PGE1 and PGA1 bind to Nurr1 and activate its transcriptional function.

The orphan nuclear receptor Nurr1 is critical for the development, maintenance and protection of midbrain dopaminergic (mDA) neurons. Here we show that prostaglandin E1 (PGE1) and its dehydrated metabolite, PGA1, directly interact with the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional function. We also report the crystallographic structure of Nurr1-LBD bound to PGA1 at 2.05 Å resolution. PGA1 couples covalently to Nurr1-LBD by forming a Michael adduct with Cys566, and induces notable conformational changes, including a 21° shift of the activation function-2 helix (H12) away from the protein core. Furthermore, PGE1/PGA1 exhibit neuroprotective effects in a Nurr1-dependent manner, prominently enhance expression of Nurr1 target genes in mDA neurons and improve motor deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse models of Parkinson's disease. Based on these results, we propose that PGE1/PGA1 represent native ligands of Nurr1 and can exert neuroprotective effects on mDA neurons, via activation of Nurr1's transcriptional function.

Speckle reduction in quantitative phase imaging by generating spatially incoherent laser field at electroactive optical diffusers.

We studied quantitative phase imaging (QPI) using coherent laser illumination coupled with static and moving optical diffusers. The spatial coherence of a continuous-wave laser was controlled by tuning the particle size and the diffusion angle of optical diffusers for speckle-reduced 3D phase imaging of transparent objects. We used a common-path QPI configuration to investigate the coherent phase mapping of polystyrene micro-beads and breast cancer cells (MCF-7) under different degrees of coherent speckles. The proposed speckle reduction method could provide an avenue for enhancing lateral resolution and suppressing coherent artifacts of the phase images from QPI.

Prostaglandin A2 Interacts with Nurr1 and Ameliorates Behavioral Deficits in Parkinson's Disease Fly Model.

The orphan nuclear receptor Nurr1 is critical for the development, maintenance, and protection of midbrain dopaminergic neurons. Recently, we demonstrated that prostaglandins E1 (PGE1) and PGA1 directly bind to the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional activation function. In this direction, here we report the transcriptional activation of Nurr1 by PGA2, a dehydrated metabolite of PGE2, through physical binding ably supported by NMR titration and crystal structure. The co-crystal structure of Nurr1-LBD bound to PGA2 revealed the covalent coupling of PGA2 with Nurr1-LBD through Cys566. PGA2 binding also induces a 21° shift of the activation function 2 (AF-2) helix H12 away from the protein core, similar to that observed in the Nurr1-LBD-PGA1 complex. We also show that PGA2 can rescue the locomotor deficits and neuronal degeneration in LRRK2 G2019S transgenic fly models.

Chloroquine modulates inflammatory autoimmune responses through Nurr1 in autoimmune diseases.

For over a half-century the anti-malarial drug chloroquine (CQ) has been used as a therapeutic agent, alone or in combination, to treat autoimmune diseases. However, neither the underlying mechanism(s) of action nor their molecular target(s) are well defined. The orphan nuclear receptor Nurr1 (also known as NR4A2) is an essential transcription factor affecting the development and maintenance of midbrain dopaminergic neurons. In this study, using in vitro T cell differentiation models, we demonstrate that CQ activates TREG cell differentiation and induces Foxp3 gene expression in a Nurr1-dependent manner. Remarkably, CQ appears to induce Nurr1 function by two distinct mechanisms: firstly, by direct binding to Nurr1's ligand-binding domain and promoting its transcriptional activity and secondly by upregulation of Nurr1 expression through the CREB signaling pathway. In contrast, CQ suppressed gene expression and differentiation of pathogenic TH17 cells. Importantly, using a valid animal model of inflammatory bowel disease (IBD), we demonstrated that CQ promotes Foxp3 expression and differentiation of TREG cells in a Nurr1-dependent manner, leading to significant improvement of IBD-related symptoms. Taken together, these data suggest that CQ ameliorates autoimmune diseases via regulating Nurr1 function/expression and that Nurr1 is a promising target for developing effective therapeutics of human inflammatory autoimmune diseases.

