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

Subditine, a new monoterpenoid indole alkaloid from bark of Nauclea subdita (Korth.) Steud. induces apoptosis in human prostate cancer cells.

In this study, a new apoptotic monoterpenoid indole alkaloid, subditine (1), and four known compounds were isolated from the bark of Nauclea subdita. Complete (1)H- and (13)C- NMR data of the new compound were reported. The structures of isolated compounds were elucidated with various spectroscopic methods such as 1D- and 2D- NMR, IR, UV and LCMS. All five compounds were screened for cytotoxic activities on LNCaP and PC-3 human prostate cancer cell-lines. Among the five compounds, the new alkaloid, subditine (1), demonstrated the most potent cell growth inhibition activity and selective against LNCaP with an IC50 of 12.24±0.19 µM and PC-3 with an IC50 of 13.97±0.32 µM, compared to RWPE human normal epithelial cell line (IC50 = 30.48±0.08 µM). Subditine (1) treatment induced apoptosis in LNCaP and PC-3 as evidenced by increased cell permeability, disruption of cytoskeletal structures and increased nuclear fragmentation. In addition, subditine (1) enhanced intracellular reactive oxygen species (ROS) production, as reflected by increased expression of glutathione reductase (GR) to scavenge damaging free radicals in both prostate cancer cell-lines. Excessive ROS could lead to disruption of mitochondrial membrane potential (MMP), release of cytochrome c and subsequent caspase 9, 3/7 activation. Further Western blot analyses showed subditine (1) induced down-regulation of Bcl-2 and Bcl-xl expression, whereas p53 was up-regulated in LNCaP (p53-wild-type), but not in PC-3 (p53-null). Overall, our data demonstrated that the new compound subditine (1) exerts anti-proliferative effect on LNCaP and PC-3 human prostate cancer cells through induction of apoptosis

An update on natural compounds in the remedy of diabetes mellitus: A systematic review

Herbal medicine, phytomedicine or botanical medicine are synonymous, utilizes plants intended for medicinal purposes. Medicinal use of herbal medicine in the treatment and prevention of diseases including diabetes has a long history compared to conventional medicine. Diabetes is one of the major public health concerns over the world. Diabetes or hyperglycemia is considered to be one of the common public health hazard; optimal control of which is still not possible. Persistent hyperglycemia or uncontrolled diabetes has the potential to cause serious complications such as kidney disease, vision loss, cardiovascular disease, and lower-limb amputations which contributed towards morbidity and mortality in diabetes. There are various approaches to treat and prevent diabetes as well as its secondary complications, one of it is herbal medicines. However, the selection of herbs might depends on several factors, which include the stage of progression of diabetes, types of comorbidities that the patients are having, availability, affordability as well as the safety profile of the herbs. This review focuses on the herbal and natural remedies that play the role in the treatment or prevention of this morbid disorder – diabetes, including their underlying mechanisms for the blood glucose-lowering property and the herbal products already been marketed for the remedial action of diabetes. Keywords: Herbal medicine, Insulin secretion, Insulin resistivity, Active component, Diabetes contro

Fast and Sensitive HPLC-ESI-MS/MS Method for Etoricoxib Quantification in Human Plasma and Application to Bioequivalence Study

Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation. The objective of the current study was to develop a sensitive, fast and high-throughput HPLC-ESI-MS/MS method to measure etoricoxib levels in human plasma using a one-step methanol protein precipitation technique. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source operated in a positive mode and multiple reaction monitoring (MRM) were used for data collection. The quantitative MRM transition ions were m/z 359.15 > 279.10 and m/z 363.10 > 282.10 for etoricoxib and IS. The linear range was from 10.00 to 4000.39 ng/mL and the validation parameters were within the acceptance limits of the European Medicine Agency (EMA) and Food and Drug Analysis (FDA) guidelines. The present method was sensitive (10.00 ng/mL with S/N > 40), simple, selective (K prime > 2), and fast (short run time of 2 min), with negligible matrix effect and consistent recovery, suitable for high throughput analysis. The method was used to quantitate etoricoxib plasma concentrations in a bioequivalence study of two 120 mg etoricoxib formulations. Incurred sample reanalysis results further supported that the method was robust and reproducible

Subditine (1) induced caspase 9, 3/7 activation in LNCaP and PC-3 cells.

<p>LNCaP and PC-3 cells were treated with subditine (12.5 µM) and caspase 8, 9, 3/7 activities were determined using bio-illuminescent assays at the indicated time point. Subditine (<b>1</b>) induced caspase 9, 3/7 activation in both LNCaP and PC-3 cells. No significant fold-change was detected in caspase 8 activity throughout 30 hours.</p

Dose-dependent effect of subditine (1) on cell membrane permeability, MMP and cytochrome c release.

<p>LNCaP and PC-3 cells were treated with subditine (<b>1</b>) for 24 h. Cells were then fixed and stained with membrane permeability dye, MMP, cytochrome c and Hoechst as described in Materials and Methods. (A) Stained cells were visualized using HSC arrayscan system to check nuclear morphology, membrane permeability, MMP integrity, cytochrome c release; Blue (nuclear), Green (Membrane permeability), Red (MMP), Cyan (cytochrome c release). (B) Bar chart showing the average fluorescent intensities of membrane permeability, MMP and cytochrome c (mean ± S.D.; *p<0.05).</p

¹H-¹H COSY and HMBC correlations of subditine (1).

<p>¹H-¹H COSY and HMBC correlations of subditine (1).</p

Chemical structure of subditine (1) angustoline (2), angustidine (3), angustine (4), nauclefine (5) isolated from the bark of <i>Nauclea subdita</i>.

<p>The structure of new compound, subditine (<b>1</b>) were elucidated using various spectroscopic method which were 1D-NMR (¹H, ¹³C, DEPT), 2D-NMR (HSQC, HMBC, NOESY), UV, IR and LCMS while the structure of the other four known compounds were confirmed through the comparison of NMR data with literature values.</p

Subditine (1) induced glutathione reductase (GR) gene expression.

<p>LNCaP and PC-3 cells were treated with subditine (<b>1</b>) (12.5 µM) for 18 h. (A) Human oxidative stress and antioxidant defence qPCR-array was used to identify genes significantly up- or down-regulated in subditine (<b>1</b>)-treated LNCaP or PC-3 cells. Gene profiling analyses were performed three times in independent experiments. (Arrow indicates location of GR in the scatter plots) (B) Transcriptional changes of GR were evaluated using quantitative real-time-PCR. Levels of GR mRNA were normalized using β-actin housekeeping gene and expressed as fold change in comparison to untreated control.</p

Western blotting analyses of apoptosis-associated molecules after subditine (1) treatment.

<p>LNCaP and PC-3 cells were treated with paclitaxel (positive control) or various concentrations of subditine (<b>1</b>) for 24 hours. Cells were lysed, subjected to SDS-PAGE and Western blotting. (A) Membranes were probed with Bcl-2, Bcl-xL and p53 antibodies. Protein loading was assessed with antibody to β-actin. Normalization for loading differences was done by dividing the densitometry values for individual bands with β-actin in the same lane (n.d.-not determined). (B) Bar charts showing densitometry quantification of Bcl-2, Bcl-xL and p53 expression in subditine (<b>1</b>)-treated cells relative to control (mean ± S.D.; *p<0.05).</p