Influenza vaccination is recognized as the most effective method for reducing morbidity and mortality due to seasonal influenza. To improve vaccine supply and to increase flexibility in vaccine ...manufacturing, cell culture-based vaccine production has emerged to overcome limitations of egg-based production. The switch of production system and the need for annual re-evaluation of vaccines for the effectiveness due to frequent viral antigenic changes call for methods for complete characterization of the hemagglutinin (HA) antigens and the final vaccine products. This study describes advanced liquid chromatography-mass spectrometry (LC-MS) methods for simultaneous identification of HA proteins and process-related impurities in a trivalent influenza candidate vaccine, comprised of purified recombinant HA (rHA) antigens produced in an insect cell-baculovirus expression vector system (BEVS). N-linked glycosylation sites and glycoforms of the three rHA proteins (corresponding to influenza A subtypes H1N1 and H3N2 and B virus, respectively) were profiled by peptide mapping using reversed-phase (RP) LC-MS(E) (data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode). The detected site-specific glycoforms were further confirmed and quantified by hydrophilic interaction LC (HILIC)-multiple reaction monitoring (MRM) assays. LC-MS(E) was used to characterize the vaccine candidate, providing both protein identities and site-specific information of glycosylation and degradations on each rHA protein. HILIC-MRM methodology was used for rapid confirming and quantifying site-specific glycoforms and potential degradations on each rHA protein. These methods can contribute to the monitoring of vaccine quality especially as it pertains to product comparability studies to evaluate the impact of production process changes.
Macro-attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopic imaging is presented as non-destructive analytical methodology with respect to the sample for the study of paint ...surfaces. This paper illustrates new opportunities offered by a conventional diamond ATR accessory coupled with a focal plane array detector to obtain chemical images of multi-layered paint cross-sections. The use of a large internal reflection element permits to increase the field of view of the imaging area up to 700
μm
×
500
μm and to obtain FT-IR images of the complete stratigraphy of the sample. This approach represents a versatile analytical tool to determine the composition of the inorganic and organic painting materials and to obtain information on their distribution within the different layers. In the optical arrangement used, a spatial resolution of approximately 15
μm is achieved without the use of infrared microscope. This study demonstrates the possibilities offered by macro-ATR-FT-IR imaging for a simple identification of the different compounds present in paint cross-sections before performing further molecular and elemental analyses which may be time consuming or require particular sample preparation.
During the last restoration (2008–2011) of the polychrome terracotta altarpiece called
Coronation of Virgin between Saints Rocco, Sebastian, Peter martyr and Antonio abbot
, located in the collegiate ...church of S. Maria Assunta in Montecassiano (Macerata, Italy), scientific investigations were carried out to acquire detailed information about the painting technique. The identification of materials allowed a correct restoration.
The altarpiece is almost entirely realized by Marco della Robbia (Fra Mattia), dates back to the first half of the XVI century and represents an interesting example of painted terracotta produced by using two different techniques: glazed polychrome terracotta and the “cold painting” technique.
The characterization of the samples’ material constituents was obtained by analysing the cross-sections and the fragments by different techniques (optical, SEM-EDS and ATR-FTIR microscopy as well as GC-MS), as the real nature of a component is often difficult to assess with one single technique.
The optical microscope examination of paint cross-sections shows the presence of many layers, indicating the complexity of the paint stratigraphic morphologies. The original polychromy of della Robbia’s masterpiece is constituted of cinnabar, red lake, red lead, orpiment, red ochre, lead white, lead tin yellow, green earth and raw umber. Two different types of gilding technique have been distinguished. The first one presents a glue mordant, and the second one shows an oil mordant composed by a mixture of red lead, red ochre, cinnabar and orpiment. The GC-MS analysis allowed the characterisation of linseed oil and a mixture of animal glue and egg as binding media stratigraphically located by the use of ATR-FTIR mapping microscopy. The analytical results of the painted terracotta integrated investigations show that original technique adopted is characterised by the application of pigments in an oil-binding medium directly applied on the substrates, probably treated with oil and animal glue.
