Cholesterol metabolism is associated with soluble amyloid precursor protein production in Alzheimer's disease

Disturbances of the cholesterol metabolism are associated with Alzheimer's disease (AD) risk and related cerebral pathology. Experimental studies found changing levels of cholesterol and its metabolites 24S‐hydroxycholesterol (24S‐OHC) and 27‐hydroxycholesterol (27‐OHC) to contribute to amyloidogenesis by increasing the production of soluble amyloid precursor protein (sAPP). The aim of this study was to evaluate the relationship between the CSF and circulating cholesterol 24S‐OHC and 27‐OHC, and the sAPP production as measured by CSF concentrations of sAPP forms in humans. The plasma and the CSF concentrations of cholesterol, 24S‐OHC and 27‐OHC, and the CSF concentrations of sAPPα, sAPPβ, and Aß1‐42 were assessed in subjects with AD and controls with normal cognition. In multivariate regression tests including age, gender, albumin ratio, and apolipoprotein E (APOE)ε4 status CSF cholesterol, 24S‐OHC, and 27‐OHC independently predicted the concentrations of sAPPα and sAPPβ. The associations remained significant when analyses were separately performed in the AD group. Furthermore, plasma 27‐OHC concentrations were associated with the CSF sAPP levels. The results suggest that high CSF concentrations of cholesterol, 24S‐OHC, and 27‐OHC are associated with increased production of both sAPP forms in AD.     

Humanization of Chicken-Derived scFv Using Yeast Surface Display and NGS Data Mining

Generation of high-affinity monoclonal antibodies by immunization of chickens is a valuable strategy, particularly for obtaining antibodies directed against epitopes that are conserved in mammals. A generic procedure is established for the humanization of chicken-derived antibodies. To this end, high-affinity binders of the epidermal growth factor receptor extracellular domain are isolated from immunized chickens using yeast surface display. Complementarity determining regions (CDRs) of two high-affinity binders are grafted onto a human acceptor framework. Simultaneously, Vernier zone residues, responsible for spatial CDR arrangement, are partially randomized. A yeast surface display library comprising ≈300 000 variants is screened for high-affinity binders in the scFv and Fab formats. Next-generation sequencing discloses humanized antibody variants with restored affinity and improved protein characteristics compared to the parental chicken antibodies. Furthermore, the sequencing data give new insights into the importance of antibody format, used during the humanization process. Starting from the antibody repertoire of immunized chickens, this work features an effective and fast high-throughput approach for the generation of multiple humanized antibodies with potential therapeutic relevance.     

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.     

Effects of Temperature, Poststaining Rinses and Ethanol-Butanol Dehydrating Mixtures on Methyl Green-Pyronin Staining

Methyl green GA (Chroma) and pyronin GS (Chroma) were used. Procedure recommended: Stain for 1 hr at 37 C in a purified 0.5% aqueous methyl green, buffered to pH 4.1 with Walpoles acetate buffer, and containing 0.2% pyronin; rinse for 1-2 sec in ice-cold distilled water; blot sections evenly, and rinse with vigorous agitation in t-butanol; dehydrate in 2 changes of t-butanol for 5 min each; clear in xylene and mount. This technique results in a consistent staining pattern for qualitative nucleic acid differentiation, whereas older methods have been only partly satisfactory. Rinsing in ice-cold water is a critical step; t-butanol was superior to n-butanol and to ethanol-butanol mixtures for dehydration. Staining at 25-27 C is feasible hut less effective.     

Suppression and augmentation of the primary in vitro immune response by different classes of antibodies

The capacity of purified γG₁ and γG₂ anti-sheep red blood cell (SRBC) antibodies to exert antigen-specific feedback regulations on the primary in vitro immune response to SRBC was studied. Antibodies were administered to the culture in the native form, as sheep erythrocyte-antibody complexes or as pepsin-derived F(ab′)₂ antibody fragments. Marked differences in the feedback regulatory effects of γG₁ and γG₂ antibodies were found. Antibodies of the γG₁ class suppressed the immune response to SRBC, whereas γG₂ antibodies isolated from the same serum exerted an augmenting effect on antibody synthesis. These opposing feedback effects on in vitro antibody synthesis were immunologically specific, relatively insensitive to changes in antigen concentrations, and could be elicited by either adding antibodies and antigen separately to the culture or as preformed antigen-antibody complexes. Experiments comparing the activities of the F(ab′)₂ antibody fragments with the parent γG₁ and γG₂ antibodies suggested that the Fc fragments may be involved in these regulatory effects on the immune response. It is concluded that the antigen-specific suppressive and augmenting effects on antibody synthesis shown here are determined by the antibody class. In addition, we suggest that these opposing antibody-mediated feedback effects may represent one of the important elements of the immune response.     

Types and Molecular Weights of Subunits of Arachin-P6

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that arachin-P6 contained nine types of subunits. The subunits were designated K15 (15, 069), K17 (17, 334), K20 (20, 000), K22 (21, 750), K24 (23, 557), K29 (29, 019), K37 (37, 161), K41 (41, 326) and K43 (43, 104) and molecular weight of each is given in parenthesis following its name. This observation was qualitatively comparable to the results of electrophoretic analysis of arachin-P6 conducted in the presence of urea.     

Hyperinsulinemia in obesity: significance of adipose tissue cell size

Adipose tissue biopsy and an oral glucose tolerance test (OGTT) (50 g) were performed in 17 non-hyperlipoproteinemic subjects without overt diabetes mellitus. All the persons were weight stable at the time of investigation. A significant correlation between fasting insulin concentration and the mean adipocyte size was observed, whereas no correlation was noted between the ideal body weight index and fasting insulin level. Persons with larger adipocytes had elevated insulin levels as well as higher and longer lasting increments following the glucose challenge. They also exhibited significantly higher mean glucose levels during the OGTT. When these patients were matched for glucose tolerance with the subgroup having smaller mean adipocyte sizes, the difference in insulin levels was still demonstrable. The importance of adipose cell enlargement regulating basal and stimulated insulin output is underlined.     

Power management by load forecasting in web server clusters

The complexity and requirements of web applications are increasing in order to meet more sophisticated business models (web services and cloud computing, for instance). For this reason, characteristics such as performance, scalability and security are addressed in web server cluster design. Due to the rising energy costs and also to environmental concerns, energy consumption in this type of system has become a main issue. This paper shows energy consumption reduction techniques that use a load forecasting method, combined with DVFS (Dynamic Voltage and Frequency Scaling) and dynamic configuration techniques (turning servers on and off), in a soft real-time web server clustered environment. Our system promotes energy consumption reduction while maintaining user’s satisfaction with respect to request deadlines being met. The results obtained show that prediction capabilities increase the QoS (Quality of Service) of the system, while maintaining or improving the energy savings over state-of-the-art power management mechanisms. To validate this predictive policy, a web application running a real workload profile was deployed in an Apache server cluster testbed running Linux.