The effects of L-DOPA on gait abnormalities in a unilateral 6-OHDA rat model of Parkinson's disease.

Parkinson's Disease (PD) is a neurodegenerative movement disorder characterized by dopamine (DA) cell loss in the substantia nigra pars compacta (SNc). As PD progresses, patients display disruptions in gait such as changes in posture, bradykinesia, and shortened stride. DA replacement via L-DOPA alleviates many PD symptoms, though its effects on gait are not well demonstrated. This study aimed to assess the relationship between DA lesion, gait, and deficit-induced reversal with L-DOPA. To do so, Sprague-Dawley rats (N = 25, 14 males, 11 females) received unilateral medial forebrain bundle (MFB) DA lesions with 6-hydroxydopamine (6-OHDA). An automated gait analysis system assessed spatiotemporal gait parameters pre- and post-lesion, and after various doses of L-DOPA (0, 3, or 6 mg/kg; s.c.). The forepaw adjusting steps (FAS) test was implemented to evaluate lesion efficacy while the abnormal involuntary movements (AIMs) scale monitored the emergence of L-DOPA-induced dyskinesia (LID). High performance liquid chromatography (HPLC) assessed changes in brain monoamines on account of lesion and treatment. Results revealed lesion-induced impairments in gait, inclusive of max-contact area and step-sequence alterations that were not reversible with L-DOPA. However, the emergence of AIMs were observed at higher doses. Post-mortem, 6-OHDA lesions induced a loss of striatal DA and norepinephrine (NE), while prefrontal cortex (PFC) displayed noticeable reduction in NE but not DA. Our findings indicate that hemiparkinsonian rats display measurable gait disturbances similar to PD patients that are not rescued by DA replacement. Furthermore, non-DA mechanisms such as attention-related NE in PFC may contribute to altered gait and may constitute a novel target for its treatment.

Deficits in basal and evoked striatal dopamine release following alpha-synuclein preformed fibril injection: An in vivo microdialysis study.

Parkinson's disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (α-syn) protein, forming intraneuronal Lewy body (LB) inclusions. The α-syn preformed fibril (PFF) model of PD recapitulates α-syn aggregation, progressive nigrostriatal degeneration and motor dysfunction; however, little is known about the time course of PFF-induced alterations in basal and evoked dopamine (DA). In vivo microdialysis is well suited for identifying small changes in neurotransmitter levels over extended periods. In the present study, adult male Fischer 344 rats received unilateral, intrastriatal injections of either α-syn PFFs or phosphate-buffered saline (PBS). At 4 or 8 months post-injection (p.i.), animals underwent in vivo microdialysis to evaluate basal extracellular striatal DA and metabolite levels, local KCl-evoked striatal DA release and the effects of systemic levodopa (l-DOPA). Post-mortem analysis demonstrated equivalent PFF-induced reductions in tyrosine hydroxylase (TH) immunoreactive nigral neurons (~50%) and striatal TH (~20%) at both time points. Compared with reduction in striatal TH, reduction in striatal dopamine transporter (DAT) was more pronounced and progressed between the 4- and 8-month p.i. intervals (36% âž” 46%). Significant PFF-induced deficits in basal and evoked striatal DA, as well as deficits in motor performance, were not observed until 8 months p.i. Responses to l-DOPA did not differ regardless of PBS or PFF treatment. These results suggest that basal and evoked striatal DA are maintained for several months following PFF injection, with loss of both associated with motor dysfunction. Our studies provide insight into the time course and magnitude of PFF-induced extracellular dopaminergic deficits in the striatum.

Broad Serotonergic Actions of Vortioxetine as a Promising Avenue for the Treatment of L-DOPA-Induced Dyskinesia.

