Investigating the Role of Substrate Stiffness on Beta-Catenin Activation in Osteoblasts

Cells are exposed to a myriad of signals from their microenvironment and the finely tuned processing of these cues regulates cell survival, proliferation, and differentiation. Traditional belief was that only soluble signaling factors governed cell behavior, but more recently, much attention has been focused on the influence of mechanical and topographical cues. Although many landmark observations have been published in this regard, still little is known about the mechanisms behind cell response to physical cues. This work stems from the previously reported observations that beta-catenin, a key cell signaling molecule involved in bone development, is activated by acute mechanical stress. The main aim of this work was to investigate the role of substrate stiffness on beta-catenin activation in osteoblasts. Here, two polyacrylamide gel platforms are adapted that vary in Young's modulus and act as adhesive substrates for cell attachment. Osteoblasts grown on these polyacrylamide gels attach through cross-linked extracellular matrix proteins and 'sense' the underlying stiffness of the substrate and change their intracellular biochemistry accordingly. It was found in this work that the amount of total beta-catenin increases after 24 hours in osteoblasts cultured on tissue culture plastic and polyacrylamide gels of 40 kPa. Moreover, the number of cell-cell contacts directly influences the amount of total beta-catenin. It was also found that osteoblasts grown on higher stiffness substrates have more active beta-catenin and less degraded beta- catenin compared to lower stiffness substrates. The novel findings reported here advance the proposal that beta-catenin is a participant in mechanosensing and also provides a new platform for investigation and further insight into a mechanism of action

Thyroid hormone receptor beta and NCOA4 regulate terminal erythrocyte differentiation

An effect of thyroid hormone (TH) on erythropoiesis has been known for more than a century but the molecular mechanism(s) by which TH affects red cell formation is still elusive. Here we demonstrate an essential role of TH during terminal human erythroid cell differentiation; specific depletion of TH from the culture medium completely blocked terminal erythroid differentiation and enucleation. Treatment with TRβ agonists stimulated premature erythroblast differentiation in vivo and alleviated anemic symptoms in a chronic anemia mouse model by regulating erythroid gene expression. To identify factors that cooperate with TRβ during human erythroid terminal differentiation, we conducted RNA-seq in human reticulocytes and identified nuclear receptor coactivator 4 (NCOA4) as a critical regulator of terminal differentiation. Furthermore, Ncoa4 −/− mice are anemic in perinatal periods and fail to respond to TH by enhanced erythropoiesis. Genome-wide analysis suggests that TH promotes NCOA4 recruitment to chromatin regions that are in proximity to Pol II and are highly associated with transcripts abundant during terminal differentiation. Collectively, our results reveal the molecular mechanism by which TH functions during red blood cell formation, results that are potentially useful to treat certain anemias. Keywords: thyroid hormone; erythropoiesis; NCOA4; nuclear receptorUnited States. Defense Advanced Research Projects Agency (Award HR0011-14-2-0005)United States. Department of Defense (Award W81WH-12-1-0449)National Heart, Lung, and Blood Institute (Grant P01 HL032262-25

Optical control of mammalian endogenous transcription and epigenetic states

A theoretical underpinning of the standard model of fundamental particles and interactions is CPT invariance, which requires that the laws of physics be invariant under the combined discrete operations of charge conjugation, parity and time reversal. Antimatter, the existence of which was predicted by Dirac, can be used to test the CPT theorem—experimental investigations involving comparisons of particles with antiparticles are numerous. Cold atoms and anti-atoms, such as hydrogen and antihydrogen, could form the basis of a new precise test, as CPT invariance implies that they must have the same spectrum. Observations of antihydrogen in small quantities and at high energies have been reported at the European Organization for Nuclear Research (CERN) and at Fermilab, but these experiments were not suited to precision comparison measurements. Here we demonstrate the production of antihydrogen atoms at very low energy by mixing trapped antiprotons and positrons in a cryogenic environment. The neutral anti-atoms have been detected directly when they escape the trap and annihilate, producing a characteristic signature in an imaging particle detector

