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

Dabrafenib Promotes Schwann Cell Differentiation by Inhibition of the MEK-ERK Pathway

Schwann cell differentiation involves a dynamic interaction of signaling cascades. However, much remains to be elucidated regarding the function of signaling molecules that differ depending on the context in which the molecules are engaged. Here, we identified a small molecule, dabrafenib, which promotes Schwann cell differentiation in vitro and exploited this compound as a pharmacological tool to understand the molecular mechanisms regulating Schwann cell differentiation. The results indicated that dabrafenib inhibited ERK phosphorylation and enhanced ErbB2 autophosphorylation and Akt phosphorylation, and the effects of dabrafenib on ErbB2 and Akt phosphorylation were phenocopied by pharmacological inhibition of the MEK-ERK signaling pathway. However, the small molecule inhibitors of MEK and ERK had no effect on the expression of Oct6 and EGR2, which are key transcription factors that drive Schwann cell differentiation. In addition, pharmacological inhibition of phosphatidylinositol-3-kinase (PI3K) almost completely interfered with dabrafenib-induced Schwann cell differentiation. These results suggest that the ErbB2-PI3K-Akt axis is required for the induction of Schwann cell differentiation by dabrafenib in vitro. Although additional molecules targeted by dabrafenib remain to be identified, our data provides insights into the crosstalk that exists between the MEK-ERK signaling pathway and the PI3K-Akt axis in Schwann cell differentiation

Energy Storage System Control Algorithm by Operating Target Power to Improve Energy Sustainability of Smart Home

As energy issues are emerging around the world, a variety of smart home technologies aimed at realizing zero energy houses are being introduced. Energy storage system (ESS) for smart home energy independence is increasingly gaining interest. However, limitations exist in that most of them are controlled according to time schedules or used in conjunction with photovoltaic (PV) generation systems. In consideration of load usage patterns and PV generation of smart home, this study proposes an ESS control algorithm that uses constant energy of energy network while making maximum use of ESS. Constant energy means that the load consumes a certain amount of power under all conditions, which translates to low variability. The proposed algorithm makes a smart home a load of energy network with low uncertainty and complexity. The simulation results show that the optimal ESS operating target power not only makes the smart home use power constantly from the energy network, but also maximizes utilization of the ESS. In addition, since the smart home is a load that uses constant energy, it has the advantage of being able to operate an efficient energy network from the viewpoint of energy providers

Effects of estrogen inhibition formula herbal mixture for danazol-induced precocious puberty in female rats: An experimental study with network pharmacology

Background: This study aimed at determining the effect of the herbal mixture estrogen inhibition formula (EIF) and its possible mechanisms by precocious puberty animal models and network pharmacology-based analysis. Methods: Precocious puberty animal models were established by a single injection of 300 μg danazol, then female rats were administered EIF, vaginal openings were monitored, uterus and pituitary indices were determined. The levels of ALP, E2, LH, and FSH were measured using ELISA kits. Real-time PCR was performed to evaluate the mRNA expression of GnRH, UNC5C, and netrin-1 in hypothalamic tissues. We applied network pharmacological analysis to predict potential targets and pathways of EIF. Results: EIF delayed danazol-induced early vaginal opening. In the onset model, EIF reduced the increased levels of serum ALP, E2, LH, and FSH; as well as mRNA expressions of GnRH, Netrin-1, and UNC5C. Moreover, long-term administration of EIF not only diminished all impaired factors but also had no effect on the normal development of the animals. The gene set enrichment analysis showed that the targets of EIF are mainly associated with the GnRH signaling and ovarian steroidogenesis pathways. Conclusion: EIF could be used in preclinical research for the treatment of precocious puberty by the inhibition of HPGA pre-maturation

Requirement of Zinc Transporter SLC39A7/ZIP7 for Dermal Development to Fine-Tune Endoplasmic Reticulum Function by Regulating Protein Disulfide Isomerase

Skin is the first area that manifests zinc deficiency. However, the molecular mechanisms by which zinc homeostasis affects skin development remain largely unknown. Here, we show that zinc-regulation transporter-/iron-regulation transporter-like protein 7 (ZIP7) localized to the endoplasmic reticulum plays critical roles in connective tissue development. Mice lacking the Slc39a7/Zip7 gene in collagen 1-expressing tissue exhibited dermal dysplasia. Ablation of ZIP7 in mesenchymal stem cells inhibited cell proliferation thereby preventing proper dermis formation, indicating that ZIP7 is required for dermal development. We also found that mesenchymal stem cells lacking ZIP7 accumulated zinc in the endoplasmic reticulum, which triggered zinc-dependent aggregation and inhibition of protein disulfide isomerase, leading to endoplasmic reticulum dysfunction. These results suggest that ZIP7 is necessary for endoplasmic reticulum function in mesenchymal stem cells and, as such, is essential for dermal development. © 2017 The Authors1

Hyperosmotic Stress Reduces Melanin Production by Altering Melanosome Formation

<div><p>Many tissues of the human body encounter hyperosmotic stress. The effect of extracellular osmotic changes on melanin production has not yet been elucidated. In this study, we determined that hyperosmotic stress induced by organic osmolytes results in reduced melanin production in human melanoma MNT-1 cells. Under hyperosmotic stress, few pigmented mature melanosomes were detected, but there was an increase in swollen vacuoles. These vacuoles were stained with an anti-M6PR antibody that recognizes late endosomal components and with anti-TA99 and anti-HMB45 antibodies, implying that melanosome formation was affected by hyperosmotic stress. Electron microscopic analysis revealed that the M6PR-positive swollen vacuoles were multi-layered and contained melanized granules, and they produced melanin when L-DOPA was applied, indicating that these vacuoles were still capable of producing melanin, but the inner conditions were not compatible with melanin production. The vacuolation phenomenon induced by hyperosmotic conditions disappeared with treatment with the PI3K activator 740 Y-P, indicating that the PI3K pathway is affected by hyperosmotic conditions and is responsible for the proper formation and maturation of melanosomes. The microarray analysis showed alterations of the vesicle organization and transport under hyperosmotic stress. Our findings suggest that melanogenesis could be regulated by physiological conditions, such as osmotic pressure.</p></div

PI3K activation inhibits hyperosmotic stress-induced vacuolation.

<p>MNT-1 cells were treated with 50 mM sucrose, 10 µM wortmannin, 10 µM YM201636, or 20 µM 740 Y-P for 24 hours. After fixation in 3% paraformaldehyde and permeabilization with 0.1% Triton X-100 in phosphate-buffered saline (PBS), the cells were stained with an antibody against M6PR and examined using confocal microscopy at a magnification of 1260×. NC, negative control; DIC, differential interference contrast.</p

Hyperosmotic stress-induced M6PR-positive swollen vacuoles contain TYRP-1 and PMEL17.

<p>MNT-1 cells were treated with 50 mM sucrose for 7 days and stained with anti-TA99 (A) or anti-HMB45 (B) antibodies. The fluorescence images were acquired using confocal microscopy at a magnification of 1260×. The insets show the magnified images. NC, negative control. (C) Electron microscopic analyses were performed on MNT-1 cells before and after 50 mM sucrose treatment for 7 days. For the in situ L-DOPA assay, the MNT-1 cells were incubated with 0.1% L-DOPA for 3 hours after treatment with 50 mM sucrose for 7 days (Sucrose + DOPA). The insets show the magnified images. The arrowheads indicate various stages of melanosomes (NC), swollen vesicles containing dark granules (Sucrose), or swollen vesicles after the in situ DOPA assay (Sucrose + DOPA). The dotted lines identify the position for the magnified images. NC, negative control.</p