Parkinson's Disease (PD) is a neurodegenerative disorder characterized by motor symptoms that result from loss of nigrostriatal dopamine (DA) cells. While L-DOPA provides symptom alleviation, its chronic use often results in the development of L-DOPA-induced dyskinesia (LID). Evidence suggests that neuroplasticity within the serotonin (5-HT) system contributes to LID onset, persistence, and severity. This has been supported by research showing 5-HT compounds targeting 5-HT1A/1B receptors and/or the 5-HT transporter (SERT) can reduce LID. Recently, vortioxetine, a multimodal 5-HT compound developed for depression, demonstrated acute anti-dyskinetic effects. However, the durability and underlying pharmacology of vortioxetine's anti-dyskinetic actions have yet to be delineated. To address these gaps, we used hemiparkinsonian rats in Experiment 1, examining the effects of sub-chronic vortioxetine on established LID and motor performance. In Experiment 2, we applied the 5-HT1A antagonist WAY-100635 or 5-HT1B antagonist SB-224289 in conjunction with L-DOPA and vortioxetine to determine the contributions of each receptor to vortioxetine's effects. The results revealed that vortioxetine consistently and dose-dependently attenuated LID while independently, 5-HT1A and 5-HT1B receptors each partially reversed vortioxetine's effects. Such findings further support the promise of pharmacological strategies, such as vortioxetine, and indicate that broad 5-HT actions may provide durable responses without significant side effects.

Pharmacological targeting of G protein-coupled receptor heteromers.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

The multimodal serotonin compound Vilazodone alone, but not combined with the glutamate antagonist Amantadine, reduces l-DOPA-induced dyskinesia in hemiparkinsonian rats.

Parkinson's disease (PD) is a progressive, neurodegenerative movement disorder caused by loss of nigrostriatal dopamine (DA) neurons. DA replacement therapy using L-3,4-dihydroxyphenylalanine (l-DOPA) improves motor function but often results in l-DOPA-induced dyskinesia (LID) typified by abnormal involuntary movements (AIMs). In states of DA depletion, striatal serotonin (5-HT) hyperinnervation and glutamate overactivity are implicated in LID. To target these co-mechanisms, this study investigated the potential anti-dyskinetic effects of FDA-approved Vilazodone (VZD), a 5-HT transport blocker and partial 5-HT1A agonist, and Amantadine (AMAT), a purported NMDA glutamate antagonist, in 6-hydroxydopamine-lesioned hemiparkinsonian Sprague-Dawley rats. Dose-response curves for each drug against l-DOPA-induced AIMs were determined to identify effective threshold doses. A second cohort of rats was tested using the threshold doses of VZD (1, 2.5 mg/kg, s.c.) and/or AMAT (40 mg/kg, s.c.) to examine their combined, acute effects on LID. In a third cohort, VZD and/or AMAT were administered daily with l-DOPA for 14 days to determine prophylactic effects on LID development. In a final cohort, rats with established LID received VZD and/or AMAT injections for 2 weeks to examine their interventional properties. Throughout experiments, AIMs were rated for dyskinesia severity and forepaw adjusting steps (FAS) were monitored l-DOPA motor efficacy. Results revealed that acute and chronic VZD + l-DOPA treatment significantly decreased AIMs and maintained FAS compared to l-DOPA alone. AMAT + l-DOPA co-administration did not exert any significant effects on AIMs or FAS, while the co-administration of VZD and AMAT with l-DOPA demonstrated intermediate effects. These results suggest that co-administration of low-dose VZD and AMAT with l-DOPA does not synergistically reduce LID in hemiparkinsonian rats. Importantly, low doses of VZD (2.5, 5 mg/kg) did reduce the development and expression of LID while maintaining l-DOPA efficacy, supporting its potential therapeutic utility for PD patients.

The effects of Vilazodone, YL-0919 and Vortioxetine in hemiparkinsonian rats.

