Real-time monitoring of biomass during Escherichia coli high-cell-density cultivations by in-line photon density wave spectroscopy

An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µ_s′ with CDW was observed in the range of 5–69 g L⁻¹ (R² = 0.98). The growth rates calculated based on µ_s′ were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µ_s′ as a function of the absorption coefficient µ_a allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.     

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.     

Circannual Variations in Blood Cholesterol Levels

The seasonality of blood cholesterol is still not well established. Some have described a seasonal pattern with highest levels during autumn and lowest in summer, whereas others have reported no change. A number of studies showed circannual variations with maximum levels in winter and minimum in summer. The aim of this study was to examine the circannual variation of cholesterol in a large cohort in Israel. In the Israeli CORDIS study, employees of 21 factories were screened for cardiovascular disease risk factors during 1985-7. As part of the information gathered, serum cholesterol and blood counts were available for 3,726 men and 1,514 women. Serum cholesterol levels fit a circannual rhythm assessed by the cosinor analysis. Highest levels of serum cholesterol were found in spring and lowest in summer. We conclude that there is a circannual change of serum cholesterol, which could be partially associated with changes in environmental temperature. The circannual variation in serum cholesterol was considerable and should be taken into account when carrying out clinical evaluation of patients.     

Trophic structure in open waters of the marginal ice zone in the Scotia-Weddell confluence region during spring (1983)

The structure of the food web was investigated in open waters adjacent to the marginal ice zone in the southern Scotia Sea in spring 1983. Diets were defined for dominant zooplankton, micronekton, and flying seabird species and then aggregated by cluster analysis into feeding groups. Most zooplankton were omnivorous, feeding on phytoplankton, protozoans, and in some cases, small metazoans (copepods). Only two species were found to be exclusively herbivorous:Calanoides acutus andRhincalanus gigas. Micronekton were carnivores with copepods being the dominant prey in all their diets. The midwater fishElectrona antarctica was the dominant food item in seven of the nine seabird species examined. Cephalopods, midwater decapod shrimps and carrion were also important in the diets of a few seabird species. Comparison (cluster analysis) of diets in spring with other seasons (winter, fall) indicated that over half the species examined (18 of 31) had similar diets in all seasons tested. The significant intraspecific shifts in diet that did occur were attributable to regional, seasonal, and interannual effects. A scheme is presented that describes the major energetic pathways through the open water ecosystem from phytoplankton to apex predators. At the base are phytoplankton and protozoans which are the principal food resource for the biomass copepods and krill. Krill and the biomass copepods are the principal forage of the midwater fishElectrona antarctica which, in turn, is the central diet component of flying seabirds as well as important food for the Antarctic fur seal and cephalopods. Krill are a major diet element for the fur seal and cephalopods, and the principal food of the minke whale.     

αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.     

POPULATION STRUCTURE OF THE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) AS DETERMINED BY RESTRICTION ENDONUCLEASE ANALYSIS OF MITOCHONDRIAL DNA

Abstract: Restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) were used to test for population subdivision in the bottlenose dolphin (Tursiops truncatus). Atlantic and Pacific dolphin mtDNA samples exhibited distinctly different haplotypes (approximately 2.4% sequence divergence), indicating a lack of gene exchange. Within the Atlantic Ocean, mtDNA samples from the Gulf of Mexico and the Atlantic Coast were also found to be distinct, with a sequence divergence of approximately 0.6%. The Atlantic Coast–Gulf of Mexico dichotomy is consistent with patterns of genetic variation from other marine and coastal organisms from this region, and supports the hypothesized role of bio-geographic events in promoting the divergence of these and other forms. Regional differentiation was identified along the Atlantic Coast, whereas low sequence divergences among haplotypes and consistent haplotype frequencies across populations suggested considerable gene exchange among Gulf of Mexico populations. A highly divergent haplotype found in two individuals from two localities in the Gulf of Mexico is best explained by dispersal from either a distinct offshore Gulf stock or an unsampled Atlantic Coast stock. Additional samples are required to test for the existence of a distinct offshore race and, if it exists, to identify its distribution and contribution to population structure.     

