Identification of anchor points for chemical modification of a small cysteine-rich protein by using a cysteine scan

Chemical modifications of proteins are increasingly important in the development of protein drugs with fine-tuned properties. Regioselective modification, such as the chemoselective alkylation of an unpaired cysteine residue, is a prerequisite for obtaining homogenous protein products. The introduction of an unpaired Cys into the Cys-rich protein, insulin, was investigated by using a Cys scan. This was challenging as the introduced Cys could interfere with insulin's three existing disulfide bonds. However, eight insulin precursors were expressed in Saccharomyces cerevisiae with good yields. Although extensive post-translational modifications of the unpaired Cys were observed, the majority could be removed by selective reduction. An example Cys(7) insulin analogue was modified with a PEGylated maleimide moiety. The new variant was active in in vitro and in vivo models. Our results show that even small Cys-rich proteins can be expressed with additional unpaired Cys in meaningful yields and further chemically modified, while maintaining their biological activity.     

Protein quality and energy density of green peas as influenced by seed size and time of harvest

Green peas (Pisum sativum L) were harvested at three different time points—3 days apart—and subsequently sorted into four fractions according to seed size (diameter > 10.2 mm; 10.2–8.75 mm; 8.75–8.2 mm and 8.2–6.0 mm). Three varieties from two years were investigated. The study comprised a total of 31 green pea samples. The average protein content of the samples was 276 g kg^?1 DM with relatively low variation (SE = 5.7). Protein content was highest in the smallest seeds. The average energy content was 18.6 MJ kg^?1DM. Dietary fibre content was 226 g kg^?1 DM, the major part (77%) as insoluble fibre. Seeds of early harvest and seeds with the smallest diameter had the lowest fibre (IDF and TDF) content and at the same time the highest energy digestibility. The average energy digestibility was 83.2%. Protein digestibility was relatively high (average 92.1%), while biological value (BV) and net protein utilisation (NPU) were moderate to low (60.6% and 55.8% respectively) due to low contents of the essential amino acids (g per 16 g N), especially tryptophan, methionine + cystine and lysine. Contents of lysine, tryptophan and methionine + cystine were more than 20% higher in the smallest seeds compared to the larger seeds. Similar differences were observed in seeds of late harvest as compared with seeds of early harvest. Correlation coefficients revealed a strong positive correlation between lysine, tryptophan and methionine + cystine content and the biological value of green pea protein. The three varieties investigated showed no differences in the chemical parameters although small differences in BV and NPU were observed.     

Identification of H₂O₂ as a major antimicrobial component in coffee

Coffee shows distinct antimicrobial activity against several bacterial genera. The present study investigated molecular mechanisms and active ingredients mediating the antimicrobial effect of coffee. Depending on concentration, roasted, but not raw coffee brew inhibited the growth of Escherichia coli and Listeria innocua. Several coffee ingredients with known antibacterial properties were tested for their contribution to the observed effect. In natural concentration, caffeine, ferulic acid and a mixture of all test compounds showed very weak, but significant activity, whereas trigonelline, 5-(hydroxymethyl)furfural, chlorogenic acid, nicotinic acid, caffeic acid, and methylglyoxal were not active. Antimicrobial activity, however, was completely abolished by addition of catalase indicating that H(2)O(2) is a major antimicrobial coffee component. In accordance with this assumption, bacterial counts during 16 h of incubation were inversely related to the H(2)O(2) concentration in the incubation solution. Pure H(2)O(2) showed slightly weaker activity. The H(2)O(2) dependent antimicrobial activity of coffee could be mimicked by a reaction mixture of d-ribose and l-lysine (30 min 120 °C) indicating that H(2)O(2) is generated in the coffee brew by Maillard reaction products. Identification of H(2)O(2) as major antimicrobial coffee component is important to evaluate the application of coffee or coffee extracts as natural preservatives.     

Heparinization of a biomimetic bone matrix: integration of heparin during matrix synthesis versus adsorptive post surface modification

This study intended to evaluate a contemporary concept of scaffolding in bone tissue engineering in order to mimic functions of the extracellular matrix. The investigated approach considered the effect of the glycosaminoglycan heparin on structural and biological properties of a synthetic biomimetic bone graft material consisting of mineralized collagen. Two strategies for heparin functionalization were explored in order to receive a three-component bone substitute material. Heparin was either incorporated during matrix synthesis by mixing with collagen prior to simultaneous fibril reassembly and mineralization (in situ) or added to the matrix after fabrication (a posteriori). Both methods resulted in an incorporation of comparable amounts of heparin, though its distribution in the matrix varied as indicated by TOF-SIMS analyses, and a similar modulation of their protein binding properties. Differential scanning calorimetry revealed that the thermal stability and thereby the degree of crosslinking of the heparinized matrices was increased. However, in contrast to the a posteriori modification, the in situ integration of heparin led to considerable changes of morphology and composition of the matrix: a more open network of collagen fibers yielding a more porous surface and a reduced mineral content were observed. Cell culture experiments with human mesenchymal stem cells (hMSC) revealed a strong influence of the mode of heparin functionalization on cellular processes, as demonstrated for proliferation and osteogenic differentiation of hMSC. Our results indicate that not only heparin per se but also the way of its incorporation into a collagenous matrix determines the cell response. In conclusion, the a posteriori modification was beneficial to support adhesion, proliferation and differentiation of hMSC.     

Effects of prenatal exposure to diesel exhaust particles on postnatal development, behavior, genotoxicity and inflammation in mice

Background

Results from epidemiological studies indicate that particulate air pollution constitutes a hazard for human health. Recent studies suggest that diesel exhaust possesses endocrine activity and therefore may affect reproductive outcome. This study in mice aimed to investigate whether exposure to diesel exhaust particles (DEP; NIST 2975) would affect gestation, postnatal development, activity, learning and memory, and biomarkers of transplacental toxicity. Pregnant mice (C57BL/6; BomTac) were exposed to 19 mg/m³ DEP (~1·10⁶ particles/cm³; mass median diameter ? 240 nm) on gestational days 9–19, for 1 h/day.