Parkinson's disease is a neurodegenerative disease often characterized by motor deficits and most commonly treated with dopamine replacement therapy. Despite its benefits, chronic use of L-DOPA results in abnormal involuntary movements known as L-DOPA-induced dyskinesia. Growing evidence shows that with burgeoning dopamine cell loss, neuroplasticity in the serotonin system leads to the development of L-DOPA-induced dyskinesia through the unregulated uptake, conversion, and release of L-DOPA-derived dopamine into the striatum. Previous studies have shown that coincident 5-HT1A agonism and serotonin transporter inhibition may have anti-dyskinetic potential. Despite this, few studies have explicitly focused on targeting both 5-HT1A and the serotonin transporter. The present study compares the 5-HT compounds Vilazodone, YL-0919, and Vortioxetine which purportedly work as simultaneous 5-HT1A receptor agonists and SERT blockers. To do so, adult female Sprague Dawley rats were rendered hemiparkinsonian and treated daily for two weeks with L-DOPA to produce stable dyskinesia. The abnormal involuntary movements and forehand adjusting step tests were utilized as measurements for L-DOPA-induced dyskinesia and motor performance in a within-subjects design. Lesion efficacy was determined by analysis of striatal monoamines via high-performance liquid chromatography. Compounds selective for 5-HT1A/SERT target sites including Vilazodone and Vortioxetine significantly reduced L-DOPA-induced dyskinesia without compromising L-DOPA pro-motor efficacy. In contrast, YL-0919 failed to reduce L-DOPA-induced dyskinesia, with no effects on L-DOPA-related improvements. Collectively, this work supports pharmacological targeting of 5-HT1A/SERT to reduce L-DOPA-induced dyskinesia. Additionally, this further provides evidence for Vilazodone and Vortioxetine, FDA-approved compounds, as potential adjunct therapeutics for L-DOPA-induced dyskinesia management in Parkinson's patients.

Nedocromil sodium and diphenhydramine HCl ameliorate exercise-induced arterial hypoxemia in highly trained athletes.

INTRODUCTION: Exercise-induced arterial hypoxemia (EIAH) has been observed in highly trained endurance athletes during near maximal exercise, which may be influenced by a histamine-mediated inflammatory response at the pulmonary capillary-alveolar membrane. In order to test this hypothesis, we examined whether the mast cell stabilizer nedocromil sodium (NS) and H1 -receptor antagonist diphenhydramine HCL (DH) would ameliorate EIAH and mitigate the drop in arterial oxyhemoglobin saturation (Sa O2 ) during intensive exercise. METHODS: Seven highly trained male cross country runners (age, 21 ± 2 years; V̇O2max , 74.7 ± 3.5 ml·kg-1 ·min-1 ) participated in the study. All subjects completed a maximal exercise treadmill test to exhaustion, followed by three 5-min constant-load exercise bouts at 70%, 80%, and 90% V̇O2max . Prior to testing, subjects received either placebo (PL), NS, or DH. RESULTS: Compared to PL, there was a significant treatment effect on Sa O2 (p < 0.001) for both NS and DH during both constant-load exercise and at V̇O2max . Post hoc tests revealed Sa O2  values, compared to PL, were significantly higher at V̇O2max and during DH trials and higher with NS at constant-load intensities except at 70% (p = 0.13). CONCLUSION: The findings provide further evidence that histamine contributes directly or indirectly to the development of EIAH during intense exercise in highly trained athletes.

Combined Knockout of Lrrk2 and Rab29 Does Not Result in Behavioral Abnormalities in vivo.

BACKGROUND: Coding mutations in the LRRK2 gene, encoding for a large protein kinase, have been shown to cause familial Parkinson's disease (PD). The immediate biological consequence of LRRK2 mutations is to increase kinase activity, suggesting that inhibition of this enzyme might be useful therapeutically to slow disease progression. Genome-wide association studies have identified the chromosomal loci around LRRK2 and one of its proposed substrates, RAB29, as contributors towards the lifetime risk of sporadic PD. OBJECTIVE: Considering the evidence for interactions between LRRK2 and RAB29 on the genetic and protein levels, we set out to determine whether there are any consequences on brain function with aging after deletion of both genes. METHODS: We generated a double knockout mouse model and performed a battery of motor and non-motor behavioral tests. We then investigated postmortem assays to determine the presence of PD-like pathology, including nigral dopamine cell count, astrogliosis, microgliosis, and striatal monoamine content. RESULTS: Behaviorally, we noted only that 18-24-month Rab29-/- and double (Lrrk2-/-/Rab29-/-) knockout mice had diminished locomotor behavior in open field compared to wildtype mice. However, no genotype differences were seen in the outcomes that represented PD-like pathology. CONCLUSION: These results suggest that depletion of both LRRK2 and RAB29 is tolerated, at least in mice, and support that this pathway might be able to be safely targeted for therapeutics in humans.

