Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Value-of-time distributions and competitive bus services

Bus industry Nash equilibrium Competition Service differentiation Transport economics

The activity of protest movements in 1956-1977 in Denmark and Sweden

Bachelor thesis "The Activity of Protest Movements in 1956-1977 in Denmark and Sweden" deals with the activities of social movements that originated in the territory of Denmark and Sweden in connection with the Western wave of protest activism and the overall radicalization of youth in the 1960s and the 1970s. It was especially a movement against nuclear weapons and the war in Vietnam and the student revolt of 1968. The paper analyzes the circumstances, the protest actions and subsequent consequences of activity of these movements in both countries and its objective is a complex comparison of the Danish and the Swedish cases. This comparison subsequently highlights the differences between the protest movements of Denmark and Sweden in various aspects, which are described in the text. One of them is the development of left-wing movement after 1956 and the emergence of new intellectual currents that influenced the direction of the protest movements. Other parts are devoted to the course and the extent of demonstrations of particular movements and their content, goals and expressions, for example through alternative culture. The thesis also provides the characteristics of the socio-political situation in Denmark and Sweden in the given period, which imply the specifics of the development of protest..

MALDI-TOF mass spectrum of the AMB-PEG fraction from RPC.

<p>The major mass peaks observed had masses corresponding to that of the AMB-PEG conjugate, thereby verifying the identity of that peak fraction. AMB-PEG mass peaks were absent from the collected unconjugated AMB fraction.</p

Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity.

<p>20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A₃₄₈/A₄₀₉ ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.</p

MIC₅₀ (μM) of various fungal species.

<p>MIC₅₀ (μM) of various fungal species.</p

LIVE/DEAD staining of HEK293 and IMR-90 cells after exposure to AMB-PEG 1, 2 and unconjugated AMB for 24 hours.

<p>Live cells are stained green and dead cells stained red. AMB-PEG did not cause cell death at concentrations of 139 μM in HEK293 cells and 277 μM in IMR-90 cells. The molar ratio of AMB to PEG did not have any visible effect on cell toxicity. Conversely, unconjugated AMB caused extensive cell death at concentrations above 4.33 μM in both cell lines. Experiment was performed twice, each time with three independently prepared AMB-PEG formulations.</p

Immunoglobulins M Survive Low-pH Conditions Used for Virus Inactivation and for Elution from Bioaffinity Columns

International audienc

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.ASTAR (Agency for Sci., Tech. and Research, S’pore)Accepted versio