Rapid antimicrobial susceptibility testing is needed to reduce prescription of inappropriate antibiotics. A rapid alternative to standard culture-based testing is to determine reductions in cell viability using the LIVE/DEAD BacLight Bacterial Viability Kit. We optimised the kit protocol for this application, focusing on simplifying the process by minimising the steps involved and on determining the optimal analytical parameters for fluorescence measurements from the dyes SYTO 9 and propidium iodide (PI). We demonstrate that for our experimental system, the intensity of emissions should be integrated from 505-515 nm for SYTO 9 and 600-610 nm for PI, and the proportion of live cells calculated from a new dye ratio formula, termed the adjusted dye ratio. We show that the pre-staining washing step is not necessary if a non-fluorescent growth media is used; however, staining must be done for each sampling as prolonged exposure to the dyes negatively impacts cell viability. The optimised methodology was able to reproducibly detect reductions in culture viability when the proportion of live cells in a sample of 1 × 10 cells/ml fell below ∼50% live in a media that supports the growth required for detecting antibiotic killing. Finally, we show that the interaction of fluorescence emission spectra from SYTO 9 and PI stained cells is influenced by the proportion of dead cells in a sample. The excitation of PI by SYTO 9 was found to occur in populations containing sufficient numbers of dead cells (>25%), whereas in populations with low numbers of dead cells the dye interaction was additive in regard to red emissions, indicating that these dye interactions may offer another dimension to live/dead analysis. Fluorescence measurements from samples established according to the optimised protocol can be taken using a flow cytometer, spectrofluorometer, microplate reader, and the Optrode, a fibre-based spectroscopic system developed at the University of Auckland.
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http://dx.doi.org/10.3389/fmicb.2019.00801 | DOI Listing |
BMC Med Genomics
January 2025
Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK.
Amyotrophic lateral sclerosis (ALS) lacks a specific biomarker, but is defined by relatively selective toxicity to motor neurons (MN). As others have highlighted, this offers an opportunity to develop a sensitive and specific biomarker based on detection of DNA released from dying MN within accessible biofluids. Here we have performed whole genome bisulfite sequencing (WGBS) of iPSC-derived MN from neurologically normal individuals.
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January 2025
Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, State Key Laboratory for Digestive Health, National Clinical Research Center of Digestive Diseases, Beijing Digestive Disease Center, Beijing, 100050, China.
Helicobacter pylori (H. pylori) infection is a well-established risk factor for gastric cancer, primarily due to its virulence factor, cytotoxin-associated gene A (CagA). Although PD-L1/PD-1-mediated immune evasion is critical in cancer development, the impact of CagA on PD-L1 regulation remains unclear.
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January 2025
State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, 518055, China.
Sterile alpha and Toll/interleukin-1 receptor motif containing 1 (SARM1), a nicotinamide adenine dinucleotide (NAD)-utilizing enzyme, mediates axon degeneration (AxD) in various neurodegenerative diseases. It is activated by nicotinamide mononucleotide (NMN) to produce a calcium messenger, cyclic ADP-ribose (cADPR). This activity is blocked by elevated NAD level.
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January 2025
Tianjian Laboratory of Advanced Biomedical Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China.
Mitochondrial oxidative phosphorylation (OXPHOS) is a therapeutic vulnerability in glycolysis-deficient cancers. Here we show that inhibiting OXPHOS similarly suppresses the proliferation and tumorigenicity of glycolytically competent colorectal cancer (CRC) cells in vitro and in patient-derived CRC xenografts. While the increased glycolytic activity rapidly replenished the ATP pool, it did not restore the reduced production of aspartate upon OXPHOS inhibition.
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January 2025
NMPA Key Laboratory for Research and Evaluation of Narcotic and Psychotropic Drugs, Xuzhou, China.
Neuroinflammation is a key factor in the pathogenesis of Parkinson's disease (PD). Activated microglia in the central nervous system (CNS) and infiltration of peripheral immune cells contribute to dopaminergic neuron loss. However, the role of peripheral immune responses, particularly triggering receptor expressed on myeloid cells-1 (TREM-1), in PD remains unclear.
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