Effects of pedunculopontine nucleus cholinergic lesion on gait and dyskinesia in hemiparkinsonian rats.

Pedunculopontine nucleus (PPN) cholinergic neurons are implicated in freezing of gait in Parkinson's disease (PD) and motor stereotypy in normal animals, but the causal role of these neurons on specific gait parameters and treatment-induced dyskinesia remains speculative. Therefore, we examined whether selective cholinergic lesion of the rostral PPN affects PD motor and gait deficits, L-DOPA-induced dyskinesia and motor improvement, and DA-agonist-induced dyskinesia. Sprague-Dawley rats were assigned to one unilaterally lesioned group: Sham lesion, PPN cholinergic lesion with diphtheria urotensin II fusion toxin, medial forebrain bundle dopamine lesion with 6-hydroxydopamine, or dual acetylcholine and dopamine lesion. We used gait analysis and forepaw adjusting steps to examine PD gait and motor deficits. Forepaw adjusting steps were also used to assess motor improvement with L-DOPA treatment. The abnormal involuntary movements scale measured L-DOPA and dopamine D1- and D2-receptor agonist-induced dyskinesia. Lesions, verified via tyrosine hydroxylase and choline acetyltransferase immunohistochemistry reduced an average of 95% of nigral dopamine neurons and 80% of PPN cholinergic neurons, respectively. Rats receiving acetylcholine and dual lesion demonstrated enhanced freezing, and acetylcholine lesioned rats exhibited increased print area and stand index. Dopamine and dual lesion produced similar forepaw adjusting steps task on and off L-DOPA. Relative to DA lesioned rats, dual lesioned rats displayed reduced L-DOPA and DA agonist-induced dyskinesia at specific time points. Our results indicate that PPN cholinergic neurons affect gait parameters related to postural stability. Therefore, therapeutically targeting PPN cholinergic neurons could reduce intractable postural instability in PD without affecting motor benefits or side effects of L-DOPA treatment.

Genetic suppression of the dopamine D3 receptor in striatal D1 cells reduces the development of L-DOPA-induced dyskinesia.

Parkinson's Disease (PD) is symptomatically managed with L-DOPA but chronic use results in L-DOPA-induced dyskinesia (LID) characterized by abnormal involuntary movements (AIMs). In LID, dopamine D3 receptors (D3R) are upregulated on D1 receptor (D1R)-bearing medium spiny neurons where the can synergistically drive downstream signaling and motor behaviors. Despite evidence implying D1R-D3R cooperativity in LID, the dyskinesiogenic role of D3R has never been directly tested. To this end, we developed a specific cre-dependent microRNA (miRNA) to irreversibly prevent D3R upregulation in D1R striatal cells. D1-Cre rats received unilateral 6-hydroxydopamine lesions. Three weeks later, rats received an adeno-associated virus expressing either D3R miRNA or a scrambled (SCR) miRNA delivered into the striatum. After 4 weeks, rats received chronic L-DOPA (6 mg/kg) or vehicle. AIMs development and motor behaviors were assayed throughout treatment. At the conclusion of the experiment, efficacy and fidelity of the miRNA strategy was analyzed using in situ hybridization (ISH). ISH analyses demonstrated that D1R+/D3R+ cells were upregulated in LID and that the selective D3R miRNA reduced D1R+/D3R+ co-expression. Importantly, silencing of D3R also significantly attenuated LID development without impacting L-DOPA efficacy or other locomotion. These data highlight a dyskinesiogenic role of D3R within D1R cells in LID and highlight aberrant D1R-D3R interactions as targets of LID management.