Chemogenomic Profiling of Human and Microbial FK506-Binding Proteins.

FK506-binding proteins (FKBPs) are evolutionarily conserved proteins that display peptidyl-prolyl isomerase activities and act as coreceptors for immunosuppressants. Microbial macrophage-infectivity-potentiator (Mip)-type FKBPs can enhance infectivity. However, developing druglike ligands for FKBPs or Mips has proven difficult, and many FKBPs and Mips still lack biologically useful ligands. To explore the scope and potential of C5-substituted [4.3.1]-aza-bicyclic sulfonamides as a broadly applicable class of FKBP inhibitors, we developed a new synthesis method for the bicyclic core scaffold and used it to prepare an FKBP- and Mip-focused library. This allowed us to perform a systematic structure-activity-relationship analysis across key human FKBPs and microbial Mips, yielding highly improved inhibitors for all the FKBPs studied. A cocrystal structure confirmed the molecular-binding mode of the core structure and explained the affinity gained as a result of the preferred substituents. The best FKBP and Mip ligands showed promising antimalarial, antileginonellal, and antichlamydial properties in cellular models of infectivity, suggesting that substituted [4.3.1]-aza-bicyclic sulfonamides could be a novel class of anti-infectives.

Stimuli-responsive multifunctional glyconanoparticle platforms for targeted drug delivery and cancer cell imaging.

Targeted bioimaging or chemotherapeutic drug delivery to achieve the desired therapeutic effects while minimizing side effects has attracted considerable research attention and remains a clinical challenge. Presented herein is a multi-component delivery system based on carbohydrate-functionalized gold nanoparticles conjugated with a fluorophore or prodrug. The system leverages active targeting based on carbohydrate-lectin interactions and release of the payload by biological thiols. Cell-type specific delivery of the activatable fluorophore was examined by confocal imaging on HepG2 cells, and displays distinct selectivity towards HepG2 cells over HeLa and NIH3T3 cells. The system was further developed into a drug delivery vehicle with camptothecin (CPT) as a model drug. It was demonstrated that the complex exhibits similar cytotoxicity to that of free CPT towards HepG2 cells, and is significantly less cytotoxic to normal HDF and NIH3T3 cells, indicating excellent specificity. The delivery vehicle itself exhibits excellent biocompatibility and offers an attractive strategy for cell-type specific delivery depending on the carbohydrates conjugated in the system.

Assembling Mn:ZnSe quantum dots-siRNA nanoplexes for gene silencing in tumor cells.

In this work, we demonstrate the use of manganese doped zinc selenide QDs (Mn:ZnSe d-dots) for gene delivery in vitro. Specifically, the d-dots were prepared as nanoplexes for facilitating the intracellular delivery of small interfering RNA (siRNA) molecules to pancreatic cancer cells (Panc-1), thereby inducing sequence-specific silencing of oncogenic K-Ras mutations in pancreatic carcinoma. For nanoplex preparation, a layer-by-layer (LBL) assembling method was adopted to modify the d-dot surface with cationic polymer poly(allylamine hydrochloride) (PAH) or polyethylenimine (PEI) for generating positive surface potential for complexing with K-Ras siRNA molecules. Owing to the unique and stable PL properties of the d-dots, siRNA transfection and the subsequent intracellular release profile from the d-dot/polymer-siRNA nanoplexes were monitored by fluorescence imaging. Quantitative results from flow cytometry study suggested that a high gene transfection efficiency was achieved. The expression of the mutant K-Ras mRNA in Panc-1 cells was observed to be significantly suppressed upon transfecting them with the nanoplex formulation. More importantly, cell viability studies showed that the d-dot/PAH nanoplexes were biocompatible and non-toxic even at concentrations as high as 160 µg mL(-1). Furthermore, the amine-terminated surface could be further modified to obtain multiple bio-functions. Based on these results, we envision that the designed d-dot nanoplexes can be developed as a flexible nanoplatform for both fundamental and practical clinical research applications.