Chd8 Mutation Leads to Autistic-like Behaviors and Impaired Striatal Circuits

Autism spectrum disorder (ASD) is a heterogeneous disease, but genetically defined models can provide an entry point to studying the molecular underpinnings of this disorder. We generated germline mutant mice with loss-of-function mutations in Chd8, a de novo mutation strongly associated with ASD, and demonstrate that these mice display hallmark ASD behaviors, macrocephaly, and craniofacial abnormalities similar to patient phenotypes. Chd8[superscript +/–] mice display a broad, brain-region-specific dysregulation of major regulatory and cellular processes, most notably histone and chromatin modification, mRNA and protein processing, Wnt signaling, and cell-cycle regulation. We also find altered synaptic physiology in medium spiny neurons of the nucleus accumbens. Perturbation of Chd8 in adult mice recapitulates improved acquired motor learning behavior found in Chd8[superscript +/–] animals, suggesting a role for CHD8 in adult striatal circuits. These results support a mechanism linking chromatin modification to striatal dysfunction and the molecular pathology of ASD.National Science Foundation (U.S.) (1122374)National Science Foundation (U.S.) (2013169249)National Institute of Mental Health (U.S.) (F31-MH111157)Howard Hughes Medical Institute (NS046789)Simons Foundation Autism Research Initiative (306063)Simons Foundation Autism Research Initiative (6927482)National Institute of Mental Health (U.S.) (5DP1-MH100706)National Institute of Mental Health (U.S.) (1R01-MH110049)Nancy Lurie Marks Family Foundation (6928117)Howard Hughes Medical Institute (NS046789

CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras[superscript G12D] mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.National Science Foundation (U.S.). Graduate Research Fellowship (Grant 1122374)Damon Runyon Cancer Research Foundation (Fellowship DRG-2117-12)Massachusetts Institute of Technology. Simons Center for the Social Brain (Postdoctoral Fellowship)European Molecular Biology Organization (Fellowship)Foundation for Polish Science (Fellowship)American Society for Engineering Education. National Defense Science and Engineering Graduate FellowshipNational Science Foundation (U.S.). Graduate Research FellowshipMassachusetts Institute of Technology (Presidential Graduate Fellowship)Human Frontier Science Program (Strasbourg, France) (Postdoctoral Fellowship)National Human Genome Research Institute (U.S.) (CEGS P50 HG006193)Howard Hughes Medical InstituteKlarman Cell ObservatoryNational Cancer Institute (U.S.) (Center of Cancer Nanotechnology Excellence Grant U54CA151884)National Institutes of Health (U.S.) (Controlled Release Grant EB000244)National Heart, Lung, and Blood Institute (Program of Excellence in Nanotechnology (PEN) Award Contract HHSN268201000045C)Massachusetts Institute of Technology (Poitras Gift 1631119)Stanley CenterSimons Foundation (6927482)Nancy Lurie Marks Family Foundation (6928117)United States. Public Health Service (National Institutes of Health (U.S.) R01-CA133404)David H. Koch Institute for Integrative Cancer Research at MIT (Marie D. and Pierre Casimir-Lambert Fund)MIT Skoltech InitiativeNational Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Institute of Mental Health (U.S.) (Director’s Pioneer Award DP1-MH100706)National Institute of Neurological Disorders and Stroke (U.S.) (Transformative R01 Grant R01-NS 07312401)National Science Foundation (U.S.) (Waterman Award)W. M. Keck FoundationKinship Foundation. Searle Scholars ProgramKlingenstein FoundationVallee FoundationMerkin Foundatio

Buildout and integration of an automated high-throughput CLIA laboratory for SARS-CoV-2 testing on a large urban campus

In 2019, the first cases of SARS-CoV-2 were detected in Wuhan, China, and by early 2020 the first cases were identified in the United States. SARS-CoV-2 infections increased in the US causing many states to implement stay-at-home orders and additional safety precautions to mitigate potential outbreaks. As policies changed throughout the pandemic and restrictions lifted, there was an increase in demand for COVID-19 testing which was costly, difficult to obtain, or had long turn-around times. Some academic institutions, including Boston University (BU), created an on-campus COVID-19 screening protocol as part of a plan for the safe return of students, faculty, and staff to campus with the option for in-person classes. At BU, we put together an automated high-throughput clinical testing laboratory with the capacity to run 45,000 individual tests weekly by Fall of 2020, with a purpose-built clinical testing laboratory, a multiplexed reverse transcription PCR (RT-qPCR) test, robotic instrumentation, and trained staff. There were many challenges including supply chain issues for personal protective equipment and testing materials in addition to equipment that were in high demand. The BU Clinical Testing Laboratory (CTL) was operational at the start of Fall 2020 and performed over 1 million SARS-CoV-2 PCR tests during the 2020-2021 academic year.Boston UniversityPublished versio

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Applications of genome editing for disease modeling in mice

