Unwinding fibril formation of medin, the peptide of the most common form of human amyloid

Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.     

Stochastic Dynamic Model for Estimation of Rate Constants and Their Variances from Noisy and Heterogeneous PET Measurements

Tissue heterogeneity, radioactive decay and measurement noise are the main error sources in compartmental modeling used to estimate the physiologic rate constants of various radiopharmaceuticals from a dynamic PET study. We introduce a new approach to this problem by modeling the tissue heterogeneity with random rate constants in compartment models. In addition, the Poisson nature of the radioactive decay is included as a Poisson random variable in the measurement equations. The estimation problem will be carried out using the maximum likelihood estimation. With this approach, we do not only get accurate mean estimates for the rate constants, but also estimates for tissue heterogeneity within the region of interest and other possibly unknown model parameters, e.g. instrument noise variance, as well. We also avoid the problem of the optimal weighting of the data related to the conventionally used weighted least-squares method. The new approach was tested with simulated time–activity curves from the conventional three compartment – three rate constants model with normally distributed rate constants and with a noise mixture of Poisson and normally distributed random variables. Our simulation results showed that this new model gave accurate estimates for the mean of the rate constants, the measurement noise parameter and also for the tissue heterogeneity, i.e. for the variance of the rate constants within the region of interest.     

Real-life use of vitamin D3-fortified bread and milk during a winter season: the effects of CYP2R1 and GC genes on 25-hydroxyvitamin D concentrations in Danish families,the VitmaD study

Common genetic variants rs10741657 and rs10766197 in CYP2R1 and rs4588 and rs842999 in GC and a combined genetic risk score (GRS) of these four variants influence late summer 25-hydroxyvitamin D (25(OH)D) concentrations. The objectives were to identify those who are most at risk of developing low vitamin D status during winter and to assess whether vitamin D₃-fortified bread and milk will increase 25(OH)D concentrations in those with genetically determined low 25(OH)D concentrations at late summer. We used data from the VitmaD study. Participants were allocated to either vitamin D₃-fortified bread and milk or non-fortified bread and milk during winter. In the fortification group, CYP2R1 (rs10741657) and GC (rs4588 and rs842999) were statistically significantly associated with winter 25(OH)D concentrations and CYP2R1 (rs10766197) was borderline significant. There was a negative linear trend between 25(OH)D concentrations and carriage of 0–8 risk alleles (p < 0.0001). No association was found for the control group (p = 0.1428). There was a significant positive linear relationship between different quintiles of total vitamin D intake and the increase in 25(OH)D concentrations among carriers of 0–2 (p = 0.0012), 3 (p = 0.0001), 4 (p = 0.0118) or 5 (p = 0.0029) risk alleles, but not among carriers of 6–8 risk alleles (p = 0.1051). Carriers of a high GRS were more prone to be vitamin D deficient compared to carriers of a low GRS. Furthermore, rs4588-AA carriers have a low but very stable 25(OH)D concentration, and interestingly, also low PTH level.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0413-7) contains supplementary material, which is available to authorized users.     

Pazopanib,a Receptor Tyrosine Kinase Inhibitor,Suppresses Tumor Growth through Angiogenesis in Dedifferentiated Liposarcoma Xenograft Models

INTRODUCTION: The rarity of dedifferentiated liposarcoma (DDLPS) and the lack of experimental DDLPS models limit the development of novel therapeutic strategies. Pazopanib (PAZ) is a tyrosine kinase inhibitor that is approved for the treatment of non-adipocytic advanced soft tissue sarcoma. The activity of this agent has not yet been properly explored in preclinical liposarcoma models nor in a randomized phase Ш clinical trial in this entity. The aim of the present study was to investigate whether PAZ had antitumor activity in DDLPS models in vivo. MATERIAL AND METHODS: We established two patient-derived DDLPS xenograft models (UZLX-STS3 and UZLX-STS5) through implantation of tumor material from sarcoma patients in athymic nude NMRI mice. An animal model of the SW872 liposarcoma cell line was also used. To investigate the efficacy of PAZ in vivo, mice bearing tumors were treated for 2 weeks with sterile water, doxorubicin (1.2 mg/kg, intraperitoneally, twice per week), PAZ [40 mg/kg, orally (p.o.), twice per day], or PAZ plus doxorubicin (same schedules as for single treatments). RESULTS: Patient-derived xenografts retained the histologic and molecular features of DDLPS. PAZ significantly delayed tumor growth by decreasing proliferation and inhibited angiogenesis in all models tested. Combining the angiogenesis inhibitor with an anthracycline did not show superior efficacy. CONCLUSION: These results suggest that PAZ has potential antitumor activity in DDLPS primarily through antiangiogenic effects and therefore should be explored in clinical trials.     