Results

Gestational parameters were similar in control and diesel groups. Shortly after birth, body weights of DEP offspring were slightly lower than in controls. This difference increased during lactation, so by weaning the DEP exposed offspring weighed significantly less than the control progeny. Only slight effects of exposure were observed on cognitive function in female DEP offspring and on biomarkers of exposure to particles or genotoxic substances.

Conclusion

In utero exposure to DEP decreased weight gain during lactation. Cognitive function and levels of biomarkers of exposure to particles or to genotoxic substances were generally similar in exposed and control offspring. The particle size and chemical composition of the DEP and differences in exposure methods (fresh, whole exhaust versus aged, resuspended DEP) may play a significant role on the biological effects observed in this compared to other studies.     

Monitoring success of remediation: seven case studies of moisture and mold damaged buildings

Based on seven case studies of buildings that underwent different degrees of moisture and mold damage remediation, we aimed to develop methodology for assessment of the success of the remediation process. Methods used in gauging the success included technical monitoring of performance of building structures and heating, ventilation and air conditioning (HVAC) systems, microbial monitoring of indoor air quality (IAQ), and health effects studies of building occupants. The assessment was based on measurable change in the situations before and after remediation. Based on technical monitoring, remediation was successful in three cases, with partial improvement noted in three cases, whereas no remediation was conducted in one case. Based on microbial monitoring, improvement was detected in one, partial improvement in two and no improvement in two cases, whereas no follow-up was conducted in two cases. Health effect studies (mainly self-reported health status) showed improvement in one case, partial improvement in two cases, and no improvement in two cases, whereas no follow-up was conducted in one case, and in one case, follow-up failed due to low response rate. The results illustrate that it is possible to monitor the effects of remediation using various metrics. However, in some cases, no improvement could be observed in IAQ or occupant health, even if the remediation was considered technically successful, i.e. the remediation was fully completed as recommended. This could be due to many reasons, including: 1) all damage may not have been addressed adequately; 2) IAQ or health may not have been perceived improved regardless of remediation; and/or 3) the methods used may not have been sensitive/specific enough to detect such improvement within the 6-12 months follow-up periods after completion of the remediation. There is a need to further develop tools for monitoring and assessment of the success of moisture damage remediation in buildings.     

“To Catch or Not to Catch”: Microcapsule‐Based Sandwich Assay for Detection of Proteins and Nucleic Acids

The development of new assays that specifically detect intermediate or final products of biotechnological processes or multiple analytes in biomedical research is important for diagnostics and biotechnology. This study presents a microcapsule‐based sandwich assay for detection of proteins and nucleic acids using flow cytometry as an optical readout. The main component of the assay are robust chemically cross‐linked microcapsules that are coated with adaptor proteins such as protein A or streptavidin. In the first approach, the ability of detecting the blood cancer biomarker beta‐2 microglobulin in the fM to pM concentration range with the help of protein A‐coated capsules is demonstrated. In the second approach, streptavidin‐coated capsules are used for detection of nucleic acids in the nM concentration range. The developed assay allows rapid quantitative analyte measurement, while providing high sensitivity and selectivity at very small sample quantities. In the future, protein A‐ and streptavidin‐coated microcapsules can be used as universal tools for detection of a broad range of analytes.     

Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver

Background

Widespread occupational exposure to carbon black nanoparticles (CBNPs) raises concerns over their safety. CBNPs are genotoxic in vitro but less is known about their genotoxicity in various organs in vivo.

Methods

We investigated inflammatory and acute phase responses, DNA strand breaks (SB) and oxidatively damaged DNA in C57BL/6 mice 1, 3 and 28 days after a single instillation of 0.018, 0.054 or 0.162 mg Printex 90 CBNPs, alongside sham controls. Bronchoalveolar lavage (BAL) fluid was analyzed for cellular composition. SB in BAL cells, whole lung and liver were assessed using the alkaline comet assay. Formamidopyrimidine DNA glycosylase (FPG) sensitive sites were assessed as an indicator of oxidatively damaged DNA. Pulmonary and hepatic acute phase response was evaluated by Saa3 mRNA real-time quantitative PCR.

Results

Inflammation was strongest 1 and 3 days post-exposure, and remained elevated for the two highest doses (i.e., 0.054 and 0.162 mg) 28 days post-exposure (P < 0.001). SB were detected in lung at all doses on post-exposure day 1 (P < 0.001) and remained elevated at the two highest doses until day 28 (P < 0.05). BAL cell DNA SB were elevated relative to controls at least at the highest dose on all post-exposure days (P < 0.05). The level of FPG sensitive sites in lung was increased throughout with significant increases occurring on post-exposure days 1 and 3, in comparison to controls (P < 0.001-0.05). SB in liver were detected on post-exposure days 1 (P < 0.001) and 28 (P < 0.001). Polymorphonuclear (PMN) cell counts in BAL correlated strongly with FPG sensitive sites in lung (r = 0.88, P < 0.001), whereas no such correlation was observed with SB (r = 0.52, P = 0.08). CBNP increased the expression of Saa3 mRNA in lung tissue on day 1 (all doses), 3 (all doses) and 28 (0.054 and 0.162 mg), but not in liver.

Conclusions

Deposition of CBNPs in lung induces inflammatory and genotoxic effects in mouse lung that persist considerably after the initial exposure. Our results demonstrate that CBNPs may cause genotoxicity both in the primary exposed tissue, lung and BAL cells, and in a secondary tissue, the liver.