Self-assembled DNA nanoparticles loaded with travoprost for glaucoma-treatment.

Lipid DNA nanoparticles (NPs) exhibit an intrinsic affinity to the ocular surface and can be loaded by hybridization with fluorophore-DNA conjugates or with the anti-glaucoma drug travoprost by hybridizing an aptamer that binds the medication. In the travoprost-loaded NPs (Trav-NPs), the drug is bound by specific, non-covalent interactions, not requiring any chemical modification of the active pharmaceutical ingredient. Fluorescently labeled Trav-NPs show a long-lasting adherence to the eye, up to sixty minutes after eye drop instillation. Biosafety of the Trav-NPs was proved and in vivo. Ex vivo and in vivo quantification of travoprost via LC-MS revealed that Trav-NPs deliver at least twice the amount of the drug at every time-point investigated compared to the pristine drug. The data successfully show the applicability of a DNA-based drug delivery system in the field of ophthalmology for the treatment of a major retinal eye disease, i.e. glaucoma.

MASP-1 and MASP-2 Do Not Activate Pro-Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors.

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(-/-) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of ∼ 3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator.

Mass-Spectrometry-Based Proteomics Reveals Organ-Specific Expression Patterns To Be Used as Forensic Evidence.

Standard forensic procedures to examine bullets after an exchange of fire include a mechanical or ballistic reconstruction of the event. While this is routine to identify which projectile hit a subject by DNA analysis of biological material on the surface of the projectile, it is rather difficult to determine which projectile caused the lethal injury--often the crucial point with regard to legal proceedings. With respect to fundamental law it is the duty of the public authority to make every endeavor to solve every homicide case. To improve forensic examinations, we present a forensic proteomic method to investigate biological material from a projectile's surface and determine the tissues traversed by it. To obtain a range of relevant samples, different major bovine organs were penetrated with projectiles experimentally. After tryptic "on-surface" digestion, mass-spectrometry-based proteome analysis, and statistical data analysis, we were able to achieve a cross-validated organ classification accuracy of >99%. Different types of anticipated external variables exhibited no prominent influence on the findings. In addition, shooting experiments were performed to validate the results. Finally, we show that these concepts could be applied to a real case of murder to substantially improve the forensic reconstruction.

Phosphorylation of serine 1137/1138 of mouse insulin receptor substrate (IRS) 2 regulates cAMP-dependent binding to 14-3-3 proteins and IRS2 protein degradation.

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, ß-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or Rp-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N(6)-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.

Optical Emission Spectroscopy for the Real-Time Identification of Malignant Breast Tissue.

Breast conserving resection with free margins is the gold standard treatment for early breast cancer recommended by guidelines worldwide. Therefore, reliable discrimination between normal and malignant tissue at the resection margins is essential. In this study, normal and abnormal tissue samples from breast cancer patients were characterized ex vivo by optical emission spectroscopy (OES) based on ionized atoms and molecules generated during electrosurgical treatment. The aim of the study was to determine spectroscopic features which are typical for healthy and neoplastic breast tissue allowing for future real-time tissue differentiation and margin assessment during breast cancer surgery. A total of 972 spectra generated by electrosurgical sparking on normal and abnormal tissue were used for support vector classifier (SVC) training. Specific spectroscopic features were selected for the classification of tissues in the included breast cancer patients. The average classification accuracy for all patients was 96.9%. Normal and abnormal breast tissue could be differentiated with a mean sensitivity of 94.8%, a specificity of 99.0%, a positive predictive value (PPV) of 99.1% and a negative predictive value (NPV) of 96.1%. For 66.6% patients all classifications reached 100%. Based on this convincing data, a future clinical application of OES-based tissue differentiation in breast cancer surgery seems to be feasible.

[Methods, applications, and future perspectives of intraoperative tissue identification]. / Methoden, Anwendungen und Zukunftsperspektiven der intraoperativen Gewebeerkennung.

Over the last century, there has been a steady development of new technologies for intraoperative tissue identification and differentiation. The applications are varied, with the core purpose being to identify target structures while preserving adjacent tissue and thereby follow a general paradigm of minimally invasive medicine. Particularly in oncology, a further asset of these technologies is the identification or classification of neoplastic tissue to support and improve therapy, for example, in breast cancer surgery.Many technologies under consideration make use of the different physical characteristics of treated tissues, such as induced fluorescence, optical coherence, and electrical impedance.Recent developments are focusing on moving from ex vivo to in situ and from asynchronous to real-time assistance of the clinicians, for example, by means of optical emission spectroscopy. Refinements of existing and the creation of new methods will include AI tools to make them more powerful while reducing the inter-operator variability in operative interventions. This talk addresses several aspects of the usage and suitability of these technologies for intraoperative, therapy-supporting application.

CLOUDING OF INTRAOCULAR SILICONE OIL IN THE ABSENCE OF EMULSIFICATION.