A Novel Fusion of IL-10 Engineered to Traffic across Intestinal Epithelium to Treat Colitis.

IL-10 is a potent anti-inflammatory cytokine capable of suppressing a number of proinflammatory signals associated with intestinal inflammatory diseases, such as ulcerative colitis and Crohn's disease. Clinical use of human IL-10 (hIL-10) has been limited by anemia and thrombocytopenia following systemic injection, side effects that might be eliminated by a gut-restricted distribution. We have identified a transcytosis pathway used by cholix, an exotoxin secreted by nonpandemic forms of the intestinal pathogen Vibrio cholerae A nontoxic fragment of the first 386 aa of cholix was genetically fused to hIL-10 to produce recombinant AMT-101. In vitro and in vivo characterization of AMT-101 showed it to efficiently cross healthy human intestinal epithelium (SMI-100) by a vesicular transcytosis process, activate hIL-10 receptors in an engineered U2OS osteosarcoma cell line, and increase cellular phospho-STAT3 levels in J774.2 mouse macrophage cells. AMT-101 was taken up by inflamed intestinal mucosa and activated pSTAT3 in the lamina propria with limited systemic distribution. AMT-101 administered to healthy mice by oral gavage or to cynomolgus monkeys (nonhuman primates) by colonic spray increased circulating levels of IL-1R antagonist (IL-1Ra). Oral gavage of AMT-101 in two mouse models of induced colitis prevented associated pathological events and plasma cytokine changes. Overall, these studies suggest that AMT-101 can efficiently overcome the epithelial barrier to focus biologically active IL-10 to the intestinal lamina propria.

Membrane Association Transforms an Inert Anti-TCRß Fab' Ligand into a Potent T Cell Receptor Agonist.

The natural peptide-major histocompatibility complex (pMHC) ligand for T cell receptors (TCRs) is inactive from solution yet capable of activating T cells at single-molecule levels when membrane-associated. This distinctive feature stems from the mechanism of TCR activation, which is thought to involve steric phosphatase exclusion as well as direct mechanical forces. It is possible to defeat this mechanism and activate T cells with solution ligands by cross-linking pMHC or using multivalent antibodies to TCR. However, these widely used strategies activate TCRs through a nonphysiological mechanism and can produce different activation profiles than natural, monovalent, membrane-associated pMHC. Here, we introduce a strictly monovalent anti-TCRß H57 Fab' ligand that, when coupled to a supported lipid bilayer via DNA complementation, triggers TCRs and activates nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) with a similar potency to pMHC in primary murine T cells. Importantly, like monovalent pMHC and unlike bivalent antibodies, monovalent Fab'-DNA triggers TCRs only when physically coupled to the membrane, and only around 100 individual Fab':TCR interactions are necessary to stimulate early T cell activation.

The evolving role of response-adapted PET imaging in Hodgkin lymphoma.

(18)F-fluorodeoxyglucose positron emission tomography with (FDG-PET) has a well-established role in the pre- and post-treatment staging of Hodgkin lymphoma (HL), however its use as a predictive therapeutic tool via responded-adapted therapy continues to evolve. There have been a multitude of retrospective and noncontrolled clinical studies showing that early (or interim) FDG-PET is highly prognostic in HL, particularly in the advanced-stage setting. Response-adapted treatment approaches in HL are attempting to diminish toxicity for low-risk patients by minimizing therapy, and conversely, intensify treatment for high-risk patients. Results from phase III noninferiority studies in early-stage HL with negative interim FDG-PET that randomized patients to chemotherapy alone versus combined modality therapy showed a continued small improvement in progression-free survival for patients who did not receive radiation. Preliminary reports of data escalating therapy for positive interim FDG-PET in early-stage HL and for de-escalation of therapy [i.e. bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone (BEACOPP)] for negative interim FDG-PET in advanced stage HL (i.e. deletion of bleomycin) have demonstrated improved outcomes. Maturation of these studies and continued follow up of all response-adapted studies are needed. Altogether, the treatment of HL remains an individualized clinical management choice for physicians and patients. Continued refinement and optimization of FDG-PET is needed, including within the context of targeted therapeutic agents. In addition, a number of new and novel techniques of functional imaging, including metabolic tumor volume and tumor proliferation, are being explored in order to enhance staging, characterization, prognostication and ultimately patient outcome.

