Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.     

The bipolar bivalve <Emphasis Type="Italic">Oxytoma (Palmoxytoma) cygnipes</Emphasis> (Young &; Bird, 1822) in the Upper Pliensbachian of Germany

The morphologically conspicuous bivalve Oxytoma (Palmoxytoma) cygnipes (Young & Bird, 1822), known for its palaeogeographically bipolar distribution, from a limestone bed in the boundary “Belemniten–Schichten”/Amaltheenton formation, Lower Jurassic, in N Germany is described. The occurrence of this palaeoceanographically significant bivalve points to an influx of cool seawater from the Arctic to the North-German Basin at the base of the Upper Pliensbachian, just before the deposition of the Amaltheenton formation. A review of previously reported occurrences on the NW European Shelf indicates two distinct stratigraphic intervals of occurrence of this taxon: the Rhaetian–Hettangian boundary and the Upper Pliensbachian. Whereas the former interval of occurrence may be related to short-term cooling in the course of the end-Triassic extinction event, the latter is interpreted as reflecting the influx of a cool water current to the eastern part of the NW European Shelf, which continued southwards parallel to the coast of the Bohemian–Vindelician High.     

Fluvival tufa formation in a hard-water creek (Deinschwanger Bach, Franconian Alb, Germany)

Summary Cyanobacteria-dominated biofilms involved in tufa deposition in the hardwater creek Deinschwanger Bach, Bavaria, were investigated with regard to their effect on the carbonate equilibrium and fabric formation. Current tufa deposition is evident by up to 1.5 mm thick crusts that have formed on substrate plates placed in the creek for 10 months. Hydrochemistry data indicate that carbonate precipitation along the creek is physicochemically driven by CO₂ degassing, whereas photosynthetic carbon assimilation is without detectable effect on the macroscale carbonate equilibrium. However, stable isotope data indicate a minor photosynthetic effect, but only for the lower creek section where the pCO₂ already drops to the two-fold of the atmospheric level. Though the initial process of external nucleation on cyanobacterial sheaths in the lower creck section might be promoted of by a photosynthetically-induced microscale pH gradient, the effect is not strong enough to cause a CaCO₃ impregnation of the sheaths. The fabric of the laminated tufa crusts in the creek reflects the temporal alternation of porous microspariticPhormidium incrustatum-Phormidium foveolarum-diatom biofilms in spring, micrite-impregnatedPhormidium incrustatum-Phormidium foveolarum-diatom biofilms in summerautumn, and detritus-rich non-calcified diatom-biofilms in winter. By contrast, exopolymer-poor surfaces of cascade tufa mosses show large, euhedral spar crystals. Non-phototrophic bacteria, which occur in large numbers inPhormidium incrustatum-Phormidium foveolarums-diatom-communities, thrive on extracellular polymeric substances (EPS) and dead cells of the cyanobacteria and are unlikely to promote CaCO₃ precipitation.     

Shoulder muscle activity during pushing, pulling, elevation and overhead throw.

PURPOSE: The aim of this study was to compare the muscle activity of recreational athletes and professional javelin throwers during pull, push, and elevation of upper extremities and during overhead throw. SCOPE: Nine professional javelin throwers and 16 recreational athletes without shoulder problems were studied. Signals were recorded by surface EMG from eight different muscles. The results obtained from the muscles of upper extremities of throwers were compared with those of recreational athletes. CONCLUSION: The different neuromuscular control of professional throwers caused a more profitable muscle activity. Differences during the overhead throw were more significant. The deltoid muscle and rotator cuff of recreational athletes showed stronger activity than those of throwers during pull, push and elevation. The deltoid muscle and the rotator cuff of professional throwers showed stronger activity during overhead throw. Studying the detailed characteristics of muscle activity pattern (differences in length of activity periods, MVC% of muscles and time broadness among peak muscle activities in percent of total time of a movement cycle) may provide a basis for better understanding improved performance and help in planning proper rehabilitation protocol.     

