Catalytic Amyloids as Novel Synthetic Hydrolases

Amyloids are supramolecular assemblies composed of polypeptides stabilized by an intermolecular beta-sheet core. These misfolded conformations have been traditionally associated with pathological conditions such as Alzheimer’s and Parkinson´s diseases. However, this classical paradigm has changed in the last decade since the discovery that the amyloid state represents a universal alternative fold accessible to virtually any polypeptide chain. Moreover, recent findings have demonstrated that the amyloid fold can serve as catalytic scaffolds, creating new opportunities for the design of novel active bionanomaterials. Here, we review the latest advances in this area, with particular emphasis on the design and development of catalytic amyloids that exhibit hydrolytic activities. To date, three different types of activities have been demonstrated: esterase, phosphoesterase and di-phosphohydrolase. These artificial hydrolases emerge upon the self-assembly of small peptides into amyloids, giving rise to catalytically active surfaces. The highly stable nature of the amyloid fold can provide an attractive alternative for the design of future synthetic hydrolases with diverse applications in the industry, such as the in situ decontamination of xenobiotics.     

Caspase-1 Inhibition Impacts the Formation of Chondrogenic Nodules,and the Expression of Markers Related to Osteogenic Differentiation and Lipid Metabolism

Caspase-1, as the main pro-inflammatory cysteine protease, was investigated mostly with respect to inflammation-related processes. Interestingly, caspase-1 was identified as being involved in lipid metabolism, which is extremely important for the proper differentiation of chondrocytes. Based on a screening investigation, general caspase inhibition impacts the expression of Cd36 in chondrocytes, the fatty acid translocase with a significant impact on lipid metabolism. However, the engagement of individual caspases in the effect has not yet been identified. Therefore, the hypothesis that caspase-1 might be a candidate here appears challenging. The primary aim of this study thus was to find out whether the inhibition of caspase-1 activity would affect Cd36 expression in a chondrogenic micromass model. The expression of Pparg, a regulator Cd36, was examined as well. In the caspase-1 inhibited samples, both molecules were significantly downregulated. Notably, in the treated group, the formation of the chondrogenic nodules was apparently disrupted, and the subcellular deposition of lipids and polysaccharides showed an abnormal pattern. To further investigate this observation, the samples were subjected to an osteogenic PCR array containing selected markers related to cartilage/bone cell differentiation. Among affected molecules, Bmp7 and Gdf10 showed a significantly increased expression, while Itgam, Mmp9, Vdr, and Rankl decreased. Notably, Rankl is a key marker in bone remodeling/homeostasis and thus is a target in several treatment strategies, including a variety of fatty acids, and is balanced by its decoy receptor Opg (osteoprotegerin). To evaluate the effect of Cd36 downregulation on Rankl and Opg, Cd36 silencing was performed using micromass cultures. After Cd36 silencing, the expression of Rankl was downregulated and Opg upregulated, which was an inverse effect to caspase-1 inhibition (and Cd36 upregulation). These results demonstrate new functions of caspase-1 in chondrocyte differentiation and lipid metabolism-related pathways. The effect on the Rankl/Opg ratio, critical for bone maintenance and pathology, including osteoarthritis, is particularly important here as well.     

Starch as a Pore‐forming and Body‐forming Agent in Ceramic Technology

Wheat and corn starch can be used for the preparation of porous alumina ceramics via the SCC (starch consolidation casting) process, resulting in porosities ranging from > 20% to approx. 50% (using nominal starch contents of 10 – 50%, v/v), with open porosity dominating (closed porosity < 6.5%). The character of porosity and the shape of the pores corresponds to the starch granules used, but the pore size is determined by a complex interplay between starch swelling (during the body‐forming step) and pore shrinkage (during sintering of the ceramic). Typically, for low starch contents (e.g. nominal starch contents of around 10%, v/v) starch swelling is a significant effect, and the pores after sintering are larger than the size of the starch granules. For higher starch contents swelling is constrained (by limited space and/ or water availability), and the matrix shrinkage during sintering overcompensates the swelling effect, so that the final pores in the ceramic can be significantly smaller than the original starch granule size. In this paper it is shown how porosity is related to pore size. In particular, it is demonstrated that the porosity indirectly determined from image analysis (via the median pore size) is closely related to the porosity directly measured via the Archimedes method. On the other hand, mercury porosimetry measures the distribution of pore throat sizes. With increasing starch content in the suspension, the pore throat size in the as‐fired ceramic materials increases, resulting in a more open microstructure.     

