Probing the Conformational Space of the Cannabinoid Receptor 2 and a Systematic Investigation of DNP-Enhanced MAS NMR Spectroscopy of Proteins in Detergent Micelles.

Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCR) and their complexes in recent years. However, understanding activation and signaling in GPCRs is still challenging due to the role of protein dynamics in these processes. Here, we show how dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance in combination with a unique pair labeling approach can be used to study the conformational ensemble at specific sites of the cannabinoid receptor 2. To improve the signal-to-noise, we carefully optimized the DNP sample conditions and utilized the recently introduced AsymPol-POK as a polarizing agent. We could show qualitatively that the conformational space available to the protein backbone is different in different parts of the receptor and that a site in TM7 is sensitive to the nature of the ligand, whereas a site in ICL3 always showed large conformational freedom.

Observation of Coherent Spin Waves in a Three-Dimensional Artificial Spin Ice Structure.

Harnessing high-frequency spin dynamics in three-dimensional (3D) nanostructures may lead to paradigm-shifting, next-generation devices including high density spintronics and neuromorphic systems. Despite remarkable progress in fabrication, the measurement and interpretation of spin dynamics in complex 3D structures remain exceptionally challenging. Here, we take a first step and measure coherent spin waves within a 3D artificial spin ice (ASI) structure using Brillouin light scattering. The 3D-ASI was fabricated by using a combination of two-photon lithography and thermal evaporation. Two spin-wave modes were observed in the experiment whose frequencies showed nearly monotonic variation with the applied field strength. Numerical simulations qualitatively reproduced the observed modes. The simulated mode profiles revealed the collective nature of the modes extending throughout the complex network of nanowires while showing spatial quantization with varying mode quantization numbers. The study shows a well-defined means to explore high-frequency spin dynamics in complex 3D spintronic and magnonic structures.

Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture.

Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB2 receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E. coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB2. Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.

Harnessing Multi-Photon Absorption to Produce Three-Dimensional Magnetic Structures at the Nanoscale.

Three-dimensional nanostructured magnetic materials have recently been the topic of intense interest since they provide access to a host of new physical phenomena. Examples include new spin textures that exhibit topological protection, magnetochiral effects and novel ultrafast magnetic phenomena such as the spin-Cherenkov effect. Two-photon lithography is a powerful methodology that is capable of realising 3D polymer nanostructures on the scale of 100 nm. Combining this with postprocessing and deposition methodologies allows 3D magnetic nanostructures of arbitrary geometry to be produced. In this article, the physics of two-photon lithography is first detailed, before reviewing the studies to date that have exploited this fabrication route. The article then moves on to consider how non-linear optical techniques and post-processing solutions can be used to realise structures with a feature size below 100 nm, before comparing two-photon lithography with other direct write methodologies and providing a discussion on future developments.

Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase.

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.

Induction of RNAi Responses by Short Left-Handed Hairpin RNAi Triggers.

Small double-stranded, left-handed hairpin (LHP) RNAs containing a 5'-guide-loop-passenger-3' structure induce RNAi responses by a poorly understood mechanism. To explore LHPs, we synthesized fully 2'-modified LHP RNAs targeting multiple genes and found all to induce robust RNAi responses. Deletion of the loop and nucleotides at the 5'-end of the equivalent passenger strand resulted in a smaller LHP that still induced strong RNAi responses. Surprisingly, progressive deletion of up to 10 nucleotides from the 3'-end of the guide strand resulted in a 32mer LHP capable of inducing robust RNAi responses. However, further guide strand deletion inhibited LHP activity, thereby defining the minimal length guide targeting length to 13 nucleotides. To dissect LHP processing, we examined LHP species that coimmunoprecipitated with Argonaute 2 (Ago2), the catalytic core of RNA-induced silencing complex, and found that the Ago2-associated processed LHP species was of a length that correlated with Ago2 cleavage of the passenger strand. Placement of a blocking 2'-OMe blocking modification at the LHP predicted Ago2 cleavage site resulted in an intact LHP loaded into Ago2 and no RNAi response. Taken together, these data argue that in the absence of a substantial loop, this novel class of small LHP RNAs enters the RNAi pathway by a Dicer-independent mechanism that involves Ago2 cleavage and results in an extended guide strand. This work establishes LHPs as an alternative RNAi trigger that can be produced from a single synthesis for potential use as an RNAi therapeutic.

Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications.

RNA interference (RNAi) has great potential to treat human disease. However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics.

Protein transduction domain delivery of therapeutic macromolecules.

Owing to their unprecedented selectivity, specific activity and potential for 1000+ fold amplification of signal, macromolecules, such as peptides, catalytic protein domains, complete proteins, and oligonucleotides, offer great potential as therapeutic molecules. However, therapeutic use of macromolecules is limited by their poor penetration in tissues and their inability to cross the cellular membrane. The discovery of small cationic peptides that cross the membrane, called Protein Transduction Domains (PTDs) or Cell Penetrating Peptides (CPPs), in the late 1980s opened the door to cellular delivery of large, bioactive molecules. Now, PTDs are widely used as research tools, and impressively, multiple clinical trials are testing PTD-mediated delivery of macromolecular drug conjugates in patients with a variety of diseases.

Epithelial p38alpha controls immune cell recruitment in the colonic mucosa.

Intestinal epithelial cells (IECs) compose the first barrier against microorganisms in the gastrointestinal tract. Although the NF-kappaB pathway in IECs was recently shown to be essential for epithelial integrity and intestinal immune homeostasis, the roles of other inflammatory signaling pathways in immune responses in IECs are still largely unknown. Here we show that p38alpha in IECs is critical for chemokine expression, subsequent immune cell recruitment into the intestinal mucosa, and clearance of the infected pathogen. Mice with p38alpha deletion in IECs suffer from a sustained bacterial burden after inoculation with Citrobacter rodentium. These animals are normal in epithelial integrity and immune cell function, but fail to recruit CD4(+) T cells into colonic mucosal lesions. The expression of chemokines in IECs is impaired, which appears to be responsible for the impaired T cell recruitment. Thus, p38alpha in IECs contributes to the host immune responses against enteric bacteria by the recruitment of immune cells.

Arginine deficiency augments inflammatory mediator production by airway epithelial cells in vitro.

BACKGROUND: Previously we showed that reduced availability of the essential amino acid tryptophan per se attenuates post-transcriptional control of interleukin (IL)-6 and IL-8 leading to hyperresponsive production of these inflammatory mediators by airway epithelial cells. Availability of the non-essential amino acid arginine in the inflamed airway mucosa of patients with asthma is reduced markedly, but it is not known whether this can also lead to an exaggerated production of IL-6 and IL-8. METHODS: IL-6 and IL-8 were determined by ELISA in culture supernatants of NCI-H292 airway epithelial-like cells and normal bronchial epithelial (NHBE) cells that were exposed to TNF-alpha, LPS or no stimulus, in medium with or without arginine. Arginine deficiency may also result from exposure to poly-L-arginine or major basic protein (MBP), which can block arginine uptake. Epithelial cells were exposed to these polycationic proteins and L-(14)C-arginine uptake was assessed as well as IL-6 and IL-8 production. To determine the mode of action, IL-6 and IL-8 mRNA profiles over time were assessed as were gene transcription and post-transcriptional mRNA degradation. RESULTS: For both NCI-H292 and NHBE cells, low arginine concentrations enhanced basal epithelial IL-6 and IL-8 production and synergized with TNF-alpha-induced IL-6 and IL-8 production. Poly-L-arginine enhanced the stimulus-induced IL-6 and IL-8 production, however, blocking arginine uptake and the enhanced IL-6 and IL-8 production appeared unrelated. The exaggerated IL-6 and IL-8 production due to arginine deficiency and to poly-L-arginine depend on a post-transcriptional and a transcriptional process, respectively. CONCLUSION: We conclude that both reduced arginine availability per se and the presence of polycationic proteins may promote airway inflammation by enhanced pro-inflammatory mediator production in airway epithelial cells, but due to distinct mechanisms.

