Methacrylation induces rapid, temperature-dependent, reversible self-assembly of type-I collagen.

Type-I collagen self-assembles into a fibrillar gel at physiological temperature and pH to provide a cell-adhesive, supportive, structural network. As such, it is an attractive, popular scaffold for in vitro evaluations of cellular behavior and for tissue engineering applications. In this study, type-I collagen is modified to introduce methacrylate groups on the free amines of the lysine residues to create collagen methacrylamide (CMA). CMA retains the properties of collagen such as self-assembly, biodegradability, and natural bioactivity but is also photoactive and can be rapidly cross-linked or functionalized with acrylated molecules when irradiated with ultraviolet light in the presence of a photoinitiator. CMA also demonstrates unique temperature-dependent behavior. For natural type-I collagen, the overall structure of the fiber network remains largely static over time scales of a few hours upon heating and cooling at temperatures below its denaturation point. CMA, however, is rapidly thermoreversible and will oscillate between a liquid macromer suspension and a semisolid fibrillar hydrogel when the temperature is modulated between 10 and 37 °C. Using a series of mechanical, scattering, and spectroscopic methods, we demonstrate that structural reversibility is manifest across multiple scales from the protein topology of the triple helix up through the rheological properties of the CMA hydrogel. Electron microscopy imaging of CMA after various stages of heating and cooling shows that the canonical collagen-like D-periodic banding ultrastructure of the fibers is preserved. A rapidly thermoreversible collagen-based hydrogel is expected to have wide utility in tissue engineering and drug delivery applications as a biofunctional, biocompatible material. Thermal reversibility also makes CMA a powerful model for studying the complex process of hierarchical collagen self-assembly.

Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors.

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transit-amplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.

The Identification of CELSR3 and Other Potential Cell Surface Targets in Neuroendocrine Prostate Cancer.

Although recent efforts have led to the development of highly effective androgen receptor (AR)-directed therapies for the treatment of advanced prostate cancer, a significant subset of patients will progress with resistant disease including AR-negative tumors that display neuroendocrine features [neuroendocrine prostate cancer (NEPC)]. On the basis of RNA sequencing (RNA-seq) data from a clinical cohort of tissue from benign prostate, locally advanced prostate cancer, metastatic castration-resistant prostate cancer and NEPC, we developed a multi-step bioinformatics pipeline to identify NEPC-specific, overexpressed gene transcripts that encode cell surface proteins. This included the identification of known NEPC surface protein CEACAM5 as well as other potentially targetable proteins (e.g., HMMR and CESLR3). We further showed that cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) knockdown results in reduced NEPC tumor cell proliferation and migration in vitro. We provide in vivo data including laser capture microdissection followed by RNA-seq data supporting a causal role of CELSR3 in the development and/or maintenance of the phenotype associated with NEPC. Finally, we provide initial data that suggests CELSR3 is a target for T-cell redirection therapeutics. Further work is now needed to fully evaluate the utility of targeting CELSR3 with T-cell redirection or other similar therapeutics as a potential new strategy for patients with NEPC. Significance: The development of effective treatment for patients with NEPC remains an unmet clinical need. We have identified specific surface proteins, including CELSR3, that may serve as novel biomarkers or therapeutic targets for NEPC.

Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.

Cytoplasmic inclusions containing alpha-synuclein (alpha-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathological hallmarks of Parkinson's disease (PD). Although alpha-Syn fibrils can be generated from recombinant alpha-Syn protein in vitro, the production of fibrillar alpha-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular alpha-Syn aggregation can be triggered by the introduction of exogenously produced recombinant alpha-Syn fibrils into cultured cells engineered to overexpress alpha-Syn. Unlike unassembled alpha-Syn, these alpha-Syn fibrils "seeded" recruitment of endogenous soluble alpha-Syn protein and their conversion into insoluble, hyperphosphorylated, and ubiquitinated pathological species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular alpha-Syn aggregation is normally limited by the number of active nucleation sites present in the cytoplasm and that small quantities of alpha-Syn fibrils can alter this balance by acting as seeds for aggregation.

Biohybrid Carbon Nanotube/Agarose Fibers for Neural Tissue Engineering.

We report a novel approach for producing carbon nanotube fibers (CNF) composed with the polysaccharide agarose. Current attempts to make CNF's require the use of a polymer or precipitating agent in the coagulating bath that may have negative effects in biomedical applications. We show that by taking advantage of the gelation properties of agarose one can substitute the bath with distilled water or ethanol and hence reduce the complexity associated with alternating the bath components or the use of organic solvents. We also demonstrate that these CNF can be chemically functionalized to express biological moieties through available free hydroxyl groups in agarose. We corroborate that agarose CNF are not only conductive and nontoxic, but their functionalization can facilitate cell attachment and response both in vitro and in vivo. Our findings suggest that agarose/CNT hybrid materials are excellent candidates for applications involving neural tissue engineering and biointerfacing with the nervous system.