PURPOSE: To describe intraocular clouding of silicone oil in the absence of emulsification. METHODS: Retrospective observational case series of patients who received silicone oil injections and developed silicone oil discoloration without emulsification after pars plana vitrectomy. Clinical examinations and physicochemical analyses were performed to find out the common cause for the opaque oil. RESULTS: Thirteen patients developed silicone oil discoloration after pars plana vitrectomy. It could be traced down that all patients had received silicone oil from one respective production batch. The silicone oil was removed as soon as possible after the changes were detected (range, 8-16 weeks). Gas chromatography flame ionization detector, size exclusion chromatography, and high-performance liquid chromatography analysis showed the absence of low-molecular-weight compounds in the opaque lot. Thermogravimetric analysis revealed the opaque lot was more temperature stable. During the follow-ups, no obvious retinal toxicity could be observed and best-recorded visual acuity improved considerably in 12 patients and was only limited by the underlying retinal pathologic conditions. CONCLUSION: This is the first report on opacification of intraocular silicone oil without emulsification. This discoloration of silicone oil may disturb vision and prevent proper fundus examination; however, it seems to be a nontoxic phenomenon without serious long-term consequences.

A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium.

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.

Metabolomics in serum of patients with non-advanced age-related macular degeneration reveals aberrations in the glutamine pathway.

Age-related macular degeneration (AMD) is a common, progressive multifactorial vision-threatening disease and many genetic and environmental risk factors have been identified. The risk of AMD is influenced by lifestyle and diet, which may be reflected by an altered metabolic profile. Therefore, measurements of metabolites could identify biomarkers for AMD, and could aid in identifying high-risk individuals. Hypothesis-free technologies such as metabolomics have a great potential to uncover biomarkers or pathways that contribute to disease pathophysiology. To date, only a limited number of metabolomic studies have been performed in AMD. Here, we aim to contribute to the discovery of novel biomarkers and metabolic pathways for AMD using a targeted metabolomics approach of 188 metabolites. This study focuses on non-advanced AMD, since there is a need for biomarkers for the early stages of disease before severe visual loss has occurred. Targeted metabolomics was performed in 72 patients with early or intermediate AMD and 72 control individuals, and metabolites predictive for AMD were identified by a sparse partial least squares discriminant analysis. In our cohort, we identified four metabolite variables that were most predictive for early and intermediate stages of AMD. Increased glutamine and phosphatidylcholine diacyl C28:1 levels were detected in non-advanced AMD cases compared to controls, while the rate of glutaminolysis and the glutamine to glutamate ratio were reduced in non-advanced AMD. The association of glutamine with non-advanced AMD corroborates a recent report demonstrating an elevated glutamine level in early AMD using a different metabolomics technique. In conclusion, this study indicates that metabolomics is a suitable method for the discovery of biomarker candidates for AMD. In the future, larger metabolomics studies could add to the discovery of novel biomarkers in yet unknown AMD pathways and expand our insights in AMD pathophysiology.

Combined Targeted Analysis of Metabolites and Proteins in Tear Fluid With Regard to Clinical Applications.

PURPOSE: To establish a robust workflow for combined mass spectrometry-based analysis of metabolites and proteins in tear fluid with regard to clinical applicability. METHODS: Tear fluid was taken from 12 healthy volunteers at different time points using specially designed Schirmer strips. Following the liquid extraction of metabolites from standardized punches, the remaining material was processed for bottom-up proteomics. Targeted metabolite profiling was performed adapting a metabolomics kit, which targets 188 metabolites from four different analyte classes. Proteomics was performed of the identical samples targeting 15 tear proteins relevant to ocular health. RESULTS: Sixty metabolites could be consistently determined in all tear samples (98 metabolites were detectable in average) covering acylcarnitines, amino acids, biogenic amines, and glycerophospholipids. Following normalization, the majority of metabolites exhibited intraindividual variances of less than 20%, both regarding different times of sampling, and the individual eye. The targeted analysis of tear proteins revealed a mean intraindividual variation of 23% for the three most abundant proteins. Even extreme differences in tear secretion rates resulted in interindividual variability below 30% for 65 metabolites and two proteins. CONCLUSIONS: The newly established workflow can be used for combined targeted detection of metabolites and proteins in one punch of a Schirmer strip in a clinical setting. TRANSLATIONAL RELEVANCE: Our data about intra- and interindividual as well as intereye variation provide a valuable basis for the design of clinical studies, and for the applicability of multiplexed "omics" to well accessible tear fluid with regard to future routine use.

Systemic and ocular fluid compounds as potential biomarkers in age-related macular degeneration.

Biomarkers can help unravel mechanisms of disease and identify new targets for therapy. They can also be useful in clinical practice for monitoring disease progression, evaluation of treatment efficacy, and risk assessment in multifactorial diseases, such as age-related macular degeneration (AMD). AMD is a highly prevalent progressive retinal disorder for which multiple genetic and environmental risk factors have been described, but the exact etiology is not yet fully understood. Many compounds have been evaluated for their association with AMD. We performed an extensive literature review of all compounds measured in serum, plasma, vitreous, aqueous humor, and urine of AMD patients. Over 3600 articles were screened, resulting in more than 100 different compounds analyzed in AMD studies, involved in neovascularization, immunity, lipid metabolism, extracellular matrix, oxidative stress, diet, hormones, and comorbidities (such as kidney disease). For each compound, we provide a short description of its function and discuss the results of the studies in relation to its usefulness as AMD biomarker. In addition, biomarkers identified by hypothesis-free techniques, including metabolomics, proteomics, and epigenomics, are covered. In summary, compounds belonging to the oxidative stress pathway, the complement system, and lipid metabolism are the most promising biomarker candidates for AMD. We hope that this comprehensive survey of the literature on systemic and ocular fluid compounds as potential biomarkers in AMD will provide a stepping stone for future research and possible implementation in clinical practice.

