Influence of Polymeric Microparticle Size and Loading Concentration on 3D Printing Accuracy and Degradation Behavior of Composite Scaffolds.

Successful employment of 3D printing for delivery of therapeutic biomolecules requires protection of their bioactivity on exposure to potentially inactivating conditions. Although intermediary encapsulation of the biomolecules in polymeric particulate delivery vehicles is a promising strategy for this objective, the inclusion of such particles in 3D printing formulations may critically impact the accuracy or precision of 3D printed scaffolds relative to their intended designed architectures, as well as the degradation behavior of both the scaffolds and the included particles. The present work aimed to elucidate the effect of poly(d,l-lactic-co-glycolic acid) particle size and loading concentration on material accuracy, machine precision, and degradation of 3D printed poly(ɛ-caprolactone)-based scaffolds. Using a main effects analysis, the sizes and loading concentrations of particle delivery vehicles investigated were found to have neither a beneficial nor disadvantageous influence on the metrics of printing quality such as material accuracy and machine precision. Meanwhile, particle loading concentration was determined to influence degradation rate, whereas printing temperature affected the trends in composite weight-average molecular weight. Neither of the two particle-related parameters (concentration nor diameter) was found to exhibit a significant effect on intra-fiber nor inter-fiber porosity. These findings evidence the capacity for controlled loading of particulate delivery vehicles in 3D printed scaffolds while preserving construct accuracy and precision, and with predictable dictation of composite degradation behavior for potential controlled release of encapsulated biomolecules.

KLF15 cistromes reveal a hepatocyte pathway governing plasma corticosteroid transport and systemic inflammation.

Circulating corticosteroids orchestrate stress adaptation, including inhibition of inflammation. While pathways governing corticosteroid biosynthesis and intracellular signaling are well understood, less is known about mechanisms controlling plasma corticosteroid transport. Here, we show that hepatocyte KLF15 (Kruppel-like factor 15) controls plasma corticosteroid transport and inflammatory responses through direct transcriptional activation of Serpina6, which encodes corticosteroid-binding globulin (CBG). Klf15-deficient mice have profoundly low CBG, reduced plasma corticosteroid binding capacity, and heightened mortality during inflammatory stress. These defects are completely rescued by reconstituting CBG, supporting that KLF15 works primarily through CBG to control plasma corticosterone homeostasis. To understand transcriptional mechanisms, we generated the first KLF15 cistromes using newly engineered Klf153xFLAG mice. Unexpectedly, liver KLF15 is predominantly promoter enriched, including Serpina6, where it binds a palindromic GC-rich motif, opens chromatin, and transactivates genes with minimal associated direct gene repression. Overall, we provide critical mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.

IRF2 is a master regulator of human keratinocyte stem cell fate.

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.

Development of a Selective CDK7 Covalent Inhibitor Reveals Predominant Cell-Cycle Phenotype.

Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G1/S transition and inhibition of E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may be useful for treatment of cancers marked by E2F misregulation.