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

Comprehensive Evaluation of Quality Traits of Hovenia acerba Germplasm Resources in Fujian Province

Hovenia acerba is a precious medicinal and edible tree. We assessed the genetic variation of H. acerba quality traits and conducted a comprehensive germplasm resource evaluation to provide a theoretical basis for breeding edible, medicinal, and edible/medicine combination varieties. We evaluated 31 H. acerba germplasm resources, including 12 infructescence and 8 fruit quality traits using correlation, principal component, and cluster analyses. The results showed that there were significant differences in all quality traits, with an average coefficient of variation greater than 0.20, an average genetic diversity greater than 1.80, and an average repeatability greater than 0.90. The average genetic variation and repeatability of quality traits in infructescence were higher than fruit. Infructescence K, Ca, Mn, Mg, and reducing sugar contents are important indicators in evaluating infructescence and fruit quality traits, and infructescence K, Mg, and reducing sugar contents are also quality innovation indices of H. acerba germplasms. Tannin, protein, and soluble sugar were the most suitable quality components for screening, followed by reducing sugar, starch, fat, total saponins, and total flavones. According to principal component factor scores and cluster analysis results, specific genotypes were selected as breeding materials for infructescence protein, tannin, flavone, reductive sugar, fruit tannin, fat, flavonoid, saponin, protein, and starch. The correlation analysis with environmental factors showed that the total amount of applied water could influence H. acerba infructescence and fruit quality. In conclusion, the variability of H. acerba germplasm resources was rich, and selection potential is large, which is beneficial to germplasm quality innovation and breeding

Synthesis and evaluation of 131I‐Skyrin as a necrosis avid agent for potential targeted radionuclide therapy of solid tumors

An innovative anticancer approach targeted to necrotic tissues, which serves as a noncancerous and generic anchor, may present a breakthrough. Necrosis avid agents with a flat conjugate aromatic structure selectively accumulate in necrotic tissues, but they easily form aggregates that undesirably distribute to normal tissues. In this study, skyrin, a dianthraquinone compound with smaller and distorted π-cores and thus decreased aggregates as compared with hypericin (Hyp), was designed to target necrosis for tumor therapy. Aggregation studies of skyrin by UV/vis spectroscopy showed a smaller self-association constant with skyrin than with Hyp. Skyrin was labeled by iodine-131 with a radiochemical purity of 98% and exhibited good stability in rat serum for 72 h. In vitro cell uptake studies showed significant difference in the uptake of 131I-skyrin by necrotic cells compared to normal cells (P < 0.05). Compared in rats with liver and muscle necrosis, radiobiodistribution, whole-body autoradiography, and SPECT/CT studies revealed higher accumulation of 131I-skyrin in necrotic liver and muscle (p < 0.05), but lower uptake in normal organs, relative to that of 131I-Hyp. In mice bearing H22 tumor xenografts treated with combretastatin A4 disodium phosphate, the highest uptake of 131I-skyrin was found in necrotic tumor. In conclusion, 131I-skyrin appears a promising agent with reduced accumulation in nontarget organs for targeted radionuclide therapy of solid tumors.status: publishe

Iron-catalyzed [4 + 2] annulation of amidines with α,β-unsaturated ketoxime acetates toward 2,4,6-trisubstituted pyrimidines

An iron-catalyzed [4 ​+ ​2] annulation of amidines with α,β-unsaturated ketoxime acetates is described. This strategy employs amidines as CN units and provides a new protocol for the construction of 2,4,6-trisubstituted pyrimidines under batch and continuous flow conditions in moderate to good yields, exhibiting good functional group tolerance, scalability and operational simplicity

Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies.

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies