote={In vitro evolution of synthetic polymers has been limited by a lack of methods for translating genetic information into synthetic libraries and for amplifying selected molecules. An in vitro selection system for peptide nucleic acids (PNA) now brings the evolution of functional PNA polymers within reach. Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template, (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures, (iii) selection of synthetic polymer libraries for desired binding or catalytic properties and (iv) amplification of template sequences that survive selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of 12 PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory.}, number={2}, journal={Nature Chemical Biology}, publisher={Springer Science and Business Media LLC}, author={Brudno, Yevgeny and Birnbaum, Michael E and Kleiner, Ralph E and Liu, David R}, year={2010}, month={Dec}, pages={148–155} }
 @article{tahiliani_koh_shen_pastor_bandukwala_brudno_agarwal_iyer_liu_aravind_et al._2009, place={New York, N.Y}, title={Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1}, volume={324}, ISSN={0036-8075 1095-9203}, url={http://dx.doi.org/10.1126/science.1170116}, DOI={10.1126/science.1170116}, abstractNote={Methylation Mediation
          
            Methylation of cytosine bases, 5-methylcytosine (5mC), in DNA plays an important regulatory role in mammalian genomes. Methylation patterns are often inherited across generations, but they can also be dynamic, suggesting that active DNA demethylation pathways exist. One such pathway, best characterized in plants, involves the removal of the 5mC base, and its replacement by C, via a DNA repair mechanism.
            Kriaucionis and Heintz
            (p.
            929
            , published online 16 April) now show that, as well as 5mC in mammalian genomes, there are also significant amounts of 5-hydroxymethylcytosine (5hmC) in DNA of Purkinje neurons, which have large nuclei with apparently very little heterochromatin.
            
              Tahiliani
              et al.
            
            (p. 930, published online 16 April) find that the protein TET1 is capable of converting 5mC into 5hmC both in vitro and in vivo. 5-Hydroxymethylcytosine is also present in embryonic stem cells, and levels of 5hmC and TET1 show correlated variation during cell differentiation.
          }, number={5929}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Tahiliani, Mamta and Koh, Kian Peng and Shen, Yinghua and Pastor, William A. and Bandukwala, Hozefa and Brudno, Yevgeny and Agarwal, Suneet and Iyer, Lakshminarayan M. and Liu, David R. and Aravind, L. and et al.}, year={2009}, month={May}, pages={930–935} }
 @article{brudno_liu_2009, title={Recent Progress Toward the Templated Synthesis and Directed Evolution of Sequence-Defined Synthetic Polymers}, volume={16}, ISSN={1074-5521}, url={http://dx.doi.org/10.1016/j.chembiol.2009.02.004}, DOI={10.1016/j.chembiol.2009.02.004}, abstractNote={Biological polymers such as nucleic acids and proteins are ubiquitous in living systems, but their ability to address problems beyond those found in nature is constrained by factors such as chemical or biological instability, limited building-block functionality, bioavailability, and immunogenicity. In principle, sequence-defined synthetic polymers based on nonbiological monomers and backbones might overcome these constraints; however, identifying the sequence of a synthetic polymer that possesses a specific desired functional property remains a major challenge. Molecular evolution can rapidly generate functional polymers but requires a means of translating amplifiable templates such as nucleic acids into the polymer being evolved. This review covers recent advances in the enzymatic and nonenzymatic templated polymerization of nonnatural polymers and their potential applications in the directed evolution of sequence-defined synthetic polymers.}, number={3}, journal={Chemistry & Biology}, publisher={Elsevier BV}, author={Brudno, Yevgeny and Liu, David R.}, year={2009}, month={Mar}, pages={265–276} }
 @article{kleiner_brudno_birnbaum_liu_2008, title={DNA-Templated Polymerization of Side-Chain-Functionalized Peptide Nucleic Acid Aldehydes}, volume={130}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja0753997}, DOI={10.1021/ja0753997}, abstractNote={The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step toward the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each building block, and functionalization densities.}, number={14}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Kleiner, Ralph E. and Brudno, Yevgeny and Birnbaum, Michael E. and Liu, David R.}, year={2008}, month={Apr}, pages={4646–4659} }