Stage-Specific Generation of Human Pluripotent Stem Cell Derived Lung Models to Measure CFTR Function

Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air-liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms. The stepwise approach directs differentiation from hPSC to definitive endoderm, anterior ventral foregut endoderm, and fetal lung progenitor cells. The article also describes the generation of immature airway epithelial spheroids in Matrigel with epithelial cells sorted by a magnetic-activated cell sorting system, and the generation of adult-like airway epithelia through air-liquid interface conditions. We demonstrate that this optimized procedure generates remarkably higher cystic fibrosis transmembrane conductance regulator (CFTR) expression and function than our previous method, and thus is uniquely suitable for CF research applications. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hESC/hiPSC differentiation to fetal lung progenitors Basic Protocol 2: Formation of airway epithelial spheroids Alternate Protocol 1: Cryopreservation of airway epithelial spheroids Basic Protocol 3: Differentiation and maturation in air-liquid interface culture Alternate Protocol 2: Differentiation and maturation of epithelial progenitors from airway epithelial spheroids in ALI culture

Fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin with gemtuzumab ozogamicin improves event-free survival in younger patients with newly diagnosed aml and overall survival in patients with npm1 and flt3 mutations

Purpose To determine the optimal induction chemotherapy regimen for younger adults with newly diagnosed AML without known adverse risk cytogenetics. Patients and Methods One thousand thirty-three patients were randomly assigned to intensified (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin [FLAG-Ida]) or standard (daunorubicin and Ara-C [DA]) induction chemotherapy, with one or two doses of gemtuzumab ozogamicin (GO). The primary end point was overall survival (OS). Results There was no difference in remission rate after two courses between FLAG-Ida + GO and DA + GO (complete remission [CR] + CR with incomplete hematologic recovery 93% v 91%) or in day 60 mortality (4.3% v 4.6%). There was no difference in OS (66% v 63%; P = .41); however, the risk of relapse was lower with FLAG-Ida + GO (24% v 41%; P < .001) and 3-year event-free survival was higher (57% v 45%; P < .001). In patients with an NPM1 mutation (30%), 3-year OS was significantly higher with FLAG-Ida + GO (82% v 64%; P = .005). NPM1 measurable residual disease (MRD) clearance was also greater, with 88% versus 77% becoming MRD-negative in peripheral blood after cycle 2 (P = .02). Three-year OS was also higher in patients with a FLT3 mutation (64% v 54%; P = .047). Fewer transplants were performed in patients receiving FLAG-Ida + GO (238 v 278; P = .02). There was no difference in outcome according to the number of GO doses, although NPM1 MRD clearance was higher with two doses in the DA arm. Patients with core binding factor AML treated with DA and one dose of GO had a 3-year OS of 96% with no survival benefit from FLAG-Ida + GO. Conclusion Overall, FLAG-Ida + GO significantly reduced relapse without improving OS. However, exploratory analyses show that patients with NPM1 and FLT3 mutations had substantial improvements in OS. By contrast, in patients with core binding factor AML, outcomes were excellent with DA + GO with no FLAG-Ida benefit

Factors governing the design, selection and cleavage of sugar-modified duplexes by ribonuclease H

The antisense principle bases its premise in the exquisite complementarity of a synthetic, chemically-modified oligonucleotide to tightly bind with a unique target RNA sequence. Rapid and selective genetic discrimination, as driven by the formation of multiple points of target contact, constitutes a central goal of oligonucleotide therapies. Most synthetic designs have, however, provided little structural insight on the role of the antisense oligonucleotide (AON) in triggering RNA cleavage of preformed hybrids, as catalyzed by a ubiquitous, intracellular enzyme known as ribonuclease H. The use of RNase H to assist AON inhibition of gene expression is crucial to mainstream antisense technologies, yet the precise mode by which this enzyme acts on AON/RNA duplexes remains unclear.To address the role of substrate structure on enzyme activation, a dominant theme of this thesis highlights the design, synthesis and structural studies of novel AONs comprised of rigid 2'-deoxy-2'-fluoroarabino (2'F-ANA) or native (DNA) nucleotides, containing interspersed flexible (e.g. "2',3'-seconucleotides") or anucleosidic (e.g. butyl) residues. This unique AON class combines both pre-organization & flexibility within the hosting heteroduplex, which on their own usually prove detrimental towards enzyme trigger. Their combination, however, synergistically activates both E. coli and human RNases H, leading to potent destruction of duplexed RNA. These compounds thus represent the first examples of modified AONs lacking deoxyribose sugars that elicit RNase H activity comparably to the native (DNA) systems. DNA-derived AONs with acyclic residues also amplify enzyme-catalyzed target degradation, suggesting the added flexibility imparted to the substrate structure to be vital for ameliorating the protein/nucleic acid interaction. Melting and circular dichroic experiments have revealed that the enhanced dynamics associated with a particular acyclic modification remain globally undetectable, indicating the acyclic residues induce only local structural deformations to the helix architecture.Intricate comparisons of the structural and biological properties of various acyclic residues (e.g. butyl, propyl and ethyl interresidue spacers) designed to locally compress or expand the AON helix backbone at a defined axial site has enabled a deeper understanding of the conformational factors that underlie the observed enhancements

Solid‐Phase Synthesis of 2′‐Deoxy‐2′‐fluoro‐ β‐D‐Oligoarabinonucleotides (2′F‐ANA) and Their Phosphorothioate Derivatives

This unit describes the chemical synthesis of 2’‐deoxy‐2’‐fluoro‐b‐D‐oligoarabinonucleotides (2’F‐ANA), both with phosphodiester and phosphorothioate linkages. The protocols described herein include araF phosphoramidite preparation, assembly on DNA synthesizers, and final deprotection and purification of oligonucleotides.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153131/1/cpnc0415.pd

Transcription factor substitution during the evolution of fungal ribosome regulation

Coordinated ribosomal protein (RP) gene expression is crucial for cellular viability, but the transcriptional network controlling this regulon has only been well characterized in the yeast Saccharomyces cerevisiae. We have used whole-genome transcriptional and location profiling to establish that, in Candida albicans, the RP regulon is controlled by the Myb domain protein Tbf1 working in conjunction with Cbf1. These two factors bind both the promoters of RP genes and the rDNA locus; Tbf1 activates transcription at these loci and is essential. Orthologs of Tbf1 bind TTAGGG telomeric repeats in most eukaryotes, and TTAGGG cis-elements are present upstream of RP genes in plants and fungi, suggesting that Tbf1 was involved in both functions in ancestral eukaryotes. In all Hemiascomycetes, Rap1 substituted Tbf1 at telomeres and, in the S. cerevisiae lineage, this substitution also occurred independently at RP genes, illustrating the extreme adaptability and flexibility of transcriptional regulatory networks.Peer reviewed: YesNRC publication: Ye

Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family

Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family