Real-time monitoring and diagnosing in wire-electro discharge machining

This paper presents the design and development of a real-time monitoring and diagnostic system for diagnosing the degraded behaviour in wire-electro discharge machining. The detection in advance of the degraded behaviour is crucial since this can lead to the breakage of the cutting tool (the wire), reducing the process productivity and the required accuracy (Ho et al., Tools Manuf 44:1247–1259, 2004). This work presents the design and development of a real-time monitoring system that alerts the degraded behaviour. It can detect different types of degraded behaviours that have been previously identified during the analysis phase. Unlike other works found in the literature review, which are focussed on proprietary hardware, the present paper proposes a flexible real-time platform based on a commercial data acquisition board that can be easily configured for different purposes. It has been applied to develop a real-time monitoring and diagnostic system that uses virtual sensors to diagnose the degradation of the process. The results of this work show a satisfactory performance of the presented approach.     

Confocal microscopy reveals Myzitiras and Vthela morphotypes as new signatures of malignancy progression

G3S1 cells are a new line derived from EM‐G3 breast cancer cells by chronic nutritional stress and treatments with 12‐O‐tetradecanoylphorbol‐13‐acetate. These cells are capable of growing in standard medium. G3S1 cells exhibited elevated invasiveness in Matrigel invasion chambers as compared with parental EM‐G3 cells. Elevated invasiveness of G3S1 cells was accompanied by higher incidence of myzitiras morphotype (sucker‐like) and newly observed vthela morphotype (leech‐like) both inducible in Hanks' Balanced Salt Solution test. Time‐lapse phase contrast microscopy showed a capacity of G3S1 cells to form lobopodial protrusions already 20 min after seeding on gelatin. These protrusions could make contact with the dish and possibly produce the vthela shape. The possible relationship of mysitiras and vthela morphotypes to an increase in malignant potential marked by enhanced invasiveness was thus indicated. SCANNING 31: 102–106, 2009. © 2009 Wiley Periodicals, Inc.     

Effect of enterocin AS-48 in combination with high-intensity pulsed-electric field treatment against the spoilage bacterium Lactobacillus diolivorans in apple juice

Enterocin AS-48 was tested in apple juice against the cider-spoilage, exopolysaccharide-producing strain Lactobacillus diolivorans 29 in combination with high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 150 Hz, 4 μs and bipolar mode). A response surface methodology was applied to study the bactericidal effects of the combined treatment, with AS-48 concentration and HIPEF treatment time as process variables. At subinhibitory bacteriocin concentrations, microbial inactivation by the combined treatment increased as the bacteriocin concentration and the HIPEF treatment time increased (from 0.5 to 2.0 μg/ml and from 100 to 1000 μs, respectively). Highest inactivation (4.87 logs) was achieved by 1000 μs HIPEF treatment in combination with 2.0 μg/ml AS-48. While application of treatments separately did not protect juice from survivors during storage, survivors to the combined treatment were inactivated within the following 24 h of storage, and the treated samples remained free from detectable lactobacilli for at least 15 days at temperatures of 4 °C as well as 22 °C. The combined treatment could be useful for inactivation of exopolysaccharide-producing L. diolivorans in apple juice.     

Determination of biogenic amines in foods using ultra-performance liquid chromatography (UPLC)

A rapid ultra-performance liquid chromatographic (UPLC) method for the determination of biogenic amines (putrescine, cadaverine, spermidine, spermine, phenylethylamine, histamine, tyramine and tryptamine) in selected food samples is described. The eight biogenic amines, which are the most important to be determined in food samples, were derivatized with dansyl chloride prior to UPLC separation. The dansylated amines were separated on an Agilent Zorbax Eclipse XDB – C18 column (50 × 4.6 mm ID, 1.8 μm) using gradient elution with a binary system of acetonitrile–water, a flow rate of 1.0 ml/min and UV detection at 225 nm. The analysis is very fast, all amines are well resolved and are eluted from the column in less than 6 min. The average repeatability of the method ranged between 1.02% and 2.14%. Limits of detection (LODs) for considered amines ranged between 0.032 and 0.098 μg/l; calibration curves showed very good linearity (r = 0.9994–1.0000). The method was applied to the analysis of amines in pork, beef, chicken and fish meat, cheese and edible mushrooms.     

