Quantitative aspects of two methods to dissolve collagen-free muscle proteins from acetone dry powders of Guelders ring sausage

Summary The purpose of this study was to find experimental conditions for the complete solubility of collagen-free muscle proteins (CFMP) using acetone powder of Guelders ring sausage. Preliminary experiments were carried out to choose the best procedure for preparing the acetone dry powder. Two different methods of acetone extraction of minced sausage were compared. The acetone dry mass (ADM) method using continuous extraction in a Soxhlet [2] apparatus gave better results than the acetone powder (ACP) method, which used a blender [1]. The ADM method was used for further investigations. ADM was extracted with two types of sodium dodecyl sulphate (SDS), containing solvents A and B. Solvent A contains a Tris-boric acid buffer (pH 8.2) with 1.5% (m/v) SDS and 0.05% (m/v) dithioerythreitol [3]. Solvent B is a borate-chloric acid buffer (pH 9.0) with 2.0% (m/v) SDS and 1.0% (m/v) mercapto-ethanol [2]. Both solvents showed a linear relationship between the quantities of CFMP in ADM and the dissolved CFMP. The linear relationships were found between quantities of 10.0 and 30.0 mg (solution A) and of 5.0 and 30.0 mg ADM (solution B) per ml solvent. The solubility of CFMP was better in solvent B than in solution A. Completely dissolved CFMP from ADM was only obtained in the case of 5.0 mg ADM in 1.0 ml solution B. These conditions will be used in liquid chromatography experiments, the results of which will be reported later.

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Quantitative Aspekte zweier Verfahren für das Auflösen kollagenfreier Muskelproteine aus acetontrockenen Pulvern der Gelderschen Rauchwurst

Zusammenfassung Der Zweck dieser Untersuchungen ist, die experimentellen Bedingungen für die vollstän-dige Löslichkeit des kollagenfreien Muskelproteins (CFMP) aus dem Acetonpulver der Gelderschen Rauchwurst zu finden. Durch Vorversuche wurde die beste Arbeitsweise für das Zubereiten des Acetonpulvers gewählt. Zwei verschiedene Extraktionsverfahren mit zerkleinertem Wurstmaterial wurden miteinander verglichen. Die Methode mit der Acetontrockenmasse (ADM) mittels kontinuierlicher Extraktion [2] führte zu besseren Ergebnissen als die Methode mit Acetonpulver (ACP), wozu ein Mischgerät [1] verwendet wurde. Die ADM-Methode wurde für weitere Untersuchungen angewendet. ADM wurde mit zwei verschiedenen Extraktionslösungen von Natrium-Dodecylsulfat (SDS) (A und B) extrahiert. Lösung A enthalt einen Tris-Borsäure Puffer (pH 8,2) mit SDS (1,5%) und Dithioerithritol (0,05%) [3]. Die Lösung B enthält einen Borat-Salzsäure Puffer (pH 9,0) mit SDS (2,0%) und Mercapto-Ethanol (1,0%) [2]. Beide Extraktionslösungen zeigen ein lineares Verhalten zwischen den Mengen vom CFMP in ADM und in aufgelöstem CFMP. Diese Linearität wurde von 10,030,0 mg ADM (Lösungsmittel A) und von 5,030,0 mg ADM (Lösungsmittel B) gefunden. Die Lös-lichkeit in Lösung B ist gegeniiber Lösung A besser. Ein vollständig gelöstes CFMP aus ADM wurde nur bei der Extraktion von 5,0 mg ADM in 1,0 ml der Losung B erhalten. Diese Bedingung soll in unseren künftigen flüssigchromatographischen Experimenten verwendet werden.

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Supported by a grant from the Hoofdinspectie Levensmiddelen of the Ministry of Welzijn, Volksgezondheid en Cultuur     

GCase and LIMP2 Abnormalities in the Liver of Niemann Pick Type C Mice

The lysosomal storage disease Niemann–Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1^−/− mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1^−/− liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1^−/− liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1^−/− liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1^−/− hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.     