α-Melanocyte Stimulating Hormone Treatment in Pigs Does Not Improve Early Graft Function in Kidney Transplants from Brain Dead Donors

Delayed graft function and primary non-function are serious complications following transplantation of kidneys derived from deceased brain dead (DBD) donors. α-melanocyte stimulating hormone (α-MSH) is a pleiotropic neuropeptide and its renoprotective effects have been demonstrated in models of acute kidney injury. We hypothesized that α-MSH treatment of the recipient improves early graft function and reduces inflammation following DBD kidney transplantation. Eight Danish landrace pigs served as DBD donors. After four hours of brain death both kidneys were removed and stored for 18 hours at 4°C in Custodiol preservation solution. Sixteen recipients were randomized in a paired design into two treatment groups, transplanted simultaneously. α-MSH or a vehicle was administered at start of surgery, during reperfusion and two hours post-reperfusion. The recipients were observed for ten hours following reperfusion. Blood, urine and kidney tissue samples were collected during and at the end of follow-up. α-MSH treatment reduced urine flow and impaired recovery of glomerular filtration rate (GFR) compared to controls. After each dose of α-MSH, a trend towards reduced mean arterial blood pressure and increased heart rate was observed. α-MSH did not affect expression of inflammatory markers. Surprisingly, α-MSH impaired recovery of renal function in the first ten hours following DBD kidney transplantation possibly due to hemodynamic changes. Thus, in a porcine experimental model α-MSH did not reduce renal inflammation and did not improve short-term graft function following DBD kidney transplantation.     

Species Identification of Archaeological Skin Objects from Danish Bogs: Comparison between Mass Spectrometry-Based Peptide Sequencing and Microscopy-Based Methods

Denmark has an extraordinarily large and well-preserved collection of archaeological skin garments found in peat bogs, dated to approximately 920 BC – AD 775. These objects provide not only the possibility to study prehistoric skin costume and technologies, but also to investigate the animal species used for the production of skin garments. Until recently, species identification of archaeological skin was primarily performed by light and scanning electron microscopy or the analysis of ancient DNA. However, the efficacy of these methods can be limited due to the harsh, mostly acidic environment of peat bogs leading to morphological and molecular degradation within the samples. We compared species assignment results of twelve archaeological skin samples from Danish bogs using Mass Spectrometry (MS)-based peptide sequencing, against results obtained using light and scanning electron microscopy. While it was difficult to obtain reliable results using microscopy, MS enabled the identification of several species-diagnostic peptides, mostly from collagen and keratins, allowing confident species discrimination even among taxonomically close organisms, such as sheep and goat. Unlike previous MS-based methods, mostly relying on peptide fingerprinting, the shotgun sequencing approach we describe aims to identify the complete extracted ancient proteome, without preselected specific targets. As an example, we report the identification, in one of the samples, of two peptides uniquely assigned to bovine foetal haemoglobin, indicating the production of skin from a calf slaughtered within the first months of its life. We conclude that MS-based peptide sequencing is a reliable method for species identification of samples from bogs. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium with the dataset identifier PXD001029.     

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies: Susceptibility vs. functionality of critical quality attributes

Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.     

Proline-glutamate chimera’s side chain conformation directs the type of β-hairpin structure

Our aim was to study the impact of two proline chimeras, containing a glutamic acid side chain in cis- or trans-configuration, on secondary structure formation. We further investigated to what extent the configuration of the side chain contributes to the overall peptide conformation. We used a 10 residue peptide (IYSNPDGTWT) that forms a β-hairpin in water. The turn-forming proline was substituted with either a cis- or trans-proline-glutamic acid chimera, resulting in the peptides IYSNP ^{cis -E} DGTWT (P1_P ^cis-E ) and IYSNP ^{trans -E} DGTWT (P1_P ^trans-E ). We studied the conformation of the modified peptides by circular dichroism (CD) and NMR-spectroscopy, and SEC/static light scattering (SLS) analysis. NMR analysis reveals that the modified peptides maintain the β-hairpin conformation in aqueous solution. At 5 °C and pH 4.3, the peptide (P1_P ^cis-E ) was found to adopt two coexisting β-hairpin conformations (2:2 β-hairpin, and 3:5 β-hairpin). In contrast to that, the peptide (P1_P ^trans-E ) adopts a 2:2 β-hairpin that exists in equilibrium with a 4:4 β-hairpin conformation. The adoption of ordered β-hairpin structures for both modified peptides could be confirmed by CD spectroscopy, while SEC/SLS analysis showed a monomeric oligomerization state for all three investigated peptides. With the combination of several NMR methods, we were able to elucidate that even small alterations in the side chain conformation of the proline-glutamate chimera (cis or trans) can significantly influence the conformation of the adopted β-hairpin.