Rapid and Sensitive Detection of Staphylococcus aureus in Food Using Selective Enrichment and Real-Time PCR Targeting a New Gene Marker

Staphylococcus aureus is a bacterial pathogen considered a principal etiological agent of food poisoning. The aim of this study was to develop and evaluate a rapid and sensitive method for the detection of S. aureus in food by using selective enrichment and a new species-specific real-time polymerase chain reaction (PCR). Specific primers and a TaqMan probe targeted to specific S. aureus gene encoding for acriflavine resistance protein were designed. The real-time PCR was highly specific for S. aureus with 100% inclusivity and 100% exclusivity determined using 83 S. aureus strains and 64 non-S.-aureus strains. PCR detection limit of 6.8 × 10¹ and 3.4 × 10¹ CFU ml⁻¹ were obtained with 100% and 70% detection probability, respectively. The single selective enrichment based on the study of different enrichment conditions was selected and a lysis by boiling was used to obtain bacterial DNA. Out of 112 food samples analyzed, 61 were positive by the PCR-based method and 53 by the standard method. Out of ten food matrices artificially contaminated at a level of 10° CFU g⁻¹, ten and six were positive by the respective methods. Moreover, 10° CFU 10 g⁻¹ was detected in all ten artificially contaminated samples after a large-scale enrichment using PCR-based detection, in contrast to seven false negative by standard detection. The developed method facilitated the detection of S. aureus on the next day after the sample reception. This method can be used for S. aureus detection as a faster, highly specific, and more sensitive alternative to microbiological method with the potential for providing of improved food-processing hygiene control.     

The correlation between 1H MRS choline concentrations and MR diffusion trace values in human brain tumors

Objective  The aim of this study was to develop a method for evaluating the spatial distribution of human brain gliomas in individual subjects by evaluating the correlation between the Choline (Cho) signal intensity and the diffusion trace (Tr_ADC) values. Materials and methods  Eleven patients with different histopathologic diagnoses and five healthy subjects were examined with diffusion-weighted EPI-trace sequence and ¹H MR spectroscopic imaging. The calculation of the correlation between choline and Tr_ADC values on a pixel-by-pixel basis and simulations estimating the influence of partial volume effects on the result were performed. Results  Statistical evaluation of the data in the patients with a glioblastoma showed that pixels corresponding to different tissue states are situated in different areas in the Cho–Tr_ADC correlation graph. Namely, points forming an inverse linear dependence interpreted as an area of an active tumor were observed. Different types of correlations were found in grade II and III gliomas. No statistically significant correlation was found in healthy subjects. Simulations proved that the observed linear dependence cannot be attributed solely to partial volume effects. Conclusion  The analysis of the correlation between Cho concentrations and Tr_ADC values on a pixel-by-pixel basis should help the regional identification of the pathological state of a tissue in patients with a glioblastoma.     

Kidney-Protector Lipidic Cilastatin Derivatives as Structure-Directing Agents for the Synthesis of Mesoporous Silica Nanoparticles for Drug Delivery

Mesoporous silica nanomaterials have emerged as promising vehicles in controlled drug delivery systems due to their ability to selectively transport, protect, and release pharmaceuticals in a controlled and sustained manner. One drawback of these drug delivery systems is their preparation procedure that usually requires several steps including the removal of the structure-directing agent (surfactant) and the later loading of the drug into the porous structure. Herein, we describe the preparation of mesoporous silica nanoparticles, as drug delivery systems from structure-directing agents based on the kidney-protector drug cilastatin in a simple, fast, and one-step process. The concept of drug-structure-directing agent (DSDA) allows the use of lipidic derivatives of cilastatin to direct the successful formation of mesoporous silica nanoparticles (MSNs). The inherent pharmacological activity of the surfactant DSDA cilastatin-based template permits that the MSNs can be directly employed as drug delivery nanocarriers, without the need of extra steps. MSNs thus synthesized have shown good sphericity and remarkable textural properties. The size of the nanoparticles can be adjusted by simply selecting the stirring speed, time, and aging temperature during the synthesis procedure. Moreover, the release experiments performed on these materials afforded a slow and sustained drug release over several days, which illustrates the MSNs potential utility as drug delivery system for the cilastatin cargo kidney protector. While most nanotechnology strategies focused on combating the different illnesses this methodology emphasizes on reducing the kidney toxicity associated to cancer chemotherapy.