PET-compatible endothelin receptor radioligands: Synthesis and first in vitro and in vivo studies

The expression and function of endothelin (ET) receptors is abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. In this study, we prepared two [¹⁸F]-fluorinated derivatives of the non-peptide ET_A receptor antagonist PD 156707 and evaluated their ET receptor binding potencies. Ex vivo as well as in vivo biodistribution studies in mice were performed, as well as the metabolism of the radiotracer, which was examined by metabolite analysis in mice and rats. All tested derivatives of PD 156707 exhibited potent in vitro pharmacological characteristics with K_i values comparable to that of the lead compound. The biodistribution studies showed a high accumulation of the tracer in bile and intestine. In vivo we were able to show that the visualization of the heart as a major target organ with high ET_AR expression is possible.     

Hook Proteins: Association with Alzheimer Pathology and Regulatory Role of Hook3 in Amyloid Beta Generation

Defects in intracellular transport are implicated in the pathogenesis of Alzheimer’s disease (AD). Hook proteins are a family of cytoplasmic linker proteins that participate in endosomal transport. In this study we show that Hook1 and Hook3 are expressed in neurons while Hook2 is predominantly expressed in astrocytes. Furthermore, Hook proteins are associated with pathological hallmarks in AD; Hook1 and Hook3 are localized to tau aggregates and Hook2 to glial components within amyloid plaques. Additionally, the expression of Hook3 is reduced in AD. Modelling of Hook3 deficiency in cultured cells leads to slowing of endosomal transport and increases β-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD.     

The Impact II,a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics

Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum—the highest proteome coverage reported with a QTOF instrument so far.Building on the fundamental advance of the soft ionization techniques electrospray ionization and matrix-assisted laser desorption/ionization (1, 2), MS-based proteomics has advanced tremendously over the last two decades (3–6). Bottom-up, shotgun proteomics is usually performed in a liquid chromatography-tandem MS (LC-MS/MS)¹ format, where nanoscale liquid chromatography is coupled through electrospray ionization to an instrument capable of measuring a mass spectrum and fragmenting the recognized precursor peaks on the chromatographic time scale. Fundamental challenges of shotgun proteomics include the very large numbers of peptides that elute over relatively short periods and peptide abundances that vary by many orders of magnitude. Developments in mass spectrometers toward higher sensitivity, sequencing speed, and resolution were needed and helped to address these critical challenges (7, 8). Especially the introduction of the Orbitrap mass analyzers has advanced the state of the art of the field because of their very high resolution and mass accuracy (9, 10). A popular configuration couples a quadrupole mass filter for precursor selection to the Orbitrap analyzer in a compact benchtop format (11–13).In addition to the improvements in MS instrumentation, there have been key advances in the entire proteomics workflow, from sample preparation through improved LC systems and in computational proteomics (14–16). Together, such advances are making shotgun proteomics increasingly comprehensive and deep analyses can now be performed in a reasonable time (13, 17–19). Nevertheless, complete analysis of all expressed proteins in a complex system remains extremely challenging and complete measurement of all the peptides produced in shotgun proteomics may not even be possible in principle (20, 21). Therefore, an urgent need for continued improvements in proteomics technology remains.Besides the Orbitrap analyzer and other ion trap technologies, the main alternative MS technology is time-of-flight, a technology that has been used for many decades in diverse fields. The configuration employed in proteomics laboratories combines a quadrupole mass filter via a collision cell and orthogonal acceleration unit to a reflectron and a multichannel plate (MCP) detector (22). TOF scans are generated in much less than a millisecond (ms), and a number of these “pulses” are added to obtain an MS or MS/MS spectrum with the desired signal to noise ratio. Our own laboratory has used such a quadrupole time-of-flight (QTOF) instrument as the main workhorse in proteomics for many years, but then switched to high-resolution trapping instruments because of their superior resolution and mass accuracy. However, TOF technology has fundamental attractions, such as the extremely high scan speed and the absence of space charge, which limits the number of usable ions in all trapping instruments. In principle, the high spectra rate makes TOF instruments capable of making use of the majority of ions, thus promising optimal sensitivity, dynamic range and hence quantification. It also means that TOF can naturally be interfaced with ion mobility devices, which typically separate ions on the ms time scale. Data independent analysis strategies such as MS^E, in which all precursors are fragmented simultaneously (23, 24) or SWATH, in which the precursor ion window is rapidly cycled through the entire mass range (25), also make use of the high scanning speed offered by QTOF instruments. It appears that QTOFs are set to make a comeback in proteomics with recent examples showing impressive depth of coverage of complex proteomes. For instance, using a variant of the MS^E method, identification of 5468 proteins was reported in HeLa cells in single shots and small sample amounts (26). In another report, employing ion mobility for better transmission of fragment ions to the detector led to the identification of up to 7548 proteins in human ovary tissue (27).In this paper, we describe the impact II™, a benchtop QTOF instrument from Bruker Daltonics, and its use in shotgun proteomics. This QTOF instrument is a member of an instrument family first introduced in 2008, which consists of the compact, the impact, and the maXis. The original impact was introduced in 2011 and was followed by the impact HD, which was equipped with a better digitizer, expanding the dynamic range of the detector. With the impact II, which became commercially available in 2014, we aimed to achieve a resolution and sequencing speed adequate for demanding shotgun proteomics experiments. To achieve this we developed an improved collision cell, orthogonal accelerator scheme, reflectron, and detector. Here we measure ion transmission characteristics of this instrument and the actually realized resolution and mass accuracy in typical proteomics experiments. Furthermore, we investigated the attainable proteome coverage in single shot analysis and we ask if QTOF performance is now sufficient for very deep characterization of complex cell line and tissue proteomes.     

Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent

An increasing number of broadly neutralizing antibodies (bnAbs) are considered leads for HIV-1 vaccine development and novel therapeutics. Here, we systematically explored the capacity of bnAbs to neutralize HIV-1 prior to and post-CD4 engagement and to block HIV-1 cell-cell transmission. Cell-cell spread is known to promote a highly efficient infection with HIV-1 which can inflict dramatic losses in neutralization potency compared to free virus infection. Selection of bnAbs that are capable of suppressing HIV irrespective of the transmission mode therefore needs to be considered to ascertain their in vivo activity in therapeutic use and vaccines. Employing assay systems that allow for unambiguous discrimination between free virus and cell-cell transmission to T cells, we probed a panel of 16 bnAbs for their activity against 11 viruses from subtypes A, B and C during both transmission modes. Over a wide range of bnAb-virus combinations tested, inhibitory activity against HIV-1 cell-cell transmission was strongly decreased compared to free virus transmission. Activity loss varied considerably between virus strains and was inversely associated with neutralization of free virus spread for V1V2- and V3-directed bnAbs. In rare bnAb-virus combinations, inhibition for both transmission modes was comparable but no bnAb potently blocked cell-cell transmission across all probed virus strains. Mathematical analysis indicated an increased probability of bnAb resistance mutations to arise in cell-cell rather than free virus spread, further highlighting the need to block this pathway. Importantly, the capacity to efficiently neutralize prior to CD4 engagement correlated with the inhibition efficacy against free virus but not cell-cell transmitted virus. Pre-CD4 attachment activity proved strongest amongst CD4bs bnAbs and varied substantially for V3 and V1V2 loop bnAbs in a strain-dependent manner. In summary, bnAb activity against divergent viruses varied depending on the transmission mode and differed depending on the window of action during the entry process, underscoring that powerful combinations of bnAbs are needed for in vivo application.     

C-Terminal Fragment of Agrin (CAF): A Novel Marker for Progression of Kidney Disease in Type 2 Diabetics

Background

Diabetes is the leading cause of CKD in the developed world. C-terminal fragment of agrin (CAF) is a novel kidney function and injury biomarker. We investigated whether serum CAF predicts progression of kidney disease in type 2 diabetics.

Methods

Serum CAF levels were measured in 71 elderly patients with diabetic nephropathy using a newly developed commercial ELISA kit (Neurotune®). Estimated glomerular filtration rate (eGFR) and proteinuria in spot urine were assessed at baseline and after 12 months follow up. The presence of end stage renal disease (ESRD) was evaluated after 24 months follow-up. Correlation and logistic regression analyses were carried out to explore the associations of serum CAF levels with GFR, proteinuria, GFR loss and incident ESRD. Renal handling of CAF was tested in neurotrypsin-deficient mice injected with recombinant CAF.

Results

We found a strong association of serum CAF levels with eGFR and a direct association with proteinuria both at baseline (r = 0.698, p<0.001 and r = 0. 287, p = 0.02) as well as after 12 months follow-up (r = 0.677, p<0.001 and r = 0.449, p<0.001), respectively. Furthermore, in multivariate analysis, serum CAF levels predicted eGFR decline at 12 months follow-up after adjusting for known risk factors (eGFR, baseline proteinuria) [OR (95%CI) = 4.2 (1.2–14.5), p = 0.024]. In mice, injected CAF was detected in endocytic vesicles of the proximal tubule.

Conclusion

Serum CAF levels reflect renal function and are highly associated with eGFR and proteinuria at several time points. Serum CAF was able to predict subsequent loss of renal function irrespective of baseline proteinuria in diabetic nephropathy. CAF is likely removed from circulation by glomerular filtration and subsequent endocytosis in the proximal tubule. These findings may open new possibilities for clinical trial design, since serum CAF levels may be used as a selection tool to monitor kidney function in high-risk patients with diabetic nephropathy.     

