MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.     

Growth of heat-treated enterotoxin-positive Clostridium perfringens and the implications for safe cooling rates

Clostridium perfringens 790-94 and 44071.C05 carrying a chromosomal and a plasmid cpe gene, respectively, were used to determine differences in heat resistance and growth characteristics between the genotypes. Heat inactivation experiments were conducted using an immersed coil apparatus. Spore germination, outgrowth, and lag phase, together named GOL time, as well as generation times were determined during constant temperatures in fluid thioglycollate (FTG) medium as well as in vacuum-packed, heat-treated minced turkey. GOL time and growth were also monitored during cooling scenarios from 65 to 10 degrees C for 3, 4, 5, 6, and 7 h in vacuum-packed, heat-treated minced turkey. Spores of strain 790-94 were approximately 10-fold more heat resistant at 85 degrees C than those of strain 44071.C05, and strain 790-94 also had a higher temperature growth range in FTG. The higher growth range for a chromosomal enterotoxin-producing CPE+ strain was confirmed using two other strains carrying a chromosomal (NCTC8239) and plasmid (945P) cpe gene. Moreover, strain 790-94 had shorter GOL times at 50 degrees C in turkey and approximately half the generation time compared with strain 44071.C05 at temperatures > or = 45 degrees C in both FTG and turkey. Strain 790-94 increased with 0.3, 1.0, 1.7, and 2.0 logs, respectively, during cooling from 65 to 10 degrees C in 4, 5, 6, and 7 h, which was significantly higher than for strain 44071.C05. A maximum acceptable cooling time of 5 h between 65 and 10 degrees C is suggested.     

Functional significance of the early component of the human blink reflex.

The relationship between the size of the first electromyographic (EMG) component of the cutaneous blink reflex (Rl) and onset of eyelid closure in human adults was determined in 4 experiments in which R1 size was varied by different means: change in stimulus intensity, paired stimulation, and warning. Two-phase lid movements were frequently seen, with an early small movement followed by a large rapid movement. All experiments showed that larger R1s were associated with shorter latencies of both movements. This covariation was general across participants and was independent of shifts in the excitability of the blink reflex pathways indexed by R1 latency, R2 latency, and R2 area (R2 is the more prolonged, later EMG component). The results indicate that R1 acts first to evoke an early lid movement and second to facilitate eyelid closure by the later R2 burst. Identification of this second behavioral function for R1 aids the interpretation of other findings and encourages its use as a model system. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.     

The influence of demographic,physical, behavioral,and dietary factors on hemoglobin adduct levels of acrylamide and glycidamide in the general U.S. population

Purpose: This study aims to better understand the individual characteristics and dietary factors that affect the relationship between estimated consumption of acrylamide and measured acrylamide hemoglobin adduct levels (HbAA) and glycidamide hemoglobin adduct levels (HbGA). Methods: Acrylamide levels in individual food items, estimated by the U.S. Food and Drug Administration, were linked to data collected in the 2003–2004 National Health and Nutrition Examination Survey. Multivariable linear regression was used to evaluate the relationship between estimated consumption of acrylamide and HbAA. Results: A significant association between acrylamide intake and HbAA was observed, after adjustment for gender, race/ethnicity, smoking status, age, and BMI (R² = 0.34). Across quartiles of acrylamide consumption, HbAA and HbGA levels increased monotonically. Among nonsmokers, an evaluation of three heavily consumed, high AA concentration foods showed a positive trend between the consumed amount of fried potatoes and HbAA in children, adolescents, and adults. A significant positive trend between the consumed amount of potato chips or coffee was indicated in adolescents, adults, and seniors. Conclusions: Consumption of some individual foods affects HbAA concentrations more strongly and in an age-dependent manner. Our results suggest that effective dietary guidelines for controlling acrylamide intake should be subpopulation specific.     

Dual mTOR/DNA-PK Inhibitor CC-115 Induces Cell Death in Melanoma Cells and Has Radiosensitizing Potential

CC-115 is a dual inhibitor of the mechanistic target of rapamycin (mTOR) kinase and the DNA-dependent protein kinase (DNA-PK) that is currently being studied in phase I/II clinical trials. DNA-PK is essential for the repair of DNA-double strand breaks (DSB). Radiotherapy is frequently used in the palliative treatment of metastatic melanoma patients and induces DSBs. Melanoma cell lines and healthy-donor skin fibroblast cell lines were treated with CC-115 and ionizing irradiation (IR). Apoptosis, necrosis, and cell cycle distribution were analyzed. Colony forming assays were conducted to study radiosensitizing effects. Immunofluorescence microscopy was performed to determine the activity of homologous recombination (HR). In most of the malign cell lines, an increasing concentration of CC-115 resulted in increased cell death. Furthermore, strong cytotoxic effects were only observed in malignant cell lines. Regarding clonogenicity, all cell lines displayed decreased survival fractions during combined inhibitor and IR treatment and supra-additive effects of the combination were observable in 5 out of 9 melanoma cell lines. CC-115 showed radiosensitizing potential in 7 out of 9 melanoma cell lines, but not in healthy skin fibroblasts. Based on our data CC-115 treatment could be a promising approach for patients with metastatic melanoma, particularly in the combination with radiotherapy.     