RISC-target interaction: cleavage and translational suppression.

Small RNA molecules have been known and utilized to suppress gene expression for more than a decade. The discovery that these small RNA molecules are endogenously expressed in many organisms and have a critical role in controlling gene expression has led to the arising of a whole new field of research. Termed small interfering RNA (siRNA) or microRNA (miRNA) these approximately 22 nt RNA molecules have the capability to suppress gene expression through various mechanisms once they are incorporated in the multi-protein RNA-Induced Silencing Complex (RISC) and interact with their target mRNA. This review introduces siRNAs and microRNAs in a historical perspective and focuses on the key molecules in RISC, structural properties and mechanisms underlying the process of small RNA regulated post-transcriptional suppression of gene expression.

TNF-alpha stimulation inhibits siRNA-mediated RNA interference through a mechanism involving poly-(A) tail stabilization.

The control of mRNA stability is a complex biological process that involves numerous factors, including microRNA (miRNA) and short interfering RNA (siRNA). Here, we show that short interfering RNA (siRNA) and microRNA share some similarities in their response to cellular stress. miR16 expedites the degradation of mRNAs containing AU-rich elements (ARE) in their 3' untranslated region (UTR). si20 is an siRNA designed to target a non-ARE sequence in the TNF 3'UTR. We found that both si20 and miR16/ARE-mediated degradation of mRNAs can be inhibited by stimulating cells with different stresses. By analyzing TNF-alpha stimulation-mediated stabilization of si20- and miR16-targeted mRNA, we show that this stabilization is not caused by modifying si20 and miR16 loading into Ago2 complexes, or mRNA targeting to Ago2, but by inhibiting mRNA deadenylation. This is the first report showing that a specific siRNA-mediated mRNA degradation can be regulated by inflammatory stimuli, and that deadenylation is involved in this siRNA-mediated mRNA decay.

Cytoskeletal architecture differentially controls post-transcriptional processing of IL-6 and IL-8 mRNA in airway epithelial-like cells.

Airway epithelial cells are critically dependent on an intact cytoskeleton for innate defense functions. There are various pathophysiological conditions that affect the cytoskeletal architecture. We studied the effect of cytoskeletal distortion in polarized airway epithelial-like NCI-H292 cells on inflammatory gene expression, exemplified by interleukin(IL)-6 and IL-8. Disruption of microtubule structure with vinblastin and of actin with cytochalasin D did not affect TNF-alpha-induced IL-6 and IL-8 gene transcription but stabilized IL-8 and IL-6 mRNA. In line with previous studies, IL-8 mRNA stabilization was paralleled by hyperresponsive IL-8 production, but surprisingly, IL-6 production was reduced despite IL-6 mRNA stabilization. Polysome profiling revealed that, in cells with a disrupted cytoskeleton, translational efficiency of IL-6 mRNA was reduced, whereas that of IL-8 mRNA remained unaffected. Our findings indicate that distortion of the cytoskeleton in airway epithelial cells differentially affects both degradation and translation of IL-6 and IL-8 mRNA, modifying inflammatory gene expression and thus their innate defense function.

E1A expression dysregulates IL-8 production and suppresses IL-6 production by lung epithelial cells.