A large number of overpaintings above the original scheme of polychromy was found, which could be ascribed to almost three different interventions; the absence of modern pigments suggests that they could be realized long ago.
Abstract
Fluctuations in the strength and direction of natural selection through time are a ubiquitous feature of life on Earth. One evolutionary outcome of such fluctuations is adaptive tracking, ...wherein populations rapidly adapt from standing genetic variation. In certain circumstances, adaptive tracking can lead to the long-term maintenance of functional polymorphism despite allele frequency change due to selection. Although adaptive tracking is likely a common process, we still have a limited understanding of aspects of its genetic architecture and its strength relative to other evolutionary forces such as drift. Drosophila melanogaster living in temperate regions evolve to track seasonal fluctuations and are an excellent system to tackle these gaps in knowledge. By sequencing orchard populations collected across multiple years, we characterized the genomic signal of seasonal demography and identified that the cosmopolitan inversion In(2L)t facilitates seasonal adaptive tracking and shows molecular footprints of selection. A meta-analysis of phenotypic studies shows that seasonal loci within In(2L)t are associated with behavior, life history, physiology, and morphological traits. We identify candidate loci and experimentally link them to phenotype. Our work contributes to our general understanding of fluctuating selection and highlights the evolutionary outcome and dynamics of contemporary selection on inversions.
Studies performed over the past decade have yielded new information related to the physiological and metabolic adjustments made in response to both short- and long-term high-altitude exposure. These ...investigations have examined the potential mechanisms responsible for the alterations observed in such key variables as heart rate, stroke volume, cardiac output, muscle blood flow, substrate utilization and mitochondrial function, both at rest and during exercise of varying intensities. Additionally, the occurrence and mechanisms related to the 'lactate paradox' continues to intrigue investigators. It is apparent that exposure to high altitude is an environmental stressor that elicits a robust sympathoadrenal response that contributes to many of the critical adjustments and adaptations mentioned above. Furthermore, as some of these important physiological adaptations are known to enhance performance, it has become popular to incorporate an aspect of altitude living/training into the training regimens of endurance athletes (e.g. 'live high-train low'). Finally, it is important to note that many factors influence the extent to which individuals adjust and adapt to the stress imposed by exposure to high altitude. Included among these are (i) the degree of hypoxia; (ii) the duration of exposure to hypoxic conditions; (iii) the exercise intensity (absolute vs relative workload); and (iv) the inter-individual variability in adapting to hypoxic environments ('responders' vs 'non-responders').
For more than 60 years, muscle mechanical efficiency has been thought to remain unchanged with acclimatization to high altitude. However, recent work has suggested that muscle mechanical efficiency ...may in fact be improved upon return from prolonged exposure to high altitude. The purpose of the present work is to resolve this apparent conflict in the literature. In a collaboration between four research centers, we have included data from independent high‐altitude studies performed at varying altitudes and including a total of 153 subjects ranging from sea‐level (SL) residents to high‐altitude natives, and from sedentary to world‐class athletes. In study A (n=109), living for 20–22 h/day at 2500 m combined with training between 1250 and 2800 m caused no differences in running economy at fixed speeds despite low typical error measurements. In study B, SL residents (n=8) sojourning for 8 weeks at 4100 m and residents native to this altitude (n=7) performed cycle ergometer exercise in ambient air and in acute normoxia. Muscle oxygen uptake and mechanical efficiency were unchanged between SL and acclimatization and between the two groups. In study C (n=20), during 21 days of exposure to 4300 m altitude, no changes in systemic or leg VO2 were found during cycle ergometer exercise. However, at the substantially higher altitude of 5260 m decreases in submaximal VO2 were found in nine subjects with acute hypoxic exposure, as well as after 9 weeks of acclimatization. As VO2 was already reduced in acute hypoxia this suggests, at least in this condition, that the reduction is not related to anatomical or physiological adaptations to high altitude but to oxygen lack because of severe hypoxia altering substrate utilization. In conclusion, results from several, independent investigations indicate that exercise economy remains unchanged after acclimatization to high altitude.
PDF