Proteasomal Inhibition by Ixazomib Induces CHK1 and MYC-Dependent Cell Death in T-cell and Hodgkin Lymphoma.

Proteasome-regulated NF-κB has been shown to be important for cell survival in T-cell lymphoma and Hodgkin lymphoma models. Several new small-molecule proteasome inhibitors are under various stages of active preclinical and clinical development. We completed a comprehensive preclinical examination of the efficacy and associated biologic effects of a second-generation proteasome inhibitor, ixazomib, in T-cell lymphoma and Hodgkin lymphoma cells and in vivo SCID mouse models. We demonstrated that ixazomib induced potent cell death in all cell lines at clinically achievable concentrations. In addition, it significantly inhibited tumor growth and improved survival in T-cell lymphoma and Hodgkin lymphoma human lymphoma xenograft models. Through global transcriptome analyses, proteasomal inhibition showed conserved overlap in downregulation of cell cycle, chromatin modification, and DNA repair processes in ixazomib-sensitive lymphoma cells. The predicted activity for tumor suppressors and oncogenes, the impact on "hallmarks of cancer," and the analysis of key significant genes from global transcriptome analysis for ixazomib strongly favored tumor inhibition via downregulation of MYC and CHK1, its target genes. Furthermore, in ixazomib-treated lymphoma cells, we identified that CHK1 was involved in the regulation of MYC expression through chromatin modification involving histone H3 acetylation via chromatin immunoprecipitation. Finally, using pharmacologic and RNA silencing of CHK1 or the associated MYC-related mechanism, we demonstrated synergistic cell death in combination with antiproteasome therapy. Altogether, ixazomib significantly downregulates MYC and induces potent cell death in T-cell lymphoma and Hodgkin lymphoma, and we identified that combinatorial therapy with anti-CHK1 treatment represents a rational and novel therapeutic approach. Cancer Res; 76(11); 3319-31. ©2016 AACR.

Extension of in vivo half-life of biologically active molecules by XTEN protein polymers.

XTEN™ is a class of unstructured hydrophilic, biodegradable protein polymers designed to increase the half-lives of therapeutic peptides and proteins. XTEN polymers and XTEN fusion proteins are typically expressed in Escherichia coli and purified by conventional protein chromatography as monodisperse polypeptides of exact length and sequence. Unstructured XTEN polypeptides have hydrodynamic volumes significantly larger than typical globular proteins of similar mass, thus imparting a bulking effect to the therapeutic payloads attached to them. Since their invention, XTEN polypeptides have been utilized to extend the half-lives of a variety of peptide- and protein-based therapeutics. Multiple clinical and preclinical studies and related drug discovery and development efforts are in progress. This review details the most current understanding of physicochemical properties and biological behavior of XTEN and XTENylated molecules. Additionally, the development path and status of several advanced drug discovery and development efforts are highlighted.

Review of Offshore Wind Farm Impact Monitoring and Mitigation with Regard to Marine Mammals.

Monitoring and mitigation reports from 19 UK and 9 other European Union (EU) offshore wind farm (OWF) developments were reviewed, providing a synthesis of the evidence associated with the observed environmental impact on marine mammals. UK licensing conditions were largely concerned with mitigation measures reducing the risk of physical and auditory injury from pile driving. At the other EU sites, impact monitoring was conducted along with mitigation measures. Noise-mitigation measures were developed and tested in UK and German waters in German government-financed projects. We highlight some of the review's findings and lessons learned with regard to noise impact on marine mammals.