Complement-Opsonized Nano-Carriers Are Bound by Dendritic Cells (DC) via Complement Receptor (CR)3, and by B Cell Subpopulations via CR-1/2, and Affect the Activation of DC and B-1 Cells

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.     

Early-Life Development of the Bifidobacterial Community in the Infant Gut

The establishment of the gut microbiota poses implications for short and long-term health. Bifidobacterium is an important taxon in early life, being one of the most abundant genera in the infant intestinal microbiota and carrying out key functions for maintaining host-homeostasis. Recent metagenomic studies have shown that different factors, such as gestational age, delivery mode, or feeding habits, affect the gut microbiota establishment at high phylogenetic levels. However, their impact on the specific bifidobacterial populations is not yet well understood. Here we studied the impact of these factors on the different Bifidobacterium species and subspecies at both the quantitative and qualitative levels. Fecal samples were taken from 85 neonates at 2, 10, 30, 90 days of life, and the relative proportions of the different bifidobacterial populations were assessed by 16S rRNA–23S rRNA internal transcribed spacer (ITS) region sequencing. Absolute levels of the main species were determined by q-PCR. Our results showed that the bifidobacterial population establishment is affected by gestational age, delivery mode, and infant feeding, as it is evidenced by qualitative and quantitative changes. These data underline the need for understanding the impact of perinatal factors on the gut microbiota also at low taxonomic levels, especially in the case of relevant microbial populations such as Bifidobacterium. The data obtained provide indications for the selection of the species best suited for the development of bifidobacteria-based products for different groups of neonates and will help to develop rational strategies for favoring a healthy early microbiota development when this process is challenged.     

Visceral Adipose Tissue Displays Unique Metabolomic Fingerprints in Obesity,Pre-Diabetes and Type 2 Diabetes

Visceral adipose tissue (VAT) metabolic profiling harbors the potential to disentangle molecular changes underlying obesity-related dysglycemia. In this study, the VAT exometabolome of subjects with obesity and different glycemic statuses are analyzed. The subjects (n = 19) are divided into groups according to body mass index and glycemic status: subjects with obesity and euglycemia (Ob+NGT, n = 5), subjects with obesity and pre-diabetes (Ob+Pre-T2D, n = 5), subjects with obesity and type 2 diabetes under metformin treatment (Ob+T2D, n = 5) and subjects without obesity and with euglycemia (Non-Ob, n = 4), used as controls. VATs are incubated in culture media and extracellular metabolite content is determined by proton nuclear magnetic resonance (¹H-NMR). Glucose consumption is not different between the groups. Pyruvate and pyroglutamate consumption are significantly lower in all groups of subjects with obesity compared to Non-Ob, and significantly lower in Ob+Pre-T2D as compared to Ob+NGT. In contrast, isoleucine consumption is significantly higher in all groups of subjects with obesity, particularly in Ob+Pre-T2D, compared to Non-Ob. Acetate production is also significantly lower in Ob+Pre-T2D compared to Non-Ob. In sum, the VAT metabolic fingerprint is associated with pre-diabetes and characterized by higher isoleucine consumption, accompanied by lower acetate production and pyruvate and pyroglutamate consumption. We propose that glucose metabolism follows different fates within the VAT, depending on the individuals’ health status.     

Gezielte Nutzung biologischer Systeme

Directed exploitation of biological systems . During the past few decades, our knowledge of molecular process responsible for genetics has increased dramatically. Discovery of the giant molecule deoxyribonucleic acid – abbreviated as DNA – as the carrier of genetic information heralded in a development which nowadays permits us to effect directed changes in the genetic material of an organism. Thus we can provide easily cultured microorganisms with genes which were previously located in a completely different genetic environment. This helps us to obtain high yields of proteins or other substances which were formerly very difficult to obtain. Higher organisms such as animals and plants can also undergo modification of their genetic equipment. This adds a new dimension to the breeding of such species. Above all, genetic engineering provides new insights into the enormously complex interplay of molecules which go to make up a living cell. The resulting understanding of life processes on a molecular level permits recognition of malfunction and therapy of the ensuring disease by new drugs. In addition to these positive aspects, genetic engineering provides scope for conducting experiments whose ethical implications demand very earnest consideration.     