BACKGROUND: The adenoviral protein E1A has been proposed to play a role in the pathophysiology of COPD, in particular by increasing IL-8 gene transcription of lung epithelial cells in response to cigarette smoke-constituents such as LPS. As IL-8 production is also under tight post-transcriptional control, we planned to study whether E1A affected IL-8 production post-transcriptionally. The production of IL-6 by E1A-positive cells had not been addressed and was studied in parallel. Based on our previous work into the regulation of IL-8 and IL-6 production in airway epithelial cells, we used the lung epithelial-like cell line NCI-H292 to generate stable transfectants expressing either E1A and/or E1B, which is known to frequently co-integrate with E1A. We analyzed IL-8 and IL-6 production and the underlying regulatory processes in response to LPS and TNF-alpha. METHODS: Stable transfectants were generated and characterized with immunohistochemistry, western blot and flow cytometry. IL-8 and IL-6 protein production was measured by ELISA. Levels of IL-8 and IL-6 mRNA were measured using specific radiolabeled probes. EMSA was used to assess transcriptional activation of relevant transcription factors. Post-transcriptional regulation of mRNA half-life was measured by Actinomycin D chase experiments. RESULTS: Most of the sixteen E1A-expressing transfectants showed suppression of IL-6 production, indicative of biologically active E1A. Significant but no uniform effects on IL-8 production, nor on transcriptional and post-transcriptional regulation of IL-8 production, were observed in the panel of E1A-expressing transfectants. E1B expression exerted similar effects as E1A on IL-8 production. CONCLUSION: Our results indicate that integration of adenoviral DNA and expression of E1A and E1B can either increase or decrease IL-8 production. Furthermore, we conclude that expression of E1A suppresses IL-6 production. These findings question the unique role of E1A protein in the pathophysiology of COPD, but do not exclude a role for adenoviral E1A/E1B DNA in modulating inflammatory responses nor in the pathogenesis of COPD.

Interleukin-17 induces hyperresponsive interleukin-8 and interleukin-6 production to tumor necrosis factor-alpha in structural lung cells.

Lung epithelial cells contribute to local inflammation by the production of pro-inflammatory mediators like interleukin (IL)-8 and IL-6. Although their production depends on gene transcription, previous studies showed that post-transcriptional mechanisms modulate IL-8 and IL-6 production. Human lung epithelial cells turn from normoresponsive into hyperresponsive IL-8- and IL-6-producing cells when their IL-8 and IL-6 mRNA degradation is reduced. We hypothesized that IL-17, a mediator predominantly released by memory T cells and present in airways of individuals with asthma, would modulate rather than induce IL-8 and IL-6 production by both human lung epithelial cells and fibroblasts. We show here for both cell types that IL-17 was a weak stimulus of IL-8 and IL-6 production, but markedly enhanced IL-8 and IL-6 responses to another stimulus, such as tumor necrosis factor-alpha. This modulatory effect of IL-17 was paralleled by a reduced IL-8 and IL-6 mRNA degradation, with no effect on IL-8 and IL-6 gene transcription. In conclusion, IL-17 particularly affects post-transcriptional regulation of IL-8 and IL-6 expression leading to enhanced IL-8 and IL-6 responses to secondary stimuli, and is only a weak proinflammatory stimulus by itself. This poses the interesting concept that by releasing IL-17 from memory T cells, the adaptive immune system instructs lung structural cells as part of the innate immune system to respond more vigorously.

Exaggerated IL-8 and IL-6 responses to TNF-alpha by parainfluenza virus type 4-infected NCI-H292 cells.

Respiratory viruses induce and potentiate airway inflammation, which is related to the induction of proinflammatory mediators such as interleukin (IL)-8 and IL-6. Here we report on mechanisms implicated in IL-8 and IL-6 production by airway epithelium-like NCI-H292 cells exposed to parainfluenza virus type 4a (PIV-4). PIV-4 readily infected NCI-H292 cells as reflected by intracellular PIV-4 antigen expression. PIV-4 infection triggered a biphasic IL-8 and IL-6 mRNA response. Transient transfection with truncated and mutated promoter constructs identified NF-kappaB and activator protein (AP)-1, and CCAAT-enhancer binding protein (C/EBP) as the relevant transcription factors for PIV-4-induced IL-8 and IL-6 gene transcription, respectively. An increase of DNA-binding activities for NF-kappaB and C/EBP paralleled the induction of the first and second IL-8 and IL-6 mRNA peaks, whereas the onset of AP-1 paralleled the first IL-8 mRNA peak only. The second mRNA peak, apparently dependent on viral replication, coincided also with a marked reduction of IL-8 and IL-6 mRNA degradation. Importantly, cells at the time of the reduced mRNA degradation displayed an exaggerated IL-8 and IL-6 protein production to a secondary stimulus, as exemplified by steeper dose-response curves to TNF-alpha. Thus PIV-4 infection enhances epithelial IL-8 and IL-6 production by transcriptional and posttranscriptional mechanisms. The previously unrecognized phase of reduced IL-8 and IL-6 mRNA degradation and the concurrent amplified epithelial IL-8 and IL-6 responses may play an important role in virus-induced potentiation of airway inflammation.

