A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.     

The dissociation of the fluid and particle phase in the forestomach as a physiological characteristic of large grazing ruminants: an evaluation of available, comparable ruminant passage data

Whether differences in digestive physiology exist between different ruminant feeding types has been an ongoing debate. In this regard, potential differences in ingesta retention have been understood to be of particular importance. We analyzed a data pool in which only mean retention time (MRT) data for the ruminoreticulum (RR) were collated that were obtained using comparable techniques with either chromium or cobalt EDTA as a fluid marker and/or with chromium-mordanted fiber of less than 2 mm in size as a particle marker. Data were compared using one averaged value per species. In general, the paucity of species in such a collection is striking and does not allow—in contrast to earlier statements—any final conclusions regarding the influence of body weight (BW) or feeding type on ruminant MRTs. In particular, there was no significant correlation between MRT_particlesRR or MRT_fluidRR and BW, neither in the interspecific nor in the intraspecific comparisons, and no difference between the feeding types. The trend that indicates longer MRT_particlesRR in grazers is based on too few species to be conclusive. Small browsers seemed to have shorter MRT_fluidRR than similar-sized grazers. In contrast, there was a trend for large grazers to have shorter MRT_fluidRR than large browsers. In direct pair-wise comparisons between cattle and the browsers giraffe, moose, and okapi, the latter difference was significant. Cattle also had the highest relative RR fluid outflow rates among the species investigated. This is in accord with the observation that grazers have larger omasa, a major function of which is water-reabsorption distal to the RR. Grazers seem to have longer MRT_particlesRR per unit MRT_fluidRR, and cattle are particular outliers in this respect. It is hypothesized that potentially shorter MRT_fluidRR in large grazers and higher relative outflow rates are linked to a higher saliva production and a lesser viscosity of both saliva and RR fluids. A constant supply of a fluid phase of low viscosity is proposed to be the prerogative for the physical mechanisms of flotation and sedimentation that result in the stratification of RR contents and its selective particle retention typical for large grazing species.     

Antitumor activity of diethynylfluorene derivatives of gold(I)

A list of diethynylfluorenes and their gold(I) derivatives have been studied for their antitumor activity as a function of their structure–activity relationships. End-capping the fluoren-9-one unit with gold(I) moieties could significantly strengthen the cytotoxic activity in vitro on three human cancer cell lines with induction of reactive oxygen species generation on Hep3B hepatocellular carcinoma cells and exhibit attractive antitumor activity from in vivo nude mice Hep3B xenograft model with limited adverse effects on vital organs including liver and kidney.     

Pattern of anterior cruciate ligament force in normal walking

The goal of this study was to calculate and explain the pattern of anterior cruciate ligament (ACL) loading during normal level walking. Knee-ligament forces were obtained by a two-step procedure. First, a three-dimensional (3D) model of the whole body was used together with dynamic optimization theory to calculate body-segmental motions, ground reaction forces, and leg-muscle forces for one cycle of gait. Joint angles, ground reaction forces, and muscle forces obtained from the gait simulation were then input into a musculoskeletal model of the lower limb that incorporated a 3D model of the knee. The relative positions of the femur, tibia, and patella and the forces induced in the knee ligaments were found by solving a static equilibrium problem at each instant during the simulated gait cycle. The model simulation predicted that the ACL bears load throughout stance. Peak force in the ACL (303 N) occurred at the beginning of single-leg stance (i.e., contralateral toe off). The pattern of ACL force was explained by the shear forces acting at the knee. The balance of muscle forces, ground reaction forces, and joint contact forces applied to the leg determined the magnitude and direction of the total shear force acting at the knee. The ACL was loaded whenever the total shear force pointed anteriorly. In early stance, the anterior shear force from the patellar tendon dominated the total shear force applied to the leg, and so maximum force was transmitted to the ACL at this time. ACL force was small in late stance because the anterior shear forces supplied by the patellar tendon, gastrocnemius, and tibiofemoral contact were nearly balanced by the posterior component of the ground reaction.     

Lunar orientation in a beetle

Many animals use the sun's polarization pattern to orientate, but the dung beetle Scarabaeus zambesianus is the only animal so far known to orientate using the million times dimmer polarization pattern of the moonlit sky. We demonstrate the relative roles of the moon and the nocturnal polarized-light pattern for orientation. We find that artificially changing the position of the moon, or hiding the moon's disc from the beetle's field of view, generally did not influence its orientation performance. We thus conclude that the moon does not serve as the primary cue for orientation. The effective cue is the polarization pattern formed around the moon, which is more reliable for orientation. Polarization sensitivity ratios in two photoreceptors in the dorsal eye were found to be 7.7 and 12.9, similar to values recorded in diurnal navigators. These results agree with earlier results suggesting that the detection and analysis of polarized skylight is similar in diurnal and nocturnal insects.     

