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

Experimental Study on the Properties of Mixed-Fiber Concrete Shield Tunnel Segments Subjected to High Temperatures

In order to study the mechanical and damage behavior of concrete shield tunnel segments under a high temperature, two self-compacting concrete and three mixed-fiber (steel and polypropylene fiber) self-compacting concrete test blocks were designed. The influence of several key factors, including fire duration, pre-loading, and concrete type, on the fire behavior of concrete shield tunnel segments were studied. The results show that the type of fiber and pre-loading have an important influence on crack development in concrete shield tunnel segments. Compared with undoped segments, cracks in segments with steel fibers and polypropylene fibers appeared later, and the average crack spacing decreased. The pre-loading has an important effect on the vertical deformation before and after the temperature rise. As the load level increases, so does the deformation after the temperature rise. The influence of the initial load level should be considered when designing the fire resistance of the segment

Bioengineered bacterial outer membrane vesicles encapsulated Polybia–mastoparan I fusion peptide as a promising nanoplatform for bladder cancer immune-modulatory chemotherapy

BackgroundNanosized bacterial outer membrane vesicles (OMVs) secreted by Gram-negative bacteria have emerged as a novel antitumor nanomedicine reagent due to their immunostimulatory properties. The encapsulated bacterial composition in OMVs can be edited via manipulating bioengineering technology on paternal bacteria, allowing us to design an ingenious antitumor platform by loading the Polybia–mastoparan I (MPI) fusion peptide into OMVs.MethodsOMVs containing the MPI fusion peptide were obtained from bioengineered Escherichia coli transformed with recombinant plasmid. The antitumor efficacy of bioengineered OMVs in vitro was verified by performing cell viability and wound-healing and apoptosis assays using MB49 and UMUC3 cells, respectively. Subcutaneous MB49 tumor-bearing mice were involved to investigate the tumor inhibition ability of bioengineered OMVs. Moreover, the activated immune response in tumor and the biosafety were also evaluated in detail.ResultsThe resulting OMVs had the successful encapsulation of MPI fusion peptides and were subjected to physical characterization for morphology, size, and zeta potential. Cell viabilities of bladder cancer cells including MB49 and UMUC3 rather than a non-carcinomatous cell line (bEnd.3) were decreased when incubated with bioengineered OMVs. In addition, bioengineered OMVs restrained migration and induced apoptosis of bladder cancer cells. With intratumor injection of bioengineered OMVs, growths of subcutaneous MB49 tumors were significantly restricted. The inherent immunostimulation of OMVs was demonstrated to trigger maturation of dendritic cells (DCs), recruitment of macrophages, and infiltration of cytotoxic T lymphocytes (CTLs), resulting in the increased secretion of pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ). Meanwhile, several lines of evidence also indicated that bioengineered OMVs had satisfactory biosafety.ConclusionBioengineered OMVs fabricated in the present study were characterized by strong bladder cancer suppression and great biocompatibility, providing a new avenue for clinical bladder cancer therapy

Comparative Analysis of Human Mesenchymal Stem Cells from Umbilical Cord, Dental Pulp, and Menstrual Blood as Sources for Cell Therapy

Although mesenchymal stem cells (MSCs) based therapy has been considered as a promising tool for tissue repair and regeneration, the optimal cell source remains unknown. Umbilical cord (UC), dental pulp (DP), and menstrual blood (MB) are easily accessible sources, which make them attractive candidates for MSCs. The goal of this study was to compare the biological characteristics, including morphology, proliferation, antiapoptosis, multilineage differentiation capacity, and immunophenotype of UC-, DP-, and MB-MSCs in order to provide a theoretical basis for clinical selection and application of these cells. As a result, all UC-, DP-, and MB-MSCs have self-renewal capacity and multipotentiality. However, the UC-MSCs seemed to have higher cell proliferation ability, while DP-MSCs may have significant advantages for osteogenic differentiation, lower cell apoptosis, and senescence. These differences may be associated with the different expression level of cytokines, including vascular endothelial growth factor, fibroblast growth factor, keratinocyte growth factor, and hepatocyte growth factor in each of the MSCs. Comprehensively, our results suggest DP-MSCs may be a desired source for clinical applications of cell therapy

Visible Light Promoted Stereoselective C(sp3)-H Glycosylation for the Synthesis of C-Glycoamino Acids and C-Glycopeptides

Glycosylative modification of peptides could improve the pharmacological properties of peptide drugs and deliver them efficiently to the target sites. Compared with O-/N-glycosides, C-glycosides exhibit more metabolic stability. We here disclose the first example of visible-light promoted and Cu-catalyzed stereoselective C-glycosylations. The mild reaction conditions are compatible with various carbohydrate substrates, including a series of mono monosaccharides and disaccharide, and is amenable to the synthesis of a wild variety of C-glycoamino acids and C-glycopeptidomimetics with good yields and excellent stereoselectivities. The dual-functional photocatalyst formed in situ via coordination of glycine derivatives and chiral phosphine Cu complex could not only catalyze the photoredox process but also control the stereoselectivity of glycosylation reaction

Site-Selective Polyfluoroaryl Modification and Unsymmetric Stapling of Unprotected Peptides

Peptide stapling is recognized as an effective strategy for improving the proteolytic stability and cell permeability of peptides. In this study, we present a novel approach for the site-selective unsymmetric perfluoroaryl stapling of Ser and Cys residues in unprotected peptides. The stapling reaction proceeds smoothly under very mild conditions, exhibiting a remarkably rapid reaction rate. It can furnish stapled products in both liquid and solid phases, and the presence of nucleophilic groups other than Cys thiol within the peptide does not impede the reaction, resulting in uniformly high yields. Importantly, the chemoselective activation of Ser β-C(sp3)–H enables the unreacted −OH to serve as a reactive handle for subsequent divergent modification of the staple moiety with various therapeutic functionalities, including a clickable azido group, a polar moiety, a lipid tag, and a fluorescent dye. In our study, we have also developed a visible-light-induced chemoselective C(sp3)–H polyfluoroarylation of the Ser β-position. This reaction avoids interference with the competitive reaction of Ser −OH, enabling the precise late-stage polyfluoroarylative modification of Ser residues in various unprotected peptides containing other highly reactive amino acid residues. The biological assay suggested that our peptide stapling strategy would potentially enhance the proteolytic stability and cellular permeability of peptides