Mutations in CSPP1 Cause Primary Cilia Abnormalities and Joubert Syndrome with or without Jeune Asphyxiating Thoracic Dystrophy

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.     

Automated Identification of Subcellular Organelles by Coherent Anti-Stokes Raman Scattering

Coherent anti-Stokes Raman scattering (CARS) is an emerging tool for label-free characterization of living cells. Here, unsupervised multivariate analysis of CARS datasets was used to visualize the subcellular compartments. In addition, a supervised learning algorithm based on the “random forest” ensemble learning method as a classifier, was trained with CARS spectra using immunofluorescence images as a reference. The supervised classifier was then used, to our knowledge for the first time, to automatically identify lipid droplets, nucleus, nucleoli, and endoplasmic reticulum in datasets that are not used for training. These four subcellular components were simultaneously and label-free monitored instead of using several fluorescent labels. These results open new avenues for label-free time-resolved investigation of subcellular components in different cells, especially cancer cells.     

Sea-level changes as controlling factor of early diagenesis: the reefal limestones of Adnet (Late Triassic,Northern Calcareous Alps,Austria)

The uppermost Rhaetian Adnet reef is part of the Dachstein carbonate platform and is situated at the transition to the intrashelf Kössen Basin. Its diagenetic evolution is investigated focusing on dissolution cavities in the Tropfbruch quarry of Adnet (near Salzburg) stratigraphically situated immediately below the Triassic–Jurassic boundary. Sea-level changes due to global eustatic trends and regional tectonics are assumed to be the controlling factors in the development of a manifold diagenetic sequence characterized by phases of meteoric dissolution, marine and burial cementation, and internal sedimentation. Despite small-scale variations of the sequence, a superordinate pattern of diagenetic phases could be elaborated. Small-scale eustatic sea-level falls subordinate to a global regression trend caused subaerial exposures of the Adnet reef in the latest Rhaetian to earliest Hettangian. The result was karstification and meteoric dissolution of aragonitic coral skeletons (Retiophyllia) leading to the formation of biomoldic porosity. Coral septa which escaped dissolution were transformed into neomorphic calcite spar under meteoric–phreatic conditions. A first generation of dog-tooth cements precipitated sporadically on the altered coral skeletons. Eustatic sea-level rise in Early to Mid-Hettangian times caused a renewed flooding of the pore space of the Adnet reef by marine water and the influx of a first generation of internal sediments (IS I), derived from the karstified host rock of the Upper Rhaetian reef limestone. These internal sediments are overgrown by radiaxial-fibrous calcites (RFCs) whose oxygen-isotopic signature (δ¹⁸O = ?1.3 (±0.7)‰) indicates precipitation in deeper (colder) water (18–21°C) due to a first phase of drowning. An intermediate phase of eustatic sea-level lowstand in the Late Hettangian is expressed by dissolution and corrosion of RFCs. Rapid drowning of the Dachstein carbonate platform due to eustatic sea-level rise and tectonic movements took place in the Early Sinemurian and a second generation of internal sediments (IS II) derived from the Lower Sinemurian Adnet Formation is washed into the dissolution cavities. Where IS II is absent, RFCs are overgrown by a second generation of dog-tooth cements with a bright-luminescent outer rim indicating the transition to negative redox conditions in the pore water during shallow burial. Burial diagenesis is represented by blocky calcite cements which occlude the remaining pore space. Depleted oxygen-isotope values and significant Fe contents indicate precipitation under reducing redox conditions and elevated temperatures of 30–50°C at burial depths of 420–870 m. Locally, replacive saddle dolomite is the latest diagenetic phase in the Adnet reef indicating crystallization under hydrothermal influences related to compressional subduction regimes of the Penninic Ocean.     

Organizing protein-DNA hybrids as nanostructures with programmed functionalities

The structural and functional information encoded in the base sequence of nucleic acids provides a means to organize hybrid protein-DNA nanostructures with pre-designed, programmed functionality. This review discusses the activation of enzyme cascades in supramolecular DNA-protein hybrid structures, the bioelectrocatalytic activation of redox enzymes on DNA scaffolds, and the programmed positioning of enzymes on 1D, 2D and 3D DNA nanostructures. These systems provide starting points towards the design of interconnected enzyme networks. Substantial progress in the tailoring of functional protein-DNA nanostructures has been accomplished in recent years, and advances in this field warrant a comprehensive discussion. The application of these systems for the control of biocatalytic transformations, for amplified biosensing, and for the synthesis of metallic nanostructures are addressed, and future prospects for these systems are highlighted.