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February 2016.Cataloged from PDF version of thesis. "September 2015."Includes bibliographical references (pages 60-66).The genome holds the blueprint of life and heredity. In the case of the mammalian genome it is comprised of billions of DNA bases grouped into elements that we are beginning to understand (ie genes) and others we know little about (ie noncoding DNA). Forward and reverse genetics in cells and animal models is key to discovering causal mechanisms relating molecular and genetic events to phenotypes. Therefore, the ability to sequence and edit DNA is fundamental to understanding of the role of genetic elements in normal biology and disease. Recently developed genome editing technologies are now making it possible to modify the genome in its endogenous context, opening up exciting possibilities for understanding its function. The RNA-guided endonuclease Cas9 from microbial type II CRISPR (clustered regularly interspaced short palindromic repeat) systems has been harnessed to facilitate facile genetic manipulations in a variety of cell types and organisms. Cas9 can be easily reprogrammed using RNA guides to generate targeted DNA double strand breaks, which can stimulate genome editing. A unique advantage of the Cas9 system is that Cas9 can be combined with multiple guide RNAs to achieve efficient multiplexed genome editing in mammalian cells, which opens up the possibility of interrogating multigenic biological processes. In this thesis, we utilize the Cas9 technology to facilitate genome editing experiments in vivo in mice. First, we create a Cas9 knockin mouse and demonstrate its utility for in vivo and ex vivo genome editing experiments. Then, we leverage the Cas9 knockin mouse and viral-mediated delivery of guide RNA to model lung adenocarcinoma to obtain pathology consistent with traditional transgenic mouse models and human patients. Finally, we utilize pronuclear injection of Cas9 mRNA and guide RNA to generate mice harboring a mutation in the autism-associated gene CHD8 and investigate the underlying behavioral and molecular phenotype. These applications broadly demonstrate the potential of Cas9 for interrogating genetic elements in vivo towards understand their role in normal biological processes and disease.by Randall Jeffrey Platt.Ph. D

Optogenetic skeletal muscle-powered adaptive biological machines

Complex biological systems sense, process, and respond to their surroundings in real time. The ability of such systems to adapt their behavioral response to suit a range of dynamic environmental signals motivates the use of biological materials for other engineering applications. As a step toward forward engineering biological machines (bio-bots) capable of nonnatural functional behaviors, we created a modular light-controlled skeletal muscle-powered bioactuator that can generate up to 300 µN (0.56 kPa) of active tension force in response to a noninvasive optical stimulus. When coupled to a 3D printed flexible bio-bot skeleton, these actuators drive directional locomotion (310 µm/s or 1.3 body lengths/min) and 2D rotational steering (2°/s) in a precisely targeted and controllable manner. The muscle actuators dynamically adapt to their surroundings by adjusting performance in response to “exercise” training stimuli. This demonstration sets the stage for developing multicellular bio-integrated machines and systems for a range of applicationsNational Science Foundation (U.S.) (Science and Technology Center Emergent Behavior of Integrated Cellular Systems (EBICS) Grant CBET-0939511)National Science Foundation (U.S.) (Graduate Research Fellowship, Grant DGE-1144245)National Science Foundation (U.S.) (NSF Cellular and Molecular Mechanics and Bionanotechnology (CMMB) Integrative Graduate Education and Research Traineeship (IGERT) at UIUC (Grant 0965918)

Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR–mediated direct in vivo screening

Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas somemutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct highthroughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adenoassociated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice. All mice that received the AAV-mTSG library developed liver cancer and diedwithin 4 months.We usedmolecular inversion probe sequencing of the sgRNA target sites to chart the mutational landscape of these tumors, revealing the functional consequence of multiple variants in driving liver tumorigenesis in immunocompetent mice. AAV-mediated autochthonous CRISPR screens provide a powerful means for mapping a provisional functional cancer genome atlas of tumor suppressors in vivo.Damon Runyon Cancer Research Foundation (DRG-2117-12; DFS-13-15)Melanoma Research Foundation (412806, 16-003524)St. Baldrick’s Foundation (426685)American Cancer Society (IRG 58-012-54)Cancer Research Institute (New York, N.Y.)Breast Cancer AllianceCancer Research Institute (New York, N.Y.) (Clinic and Laboratory Integration Program)American Association for Cancer Research (499395)United States. Department of Defense (W81XWH-17-1-0235)National Cancer Institute (U.S.) (1U54CA209992)National Cancer Institute (U.S.) (5P50CA196530-A10805)National Cancer Institute (U.S.) (4P50CA121974-A08306)National Institutes of Health (U.S.) (R01-CA133404)National Institutes of Health (U.S.) (R01-GM034277)National Institutes of Health (U.S.) (CCNE)Skolkovo FoundationCasimir-Lambert FundNational Institutes of Health (U.S.) (grant 1R01-HG009761)National Institutes of Health (U.S.) (grant R01-MH110049)National Institutes of Health (U.S.) (grant DP1-HL141201)Howard Hughes Medical InstituteNew York Stem Cell FoundationSimons, Paul G. Allen FamilyVallee FoundationsPoitras Center for Affective Disorders Research at MITHock E. Tan and K. Lisa Yang Center for Autism Research at MI