Spirocyclic Thiohydantoin Antagonists of F877L and Wild-Type Androgen Receptor for Castration-Resistant Prostate Cancer.

Androgen receptor (AR) transcriptional reactivation plays a key role in the development and progression of lethal castration-resistant prostate cancer (CRPC). Recurrent alterations in the AR enable persistent AR pathway signaling and drive resistance to the treatment of second-generation antiandrogens. AR F877L, a point mutation in the ligand binding domain of the AR, was identified in patients who acquired resistance to enzalutamide or apalutamide. In parallel to our previous structure-activity relationship (SAR) studies of compound 4 (JNJ-pan-AR) and clinical stage compound 5 (JNJ-63576253), we discovered additional AR antagonists that provide opportunities for future development. Here we report a highly potent series of spirocyclic thiohydantoins as AR antagonists for the treatment of the F877L mutant and wild-type CRPC.

Discovery of JNJ-63576253: A Clinical Stage Androgen Receptor Antagonist for F877L Mutant and Wild-Type Castration-Resistant Prostate Cancer (mCRPC).

Persistent androgen receptor (AR) activation drives therapeutic resistance to second-generation AR pathway inhibitors and contributes to the progression of advanced prostate cancer. One resistance mechanism is point mutations in the ligand binding domain of AR that can transform antagonists into agonists. The AR F877L mutation, identified in patients treated with enzalutamide or apalutamide, confers resistance to both enzalutamide and apalutamide. Compound 4 (JNJ-pan-AR) was identified as a pan-AR antagonist with potent activity against wild-type and clinically relevant AR mutations including F877L. Metabolite identification studies revealed a latent bioactivation pathway associated with 4. Subsequent lead optimization of 4 led to amelioration of this pathway and nomination of 5 (JNJ-63576253) as a clinical stage, next-generation AR antagonist for the treatment of castration-resistant prostate cancer (CRPC).

Discovery of JNJ-63576253, a Next-Generation Androgen Receptor Antagonist Active Against Wild-Type and Clinically Relevant Ligand Binding Domain Mutations in Metastatic Castration-Resistant Prostate Cancer.

Numerous mechanisms of resistance arise in response to treatment with second-generation androgen receptor (AR) pathway inhibitors in metastatic castration-resistant prostate cancer (mCRPC). Among these, point mutations in the ligand binding domain can transform antagonists into agonists, driving the disease through activation of AR signaling. To address this unmet need, we report the discovery of JNJ-63576253, a next-generation AR pathway inhibitor that potently abrogates AR signaling in models of human prostate adenocarcinoma. JNJ-63576253 is advancing as a clinical candidate with potential effectiveness in the subset of patients who do not respond to or are progressing while on second-generation AR-targeted therapeutics.

Modulating Androgen Receptor-Driven Transcription in Prostate Cancer with Selective CDK9 Inhibitors.

Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.

Contrasting expression of canonical Wnt signaling reporters TOPGAL, BATGAL and Axin2(LacZ) during murine lung development and repair.

Canonical WNT signaling plays multiple roles in lung organogenesis and repair by regulating early progenitor cell fates: investigation has been enhanced by canonical Wnt reporter mice, TOPGAL, BATGAL and Axin2(LacZ). Although widely used, it remains unclear whether these reporters convey the same information about canonical Wnt signaling. We therefore compared beta-galactosidase expression patterns in canonical Wnt signaling of these reporter mice in whole embryo versus isolated prenatal lungs. To determine if expression varied further during repair, we analyzed comparative pulmonary expression of beta-galactosidase after naphthalene injury. Our data show important differences between reporter mice. While TOPGAL and BATGAL lines demonstrate Wnt signaling well in early lung epithelium, BATGAL expression is markedly reduced in late embryonic and adult lungs. By contrast, Axin2(LacZ) expression is sustained in embryonic lung mesenchyme as well as epithelium. Three days into repair after naphthalene, BATGAL expression is induced in bronchial epithelium as well as TOPGAL expression (already strongly expressed without injury). Axin2(LacZ) expression is increased in bronchial epithelium of injured lungs. Interestingly, both TOPGAL and Axin2(LacZ) are up regulated in parabronchial smooth muscle cells during repair. Therefore the optimal choice of Wnt reporter line depends on whether up- or down-regulation of canonical Wnt signal reporting in either lung epithelium or mesenchyme is being compared.

Shear stress and plaque development.

Although traditional cardiovascular risk factors 'prime the soil' for atherogenesis systemically, atherosclerosis primarily occurs in a site-specific manner with a predilection towards the inner wall of curvatures and outer wall of bifurcations with sparing of flow-dividers. Wall shear stress is a frictional force exerted parallel to the vessel wall that leads to alteration of the endothelial phenotype, endothelial cell signaling, gene and protein expression leading to a proinflammatory phenotype, reduced nitric oxide availability and disruption of the extracellular matrix, which in turn leads to plaque development. Clinical and experimental data are emerging that suggest the pathobiology associated with abnormal wall shear stress results in atherosclerotic plaque development and progression.