The Human Mast Cell Chymase Gene (CMA1): Mapping to the Cathepsin G/Granzyme Gene Cluster and Lineage-Restricted Expression

Genes encoding T-cell-receptor α/δ chains, neutrophil cathepsin G, and lymphocyte CGL/granzymes are closely linked on chromosomal band 14q11.2. The current work identifies the human mast cell chymase gene (CMA1) as the fourth protease in this cluster and maps the gene to within 150 kb of the cathepsin G gene. The gene order is centromere-T cell receptor α/δ-CGL-1/granzyme B-CGL-2/granzyme H-cathepsin G-chymase. Chymase and cathepsin G genes are shown to be cotranscribed in the human mast cell line HMC-1 and in U-937 cells. Other cells transcribe cathepsin G or CGL/granzyme genes, but not chymase genes, suggesting a capacity for independent regulation. Comparison of the 5′ flank of the chymase gene with those of cathepsin G and CGL/granzymes reveals little overall homology. Only short regions of the 5′ flanks of the human and murine chymase genes sequenced to date are similar, suggesting that they are more distantly related than human and rodent CGL-1/granzyme B, the flanks of which are highly homologous. The expression patterns and clustering of genes provide possible clues to the presence of locus control regions that orchestrate lineage-restricted expression of leukocyte and mast cell proteases.     

Recognition of errors in three-dimensional structures of proteins

A major problem in the determination of the three-dimensional structure of proteins concerns the quality of the structural models obtained from the interpretation of experimental data. New developments in X-ray crystallography and nuclear magnetic resonance spectroscopy have acceleratedd the process of structure determination and the biological community is confronted with a steadily increasing number of experimentally determined protein folds. However, in the recent past several experimentally determined protein structures have been proven to contain major errors, indicating that in some cases the interpretation of experimental data is difficult and may yield incorrect models. Such problems can be avoided when computational methods are employed which complement experimental structure determinations. A prerequisite of such computational tools is that they are independent of the parameters obtained from a particular experiment. In addition such techniques are able to support and accelerate experimental structure determinations. Here we present techniques based on knowledge based mean fields which can be used to judge the quality of protein folds. The methods can be used to identify misfolded structures as well as faulty parts of structural models. The techniques are even applicable in cases where only the C_α trace of a protein conformation is available. The capabilities of the technique are demonstrated using correct and incorrect protein folds. © 1993 Wiley-Liss, Inc.     

Chiral recognition by 1H-NMR,EPR, and ENDOR spectroscopy

Chiral recognition with magnetic methods requires the formation of diastereomers. Due to the variety of appropriate reactions, hydrogen bond formation, esterification, and acetalization as well as host–guest interactions were chosen for basic investigations. The results obtained indicate that in the case of diamagnetic compounds the chemical shifts and for paramagnetic compounds the β-proton coupling constants are the most useful parameters. By combination of both pieces of information, assignment of the absolute configuration was achieved. © 1993 Wiley-Liss, Inc.     

Chiral symmetry breaking: Experimental results and computer analysis of a liquid-phase autoxidation

The autoxidation of tetralin is treated as a model reaction system to define the applicability of stereospecific autocatalysis. This concept, predicting a spontaneous amplification of enantiomeric excess generated by an autocatalytic chemical reaction, is used in several theoretical models as an explanation for the origin of natural optical activity. The reaction system investigated obeys the basic criteria of these models: a chiral intermediate (tetralin hydroperoxide) is produced from an achiral substrate (tetralin) via an autocatalytic pathway where the feedback mechanism is expected to generate a state of broken chiral symmetry. In order to test the amplification capacity of this reaction a computer analysis of the kinetic scheme is performed. This simulation is derived from the known kinetic scheme of autoxidation and is validated by fitting the experimentally observed data of hydroperoxide evolution. Calculations show that this model allows powerful amplification of enantiomeric excess and a transient amplification of the optical rotation. It is also demonstrated that the model system exhibits pronounced sensitivity toward any loss of absolute configuration of the involved chiral species. Since an amplification effect results exclusively at a high degree of stereoselectivity, it is concluded that stereospecific autocatalysis is possible in systems which show template reactions, crystallization, or colloidal effects. © 1993 Wiley-Liss, Inc.