“入时”年末亮相厦门 “世界500强”企业进军时尚配饰市场

鉴于时尚配饰行业正随着国民对于美好生活的需求不断提升而飞速发展,“建发星光”将“中国国际配饰设计大赛活动”升级打造为“入时·时尚配饰设计师服务平台”,帮助原创设计师和企业拓展更多商业发展空间。     

Investigation of creep behaviours of gypsum specimens with flaws under different uniaxial loads

The aim of this study is to identify the influence of the dip angle of a pre-existing macrocrack on the lifetime and ultimate deformation of rock-like material. Prediction of lifetime has been studied for three groups of specimens under axial static compressive load levels. The specimens were investigated from 65% to 85% of UCS(uniaxial compressive strength) at an interval of 10% of UCS for the groups of specimens with a single modelled open flaw with a dip angle to the loading direction of 30°(first group), at an interval of 5% of UCS increment for the groups of specimens with single(second group), and double sequential open flaws with a dip angle to the loading direction of 60°(third group). This study shows that crack propagation in specimens with a single flaw follows the same sequences. At first, wing cracks appear, and then shear crack develops from the existing wing cracks. Shear cracking is responsible for specimen failure in all three groups. A slip is expected in specimens from the third group which connects two individual modelled open flaws. The moment of the slip is noticed as a characteristic rise in the axial deformation at a constant load level. It is also observed that axial deformation versus time follows the same pattern, irrespective of local geometry. Specimens from the first group exhibit higher axial deformation under different load levels in comparison with the specimens from the second and third groups.     

Porous Titanium Coatings Through Electrophoretic Deposition of TiH2 Suspensions

In the biomedical field, modification of titanium surfaces to improve the osteoinductive and antibacterial behavior is widely investigated. This functionalization can be further ameliorated by providing a porous coating with high loading capacity for bioactive materials and drug delivery carriers at the implant surface. In this work, a new powder metallurgical processing route used to deposit such porous pure titanium coatings on Ti based substrates is presented. The coatings were prepared by electrophoretic deposition (EPD) of TiH₂ powder suspensions followed by dehydrogenation and sintering in vacuum. The use of hydrides allowed to lower the sintering temperature below that of the α–β transition of the Ti6Al4V substrate. Measurement of the tensile bond strength confirmed a strong adhesion of the porous coating. Deposition of powders with different grain sizes resulted in porous titanium coatings with varying thickness, pore morphology, and surface roughness. The possibility to extend this coating technique to complex shaped implants is highlighted.     

A Printable Photopolymerizable Thermosensitive p(HPMAm‐lactate)‐PEG Hydrogel for Tissue Engineering

Bioprinting is a new technology in regenerative medicine that allows the engineering of tissues by specific placement of cells in biomaterials. Importantly, the porosity and the relatively small dimensions of the fibers allow rapid diffusion of nutrients and metabolites. This technology requires the availability of hydrogels that ensure viability of encapsulated cells and have adequate mechanical properties for the preparation of structurally stable and well‐defined three‐dimensional constructs. The aim of this study is to evaluate the suitability of a biodegradable, photopolymerizable and thermosensitive A–B–A triblock copolymer hydrogel as a synthetic extracellular matrix for engineering tissues by means of three dimensional fiber deposition. The polymer is composed of poly(N‐(2‐hydroxypropyl)methacrylamide lactate) A‐blocks, partly derivatized with methacrylate groups, and hydrophilic poly(ethylene glycol) B‐blocks of a molecular weight of 10 kDa. Gels are obtained by thermal gelation and stabilized with additional chemical cross‐links by photopolymerization of the methacrylate groups coupled to the polymer. A power law dependence of the storage plateau modulus of the studied hydrogels on polymer concentration is observed for both thermally and chemically cross‐linked hydrogels. The hydrogels demonstrated mechanical characteristics similar to natural semi‐flexible polymers, including collagen. Moreover, the hydrogel shows suitable mechanical properties for bioprinting, allowing subsequent layer‐by‐layer deposition of gel fibers to form stable constructs up to at least 0.6 cm (height) with different patterns and strand spacing. The resulting constructs have reproducible vertical porosity and the ability to maintain separate localization of encapsulated fluorescent microspheres. Moreover, the constructs show an elastic modulus of 119 kPa (25 wt% polymer content) and a degradation time of approximately 190 days. Furthermore, high viability is observed for encapsulated chondrocytes after 1 and 3 days of culture. In summary, we conclude that the evaluated hydrogel is an interesting candidate for bioprinting applications.