A new perspective on lipid research in age-related macular degeneration.

There is an urgency to find new treatment strategies that could prevent or delay the onset or progression of AMD. Different classes of lipids and lipoproteins metabolism genes have been associated with AMD in a multiple ways, but despite the ever-increasing knowledge base, we still do not understand fully how circulating lipids or local lipid metabolism contribute to AMD. It is essential to clarify whether dietary lipids, systemic or local lipoprotein metabolismtrafficking of lipids in the retina should be targeted in the disease. In this article, we critically evaluate what has been reported in the literature and identify new directions needed to bring about a significant advance in our understanding of the role for lipids in AMD. This may help to develop potential new treatment strategies through targeting the lipid homeostasis.

Identification and characterization of secreted effector proteins of Chlamydophila pneumoniae TW183.

We report the expression of several chlamydial effector proteins in Chlamydophila pneumoniae, as well as their time-dependent secretion into the inclusion membrane. Localization of the respective genes within type III secretion gene clusters as well as bioinformatic analysis suggest that the identified proteins are type III-secreted effector proteins. Immunocytochemistry with antisera raised against CpMip (C. pneumoniae macrophage infectivity potentiator, Cpn0661), Pkn5 (Cpn0703), Cpn0709, Cpn0712 and Cpn0827 showed secretion of the respective proteins into the inclusion membrane at 20 h postinfection (hpi). CpMip was detected within the inclusion membrane from 20 to 72 hpi, whereas Cpn0324 (CopN) was located in this compartment at 72 hpi only. This was confirmed by co-localization of the respective proteins with IncA, an inclusion membrane marker protein. These data illustrate the fact that different effectors are being expressed and secreted during different time intervals of the infection cycle. Proteins Cpn0706 and Cpn0808 were not secreted by C. pneumoniae. The immunophilin FK506, known to inhibit the activity of Legionella, C. trachomatis and C. psittaci Mip proteins, was shown to interfere with chlamydial infection. Here we report the putatively type III-dependent secretion of CpMip into the inclusion membrane as well as the effect of its inhibition on C. pneumoniae infection of HEp-2 cells.

Circulating lysophosphatidylcholines are markers of a metabolically benign nonalcoholic fatty liver.

OBJECTIVE: Nonalcoholic fatty liver (NAFL) is thought to contribute to insulin resistance and its metabolic complications. However, some individuals with NAFL remain insulin sensitive. Mechanisms involved in the susceptibility to develop insulin resistance in humans with NAFL are largely unknown. We investigated circulating markers and mechanisms of a metabolically benign and malignant NAFL by applying a metabolomic approach. RESEARCH DESIGN AND METHODS: A total of 265 metabolites were analyzed before and after a 9-month lifestyle intervention in plasma from 20 insulin-sensitive and 20 insulin-resistant subjects with NAFL. The relevant plasma metabolites were then tested for relationships with insulin sensitivity in 17 subjects without NAFL and in plasma from 29 subjects with liver tissue samples. RESULTS: The best separation of the insulin-sensitive from the insulin-resistant NAFL group was achieved by a metabolite pattern including the branched-chain amino acids leucine and isoleucine, ornithine, the acylcarnitines C3:0-, C16:0-, and C18:0-carnitine, and lysophosphatidylcholine (lyso-PC) C16:0 (area under the ROC curve, 0.77 [P = 0.00023] at baseline and 0.80 [P = 0.000019] at follow-up). Among the individual metabolites, predominantly higher levels of lyso-PC C16:0, both at baseline (P = 0.0039) and at follow-up (P = 0.001), were found in the insulin-sensitive compared with the insulin-resistant subjects. In the non-NAFL groups, no differences in lyso-PC C16:0 levels were found between the insulin-sensitive and insulin-resistant subjects, and these relationships were replicated in plasma from subjects with liver tissue samples. CONCLUSIONS: From a plasma metabolomic pattern, particularly lyso-PCs are able to separate metabolically benign from malignant NAFL in humans and may highlight important pathways in the pathogenesis of fatty liver-induced insulin resistance.

Critical role of novel Thr-219 autophosphorylation for the cellular function of PKCtheta in T lymphocytes.

Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)theta. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCtheta in primary CD3+ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCtheta to mediate NF-kappaB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCtheta's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCtheta with Akt/PKBalpha in NF-kappaB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCtheta in activated T cells. The PKCthetaT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCtheta at Thr-219 plays an important role in the correct targeting and cellular function of PKCtheta upon antigen receptor ligation.