A morphologic study of bronchus-associated lymphoid tissue in turkeys

Bronchus-associated lymphoid tissue (BALT) in normal turkeys of ages 1 day and 1, 2, 3, 4, 8, and 18 weeks was examined by light microscopy and by scanning and transmission electron microscopy. Turkey BALT resembled other mucosa-associated lymphoid tissues; it was made up of a population of lymphocytes covered by a specialized epithelium different from typical pseudostratified ciliated columnar bronchial epithelium. There were distinct age-related differences in BALT structure. Bronchus-associated lymphoid nodules were larger and more numerous in older turkeys. In 1-day- to 2-week-old turkeys, the primary cell type of BALT epithelium was nonciliated cuboidal; in 2-week old turkeys it was squamous; and in turkeys older than 4-weeks of age, the epithelium was primarily ciliated columnar. In 1- to 4-week old turkeys, large numbers of intraepithelial lymphocytes disrupted the normal organization of the epithelium. In older turkeys, epithelial and lymphoid cells were in discrete compartments separated by connective tissue. Lymphocytes in 1-day-old turkeys were found in loose aggregates around venules and within the epithelium. In 1-week old turkeys, lymphocytes were organized into compartments of morphologically similar cells. By 3-weeks of age, lymphocytes were present in distinct germinal centers. Epithelial cells of BALT did not have large numbers of apical vesicles and thus were not structurally specialized for antigen uptake by endocytosis. However, the epithelial barrier appeared to be disrupted over lymphoid nodules, suggesting that antigen would be readily available to lymphocytes and phagocytes in BALT. Age-related differences in turkey BALT structure may have functional consequences with respect to the respiratory immune response.     

Moldy walnut toxicosis in a dog, caused by the mycotoxin, penitrem A

Penitrem A was found in moldy walnuts that were involved in an intoxication of a dog in California.Penicillium crustosum was isolated from the walnuts and penitrem A was isolated from extracts of the mycelium of this fungus when it was grown on a synthetic medium.     

Difference spectroscopy with respiratory membranes of an H2-oxidizing microorganism layered between two gas-permeable, plastic foils: a procedure that allows measurements in the ultraviolet range

A method for recording redox difference spectra of respiratory membranes which minimizes light scattering and results in increased sensitivity and spectral range when compared to the traditional technique of recording spectra of respiratory particles in suspension is described. Membranes were sandwiched between two gas-permeable, plastic foils, placed in a sealed cuvette, and gassed with H2 as reductant or O2 as oxidant. The membranes of the H2-oxidizing microorganism (Alcaligenes eutrophus H16) used in this work contain a hydrogenase which is coupled to the electron transport chain, thereby allowing H2 to serve as a source of electrons. With this procedure, spectra of both cytochromes and quinones were recorded. By using mixtures of H2 and O2, spectra of cytochromes between the fully oxidized and fully reduced states were recorded.     

Tissue layer specific regulation of leaf length and width in Arabidopsis as revealed by the cell autonomous action of ANGUSTIFOLIA

In vascular plants the shoot apical meristem consists of three tissue layers, L1, L2 and the L3, that are kept separate during organ formation and give rise to the epidermis (L1) and the subepidermal tissues (L2, L3). For proper organ development these different tissue layers must interact with each other, though their relative contributions are a matter of debate. Here we use ANGUSTIFOLIA (AN), which controls cell polarity and leaf shape, to study its morphogenetic function in the epidermis and the subepidermis of Arabidopsis thaliana. We show that ANGUSTIFOLIA expression in the subepidermis cannot rescue epidermal cell polarity defects, indicating a cell‐autonomous molecular function. We demonstrate that leaf width is only rescued by subepidermal AN expression, whereas leaf length is also rescued by epidermal expression. Strikingly, subepidermal rescue of leaf width is accompanied by increased cell number in the epidermis, indicating that AN can trigger cell divisions in a non‐autonomous manner.