Enhanced Human Tissue Microdialysis Using Hydroxypropyl-?-Cyclodextrin as Molecular Carrier

Microdialysis sampling of lipophilic molecules in human tissues is challenging because protein binding and adhesion to the membrane limit recovery. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) forms complexes with hydrophobic molecules thereby improving microdialysis recovery of lipophilic molecules in vitro and in rodents. We tested the approach in human subjects. First, we determined HP-ß-CD influences on metabolite stability, delivery, and recovery in vitro. Then, we evaluated HP-ß-CD as microdialysis perfusion fluid supplement in 20 healthy volunteers. We placed 20 kDa microdialysis catheters in subcutaneous abdominal adipose tissue and in the vastus lateralis muscle. We perfused catheters with lactate free Ringer solution with or without 10% HP-ß-CD at flow rates of 0.3–2.0 µl/min. We assessed tissue metabolites, ultrafiltration effects, and blood flow. In both tissues, metabolite concentrations with Ringer+HP-ß-CD perfusate were equal or higher compared to Ringer alone. Addition of HP-ß-CD increased dialysate volume by 10%. Adverse local or systemic reactions to HP-ß-CD did not occur and analytical methods were not disturbed. HP-ß-CD addition allowed to measure interstitial anandamide concentrations, a highly lipophilic endogenous molecule. Our findings suggest that HP-ß-CD is a suitable supplement in clinical microdialysis to enhance recovery of lipophilic molecules from human interstitial fluid.     

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.     

PCSK9 Plasma Concentrations Are Independent of GFR and Do Not Predict Cardiovascular Events in Patients with Decreased GFR

Background

Impaired renal function causes dyslipidemia that contributes to elevated cardiovascular risk in patients with chronic kidney disease (CKD). The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a regulator of the LDL receptor and plasma cholesterol concentrations. Its relationship to kidney function and cardiovascular events in patients with reduced glomerular filtration rate (GFR) has not been explored.

Methods

Lipid parameters including PCSK9 were measured in two independent cohorts. CARE FOR HOMe (Cardiovascular and Renal Outcome in CKD 2–4 Patients—The Forth Homburg evaluation) enrolled 443 patients with reduced GFR (between 90 and 15 ml/min/1.73 m²) referred for nephrological care that were prospectively followed for the occurrence of a composite cardiovascular endpoint. As a replication cohort, PCSK9 was quantitated in 1450 patients with GFR between 90 and 15 ml/min/1.73 m² enrolled in the Ludwigshafen Risk and Cardiovascular Health Study (LURIC) that were prospectively followed for cardiovascular deaths.

Results

PCSK9 concentrations did not correlate with baseline GFR (CARE FOR HOMe: r = -0.034; p = 0.479; LURIC: r = -0.017; p = 0.512). 91 patients in CARE FOR HOMe and 335 patients in LURIC reached an endpoint during a median follow-up of 3.0 [1.8–4.1] years and 10.0 [7.3–10.6] years, respectively. Kaplan-Meier analyses showed that PCSK9 concentrations did not predict cardiovascular events in either cohort [CARE FOR HOMe (p = 0.622); LURIC (p = 0.729)]. Sensitivity analyses according to statin intake yielded similar results.

Conclusion

In two well characterized independent cohort studies, PCSK9 plasma levels did not correlate with kidney function. Furthermore, PCSK9 plasma concentrations were not associated with cardiovascular events in patients with reduced renal function.     

Amyloid beta impairs mitochondrial anterograde transport and degenerates synapses in Alzheimer's disease neurons

Loss of synapses and synaptic damage are the best correlates of cognitive decline identified in patients with Alzheimer's disease (AD), and mitochondrial oxidative damage and synaptic pathology have been identified as early events in the progression of AD. The progressive accumulation of amyloid beta (Aβ) in synapses and synaptic mitochondria are hypothesized to cause synaptic degeneration and cognitive decline in patients with AD. However, the precise mechanistic link between Aβ and mitochondria is not well understood. The purpose of this study was to better understand the effects of Aβ on mitochondrial axonal transport and synaptic alterations in AD. Using mouse hippocampal neurons and Aβ_25-35 peptide, we studied axonal transport of mitochondria, including mitochondrial motility, mitochondrial length and size, mitochondrial index per neurite, and synaptic alterations of the hippocampal neurons. In the PBS-treated neurons, 36.4 ± 4.7% of the observed mitochondria were motile, with 21.0 ± 1.3% moving anterograde and 15.4 ± 3.4% moving retrograde and the average speed of movement was 12.1 ± 1.8 μm/min. In contrast, in the Aβ-treated neurons, the number of motile mitochondria were significantly less, at 20.4 ± 2.6% (P < 0.032), as were those moving anterograde (10.1 ± 2.6%, P < 0.016) relative to PBS-treated neurons, suggesting that the Aβ_25-35 peptide impairs axonal transport of mitochondria in AD neurons. In the Aβ-treated neurons, the average speed of motile mitochondria was also less, at 10.9 ± 1.9 μm/min, and mitochondrial length was significantly decreased. Further, synaptic immunoreactivity was also significantly less in the Aβ-treated neurons relative to the PBS-treated neurons, indicating that Aβ affects synaptic viability. These findings suggest that, in neurons affected by AD, Aβ is toxic, impairs mitochondrial movements, reduces mitochondrial length, and causes synaptic degeneration.