Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation

Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advates, Helixate NexGens and Refactos), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGens showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refactos showed glutathione S-transferase and b-lactamase, whereas Advates apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants.L'artcoo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com

Proteomics, pigment composition, and organization of thylakoid membranes in iron-deficient spinach leaves

The changes induced in the photosynthetic apparatus of spinach (Spinacia oleracea L.) seedlings exposed to iron deficiency shortly after germination were characterized with two proteomic approaches coupled with chlorophyll and xanthophyll analysis and in vivo measurements of photosynthesis. During the first 10 d of iron deficiency the concentrations of chlorophyll b and violaxanthin were greatly reduced, but all xanthophylls recovered after 13-17 d of iron deficiency, when both chlorophylls were negatively affected. No new protein was formed in iron-deficient leaves, and no protein disappeared altogether. Photosystem I (PSI) proteins were largely reduced, but the stoichiometry of the antenna composition of PSI was not compromised. On the contrary, PSII proteins were less affected by the stress, but the specific antennae Lhcb4 and Lhcb6, Lhcb2 and its isoform Lhcb1.1 were all reduced, while the concentration of Lhcb3 increased. A strong reduction in thylakoid bending and an altered distribution pattern for the reduced PSI and PSII complexes were observed microscopically in iron-deficient leaves. Supercomplex organization was also affected by the stress. The trimeric organization of Lhcb and the dimerization of Lhca were reduced, while monomerization of Lhcb increased. However, the trimerization of Lhcb was partially recovered after 13-17 d of iron deficiency. In iron-deficient leaves, photosynthesis was strongly inhibited at different light intensities, and a high de-epoxidation status of the xanthophylls was observed, in association with a strong impairment of photochemical efficiency and an increase of heat dissipation as monitored by the non-photochemical quenching of fluorescence. All these negative effects of iron deficiency were attenuated but not fully reversed after again supplying iron to iron-deficient leaves for 7-13 d. These results indicate that iron deficiency has a strong impact on the proteomic structure of spinach photosystems and suggest that, in higher plants, adaptive mechanisms common in lower organisms, which allow rapid changes of the photosystem structure to cope with iron stress, are absent. It is speculated that the observed changes in the monomer-trimer equilibrium of major PSII antennae, which is possibly the result of xanthophyll fluctuations, is a first adaptative adjustment to iron deficiency, and may eventually play a role in light dissipation mechanisms

From Targeted Quantification to Untargeted Metabolomics

Metabolomics is an emerging and rapidly evolving technology tool, which involves quantitative and qualitative metabolite assessments science. It offers tremendous promise for different applications in various fields such as medical, environmental, nutrition, and agricultural sciences. Metabolomic approach is based on global identification of a high number of metabolites present in a biological fluid. This allows to characterize the metabolic profile of a given condition and to identify which metabolites or metabolite patterns may be useful in the discrimination between different groups. The use of one mass spectrometry (MS) platform from targeted quantification to untargeted metabolomics will make more efficient workflows in many fields and should allow projects to be more easily undertaken and realized. Metabolomics can be divided into non-targeted and targeted. The first one can analyze metabolites derived from the organisms comprehensively and systematically, so it is an unbiased metabolomics analysis that can discover new biomarkers. Targeted metabolomics, on the other hand, is the study and analysis of specific metabolites. Both have their own advantages and disadvantages, and are often used in combination for discovery and accurate weight determination of differential metabolites, and allow in-depth research and analysis of subsequent metabolic molecular markers. Targeted and non-targeted metabolomics are involved in food identification, disease research, animal model verification, biomarker discovery, disease diagnosis, drug development, drug screening, drug evaluation, clinical plant metabolism and microbial metabolism research. The aim of this chapter is to highlight the versatility of metabolomic analysis due to both the enormous variety of samples and the no strict barriers between quantitative and qualitative analysis. For this purpose, two examples from our group will be considered. Using non-targeted metabolomics in opposite Antarctic cryptoendolytic communities exposed to the sun, we revealed specific adaptations. Instead, through the targeted metabolomics applied to the urine during childbirth, we identified a different distribution of specific metabolites and the metabolic differences allowed us to discriminate between the two phases of labor, highlighting the metabolites most involved in the discrimination. The choice of these two approaches is to highlight that metabolomic analysis can be applied to any sample, even physiologically and metabolomically very distant, as can be microorganisms living on Antarctic rocks and biological fluids such as urine

Comparison of Milk Fat Globule Membrane (MFGM) Proteins of Chianina and Holstein Cattle Breed Milk Samples Through Proteomics Methods

Identification of proteins involved in milk production is important to understand the biology of lactation. Many studies have advanced the understanding of mammary function and milk secretion, but the critical molecular mechanisms implicated in milk fat secretion is still incomplete. Milk Fat Globules are secreted from the apical surface of the mammary cells, surrounded by a thin membrane bilayer, the Milk Fat Globule Membrane (MFGM), formed by proteins which have been suggested to be holesterolemia-lowering factors, inhibitors of cancer cell growth, vitamin binders, bactericidal, suppressors of multiple sclerosis. Using a proteomic approach, we compared MFGM from milk samples of individuals belonging to two different cattle breeds, Chianina and Holstein,representative of selection for milk and meat traits, respectively. We were able to isolate some of the major MFGM proteins in the examined samples and to identify differences between the protein fractions of the two breeds. We detected differences in the amount of proteins linked to mammary gland development and lipid droplets formation, as well as host defence mechanisms. We have shown that proteomics is a suitable, unbiased method for the study of milk fractions proteins and a powerful tool in nutritional genomics