Characterisation of mortar morphology in thin sections by digital image processing

A digital imaging approach was applied to investigate mortar morphology in thin sections; in particular, the binder/aggregate ratio and the grading curve of five mortar bars were attained by digital image processing (DIP), using the Image Pro Plus 4.1 software package. The imaging procedure employed image segmentation, to extract mortar aggregate, and image filtering, to fix grain boundaries. The results show that digital image processing may be considered as an alternative method to mechanical sieving for the characterisation of mortar morphology, as it appears to be quicker and more accurate than the traditional method. However, digital image processing exhibits limits, which are discussed in the text.     

(CO2 + 2‐propanol) mixture as a foaming agent for polystyrene: A simple thermodynamic model for the high pressure VLE‐phase diagrams taking into account the foam vitrification

The equation of state model developed by Lacombe and Sanchez (J Phys Chem 1976, 80, 2352) is used in the form proposed later by Sanchez and Stone (Polymer Blends, Vol. 1: Formulation, 2000; Chapter 2) to correlate experimental vapor‐liquid equilibrium (VLE) data for the three binaries and the ternary systems. Experimental data from the binary systems carbon dioxide‐isopropyl alcohol (CO₂‐IPrOH), isopropyl alcohol‐polystyrene (IPrOH‐PS), and carbon dioxide‐polystyrene (CO₂‐PS) are used to calculate VLE properties for the ternary system CO₂‐IPrOH‐PS. Two‐dimensional VLE‐phase diagrams were calculated and used to describe from a thermodynamic point of view the pressure, volume, and temperature values that characterize a thermoplastic foam evolution process, from the extruder to the foaming die. For different initial mixture CO₂ + IPrOH concentrations, pressure reduction produces liquid foaming until the vitrification curve arrests the final foam volume expansion. The dependence of the vitreous transition with the system CO₂ + IPrOH concentration while foaming is represented by the Chow (Macromolecules 1980, 13, 362) equation. The calculation procedure is proposed as a design tool to reduce the amount of experimental data usually needed as a requirement previous to the design stage. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 2663–2671, 2007     

A low-cost real time virtual system for postural stability assessment at home

Background and objective

The degeneration of the balance control system in the elderly and in many pathologies requires measuring the equilibrium conditions very often. In clinical practice, equilibrium control is commonly evaluated by using a force platform (stabilometric platform) in a clinical environment.In this paper, we demonstrate how a simple movement analysis system, based on a 3D video camera and a 3D real time model reconstruction of the human body, can be used to collect information usually recorded by a physical stabilometric platform.

Methods

The algorithm used to reconstruct the human body model as a set of spheres is described and discussed. Moreover, experimental measurements and comparisons with data collected by a physical stabilometric platform are also reported. The measurements were collected on a set of 6 healthy subjects to whom a change in equilibrium condition was stimulated by performing an equilibrium task.

Results

The experimental results showed that more than 95% of data collected by the proposed method were not significantly different from those collected by the classic platform, thus confirming the usefulness of the proposed system.

Conclusions

The proposed virtual balance assessment system can be implemented at low cost (about 500$) and, for this reason, can be considered a home use medical device. On the contrary, astabilometric platform has a cost of about 10,000$ and requires periodical calibration. The proposed system does not require periodical calibration, as is necessary for stabilometric force platforms, and it is easy to use. In future, the proposed system with little integration can be used, besides being an emulator of a stabilometric platform, also to recognize and track, in real time, head, legs, arms and trunk, that is to collect information actually obtained by sophisticated optoelectronic systems.