Structural transition in Bcl-xL and its potential association with mitochondrial calcium ion transport.

Bcl-2 family proteins are key regulators for cellular homeostasis in response to apoptotic stimuli. Bcl-xL, an antiapoptotic Bcl-2 family member, undergoes conformational transitions, which leads to two conformational states: the cytoplasmic and membrane-bound. Here we present the crystal and small-angle X-ray scattering (SAXS) structures of Bcl-xL treated with the mild detergent n-Octyl ß-D-Maltoside (OM). The detergent-treated Bcl-xL forms a dimer through three-dimensional domain swapping (3DDS) by swapping helices α6-α8 between two monomers. Unlike Bax, a proapoptotic member of the Bcl-2 family, Bcl-xL is not converted to 3DDS homodimer upon binding BH3 peptides and ABT-737, a BH3 mimetic drug. We also designed Bcl-xL mutants which cannot dimerize and show that these mutants reduced mitochondrial calcium uptake in MEF cells. This illustrates the structural plasticity in Bcl-xL providing hints toward the probable molecular mechanism for Bcl-xL to play a regulatory role in mitochondrial calcium ion transport.

Biodegradable nanoparticle-mediated K-ras down regulation for pancreatic cancer gene therapy.

RNA interference (RNAi) targeting the K-ras oncogene mutation in pancreatic cancer mediated by small interfering RNA (siRNA) transfection is a very promising treatment. However, the rapid degradation and negative charge of naked siRNAs restrict their direct delivery into cells. In this contribution, we propose a safe and effective transmembrane transport nanocarrier formulation based on a newly developed biodegradable charged polyester-based vector (BCPV) for K-ras siRNA delivery into pancreatic cancer cells. Our results have shown that these biodegradable and biocompatible vectors are able to transfect siRNAs targeting mutant K-ras into MiaPaCa-2 cells with high transfection and knockdown efficiency. More importantly, the RNAi process initiated a cascade gene regulation of the downstream proteins of K-ras associated with cell proliferation, migration, invasion and apoptosis. We observed that after the mutant K-ras siRNA transfection, the growth, migration and invasion of the MiaPaCa-2 cells were significantly reduced; also, the apoptosis of the pancreatic cancer cells was promoted. Although in vivo testing data are limited, we propose that the BCPV based nanoparticle formulation could be a promising candidate as non-viral vectors for gene therapy in clinical settings.

Interleukin-8 gene silencing on pancreatic cancer cells using biodegradable polymer nanoplexes.

Pancreatic cancer is one of the deadliest cancers throughout the world with rarely efficient therapies currently available. Gene therapy on pancreatic cancer through small interfering RNA (siRNA)-based RNA interference (RNAi) has shown great potential and attracted much attention. However, due to the fragile nature of nucleic acid, the application of RNAi as a safe and efficient carrier faces great challenges. In this contribution, a self-assembly regime, which is based on well-defined cationic polylactides (CPLAs) with tertiary amine groups, has been used to encapsulate and protect siRNAs from fast degradation. CPLA is a safe and degradable formulation that allowed us to deliver siRNAs targeting the proangiogenic chemokine interleukin-8 (IL-8) to pancreatic cancer cells for gene therapy. Stable IL-8 siRNA-CPLA nanoplexes were successfully formed by electrostatic force and high gene transfection efficiencies were shown on two pancreatic cancer cell lines. We did not observe any cytotoxicity from these CPLAs over a large concentration range via cell viability evaluations. More importantly, the silencing of IL-8 gene expression significantly attenuated the proliferation of pancreatic cancer cells. Our preliminary results support the future development of gene therapy that might provide an effective and safe treatment approach towards pancreatic cancer.