The impact of height-adjustable desks and classroom prompts on classroom sitting time, social, and motivational factors among adolescents

Purpose: This quasi-experimental study examined the impact of height-adjustable desks in combination with prompts to break up prolonged sitting time during class time and identified social and motivational factors associated with breaking up sitting time among adolescents. Teachers’ perceptions of strategies were also examined. Methods: Over 17 weeks, 1 classroom in a government secondary school in Melbourne, Australia, was equipped with 27 height-adjustable desks and prompts (posters and desk stickers) to break up classroom sitting time. Teachers received professional development in the use of the desks and prompts. One group of adolescents (n = 55) had 2–5 lessons/week using the height-adjustable desks in an intervention classroom, and a comparison group matched by year level and subject (n = 50) was taught in traditional “seated” classrooms. Adolescents wore an activPAL monitor at baseline (T0), 4 weeks (T1), and 17 weeks (T2) and completed a survey at T0 and T2. Six teachers participated in interviews at T2. Effect sizes were calculated (d). Results: Linear mixed models found that, compared to the traditional “seated” classrooms, the adolescents in the intervention classroom had significantly lower sitting time (T1: –9.7 min/lesson, d = –0.96; T2: –6.7 min/lesson, d = –0.70) and time spent in sitting bouts >15 min (T2: –11.2 min/lesson, d = –0.62), and had significantly higher standing time (T1: 7.3 min/lesson, d = 0.84; T2: 5.8 min/lesson, d = 0.91), number of breaks from sitting (T1: 1.3 breaks/lesson, d = 0.49; T2: 1.8 breaks/lesson, d = 0.67), and stepping time (T1: 2.5 min/lesson, d = 0.66). Intervention classroom adolescents reported greater habit strength (d = 0.58), self-efficacy for breaking up sitting time (d = 0.75), and indicated that having a teacher/classmate remind them to stand as helpful (d = 0.50). Conclusion: This intervention shows promise for targeting sitting behaviors in the classroom and indicates that incorporating social and motivational strategies may further enhance outcomes

High resolution preparation of monocyte-derived macrophages (MDM) protein fractions for clinical proteomics

<p>Abstract</p> <p>Background</p> <p>Macrophages are involved in a number of key physiological processes and complex responses such as inflammatory, immunological, infectious diseases and iron homeostasis. These cells are specialised for iron storage and recycling from senescent erythrocytes so they play a central role in the fine tuning of iron balancing and distribution. The comprehension of the many physiological responses of macrophages implies the study of the related molecular events. To this regard, proteomic analysis, is one of the most powerful tools for the elucidation of the molecular mechanisms, in terms of changes in protein expression levels.</p> <p>Results</p> <p>Our aim was to optimize a protocol for protein fractionation and high resolution mapping using human macrophages for clinical studies. We exploited a fractionation protocol based on the neutral detergent Triton X-114. The 2D maps of the fractions obtained showed high resolution and a good level of purity. Western immunoblotting and mass spectrometry (MS/MS analysis) indicated no fraction cross contamination. On 2D-PAGE mini gels (7 × 8 cm) we could count more than five hundred protein spots, substantially increasing the resolution and the number of detectable proteins for the macrophage proteome. The fractions were also evaluated, with preliminary experiments, using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS).</p> <p>Conclusion</p> <p>This relatively simple method allows deep investigation into macrophages proteomics producing discrete and accurate protein fractions, especially membrane-associated and integral proteins. The adapted protocol seems highly suitable for further studies of clinical proteomics, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions.</p

Impact of an 8-month trial using height-adjustable desks on children\u27s classroom sitting patterns and markers of cardio-metabolic and musculoskeletal health

During school hours, children can sit for prolonged and unbroken periods of time. This study investigated the impact of an 8-month classroom-based intervention focusing on reducing and breaking-up sitting time on children\u27s cardio-metabolic risk factors (i.e., body mass index, waist circumference, blood pressure) and perceptions of musculoskeletal discomfort. Two Year-6 classes (24 students per class) in one primary school were assigned to either an intervention or control classroom. The intervention classroom was equipped with height-adjustable desks and the teacher was instructed in the delivery of pedagogical strategies to reduce and break-up sitting in class. The control classroom followed standard practice using traditional furniture. At baseline, and after 8-months, time spent sitting, standing, stepping, and sitting-bouts (occasions of continuous sitting) as well as the frequency of sit-to-stand transitions were obtained from activPAL inclinometers and the time spent in light-intensity physical activity was obtained from ActiGraph accelerometers. Demographics and musculoskeletal characteristics were obtained from a self-report survey. Hierarchical linear mixed models found that during class-time, children\u27s overall time spent sitting in long bouts (&gt;10 min) were lower and the number of sit-to-stand transitions were higher in the intervention group compared to the control group, while no changes were observed for musculoskeletal pain/discomfort. No significant intervention effects were found for the anthropometrics measures and blood pressure. Height-adjustable desks and pedagogical strategies to reduce/break-up sitting can positively modify classroom sitting patterns in children. Longer interventions, larger and varied sample size may be needed to show health impacts; however, these desks did not increase musculoskeletal pain/discomfort