Lenalidomide in non-Hodgkin lymphoma: biological perspectives and therapeutic opportunities.

Lenalidomide is an immunomodulatory drug (IMiD) with activity in lymphoid malignancies occurring primarily through immune modulation (eg, T-cell immune synapse enhancement and NK-cell/T-cell effector augmentation) and antiproliferative effects. Food and Drug Administration-approved for bortezomib-resistant, relapsed/refractory mantle-cell lymphoma, lenalidomide has demonstrated efficacy in several additional lymphoma subtypes. There are many ongoing clinical trials examining the use of lenalidomide alone or in combinatorial therapy. It will be important in these studies to delineate reliable, predictive biomarkers to optimally integrate lenalidomide into lymphoma treatment paradigms.

DNA-mediated assembly of protein heterodimers on membrane surfaces.

We present a method based on self-assembling oligonucleotides to anchor proteins to a supported membrane surface. This anchoring method allows control of the surface density of multiple proteins. By incorporating additional recognition sequences into the DNA linkers, defined heterodimers can be produced upon the addition of a heterospecific DNA cross-linking strand. Characterization by fluorescence cross-correlation spectroscopy (FCCS) confirmed lateral mobility and the formation of specific heterodimers. We further demonstrate that proteins linked in this manner as either monomers or dimers can form functional interfaces with living cells.

Controlled integration of gold nanoparticles and organic fluorophores using synthetically modified MS2 viral capsids.

The placement of fluorophores in close proximity to metal nanoparticle surfaces is proposed to enhance several photophysical properties of the dyes, potentially leading to improved quantum yields and decreased photobleaching. It is difficult in practice, however, to establish and maintain the nanoscale distances that are required to maximize these effects. The type of metal, size, and shape of the nanoparticle, the physical distance separating the metal nanoparticle from the organic dye, and the spectral properties of the fluorophore itself are all proposed to influence the quantum yield and lifetime. This results in a complex behavior that can lead to either enhanced or quenched fluorescence in different contexts. In this report, we describe a well-defined system that can be used to explore these effects, while physically preventing the fluorophores from contacting the nanoparticle surfaces. The basis of this system is the spherical protein capsid of bacteriophage MS2, which was used to house gold particles within its interior volume. The exterior surface of each capsid was then modified with Alexa Fluor 488 (AF 488) labeled DNA strands. By placing AF 488 dyes at distances of 3, 12, and 24 bp from the surface of capsids containing 10 nm gold nanoparticles, fluorescence intensity enhancements of 2.2, 1.2, and 1.0 were observed, respectively. A corresponding decrease in fluorescence lifetime was observed for each distance. Because of its well-defined and modular nature, this architecture allows the rapid exploration of the many variables involved in metal-controlled fluorescence, leading to a better understanding of this phenomenon.

Supported membranes embedded with fixed arrays of gold nanoparticles.

We present a supported membrane platform consisting of a fluid lipid bilayer membrane embedded with a fixed array of gold nanoparticles. The system is realized by preforming a hexagonal array of gold nanoparticles (∼5-7 nm) with controlled spacing (∼50-150 nm) fixed to a silica or glass substrate by block copolymer lithography. Subsequently, a supported membrane is assembled over the intervening bare substrate. Proteins or other ligands can be associated with the fluid lipid component, the fixed nanoparticle component, or both, providing a hybrid interface consisting of mobile and immobile components with controlled geometry. We test different biochemical coupling strategies to bind individual proteins to the particles surrounded by a fluid lipid membrane. The coupling efficiency to nanoparticles and the influence of nanoparticle arrays on the surrounding membrane integrity are characterized by fluorescence imaging, correlation spectroscopy, and super-resolution fluorescence microscopy. Finally, the functionality of this system for live cell experiments is tested using the ephrin-A1-EphA2 juxtacrine signaling interaction in human breast epithelial cells.