Ingrowth of aorta wall into stent grafts impregnated with basic fibroblast growth factor: a porcine in vivo study of blood vessel prosthesis healing.

OBJECTIVE: Endovascular aneurysm repair is an alternative treatment of abdominal aortic aneurysm. The procedure is less invasive, and morbidity and most probably mortality are reduced. However, some problems, such as endoleakage, are yet to be resolved. Endoleakage can occur after graft migration, as a result of insufficient fixation of the stent graft. One cause is deficient healing between the aortic neck and the stent graft. We hypothesize that better healing, achieved by induction of vascular cell ingrowth into the graft material, results in better graft fixation. Previously we demonstrated ingrowth of neointima into the graft material if the stent graft is impregnated with a coat of basic fibroblast growth factor (bFGF), heparin, and collagen. In this study we evaluated healing with bFGF-heparin-collagen-coated stent grafts in vivo. METHODS: In 4 pigs, 32 endovascular stent grafts, manufactured from standard Dacron and Gianturco Z-stents, were placed in the aorta. The stent grafts were impregnated with either bFGF-heparin containing collagen (n=16) or control collagen (n=16). After 4 and 8 weeks animals were killed, and ingrowth and healing of the stent grafts were macroscopically and electron microscopically evaluated. RESULTS: After 8 weeks all bFGF-impregnated stent grafts demonstrated ingrowth of tissue and healing between the graft and the aorta, whereas the control nonimpregnated stent grafts showed no ingrowth. Microscopic evaluation demonstrated alpha-smooth muscle actin-positive cells, most probably smooth muscle cells or myofibroblasts, growing from the vascular wall through the graft material. CONCLUSION: A Dacron prosthesis impregnated with collagen, heparin, and bFGF induced graft healing in an in vivo pig model, in contrast to nonimpregnated stent grafts. This in vivo study confirms our previous findings in vitro. These results indicate that healing between Dacron and the aorta can be achieved, and suggest that type I endoleakage may be resolved by inducing healing between the aortic wall and the prosthesis with graft material containing growth factor.

Ingrowth of aorta vascular cells into basic fibroblast growth factor-impregnated vascular prosthesis material: a porcine and human in vitro study on blood vessel prosthesis healing.

OBJECTIVE: One of the most life-threatening vascular diseases is rupture of an abdominal aneurysm. The conventional treatment is based on surgical reconstruction. An alternative treatment is endovascular aneurysm repair (EVAR). Despite many advantages, one of the problems of EVAR is endoleakage from deficient healing between the aortic neck and the fabric of the endograft. We hypothesize that better healing, achieved with induction of vascular cell ingrowth into the graft material, would lead to better graft healing. METHODS: Both pig aorta and human normal and aneurysmal aortic wall were used for organ cultures. Various growth factors were evaluated for the potential to induce intimal hyperplasia (ie, platelet-derived growth factor, vascular endothelial growth factor, and basic fibroblast growth factor [bFGF]). After the most potent growth factor had been selected, a vascular prosthetic material (Dacron fabric) impregnated with collagen and heparin was incubated with this growth factor. Impregnated pieces of Dacron were fixated on top of the aortic organ cultures for study of ingrowth of the neointima formation into the graft material. RESULTS: bFGF was the most potent growth factor to induce neointima in aortic organ cultures. The pieces of impregnated Dacron had a release of 5 ng/24 h of bFGF for a period of at least 28 days. With fixation on top of the aortic organ cultures, the impregnated Dacron was capable of inducing neointima formation and ingrowth of the neointima into the graft material after 28 days. CONCLUSION: We showed that a Dacron prosthesis impregnated with collagen, heparin, and bFGF is capable of inducing graft healing in our in vitro model, the aortic organ cultures of pig and human aortas. These results suggest that the problem of endoleakage with EVAR may be solved with a perfect proximal healing between the aortic wall and the prosthesis.