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

Cognitive and behavioral characteristics of chronic primary insomnia in Hong Kong : a qualitative and quantitative study

Introduction: Sleep-related cognitive and behavioral characteristics play an important role in the maintenance of insomnia. Culture affects individuals’ belief system and behaviors, but few studies have examined how Chinese subjects describe their experiences of insomnia and how they deal with it. This study explored the topic using qualitative and quantitative approaches. Methods: Participants were recruited from the community via advertisements. Their DSMIV diagnosis of primary insomnia for at least 6 months was confirmed by a sleep specialist with the help of the Structured Clinical Interview for DSM-IV and sleep-wake questionnaires. A focus group approach was used to elicit participants’ insomnia experiences. They were also asked to keep a 1-week insomnia experience diary prior to attending the focus group and to complete a set of sleepwake questionnaires, including the 30-item Dysfunctional Beliefs and Attitudes about Sleep Scale after the group meeting. Each focus group comprised 6 to 8 participants. Discussion was led by a facilitator with a pre-determined question route and it was audiotaped, transcribed verbatim, and managed with NVivo software to facilitate coding and analysis. Focus group data collection ceased when data saturation was achieved. All data were fragmented into meaningful units, compared iteratively, and assigned with descriptive codes to condense the emerging meanings. Codes pertaining to the same phenomena were grouped together and a coding framework was built. The findings from the qualitative study were then validated in a quantitative questionnaire survey of a separate group of participants with chronic primary insomnia and good sleepers. Results: A total of 6 focus groups were arranged, involving 31 women and 12 men, with an average age of 51 years. Participants had a mean duration of insomnia of 11.81 years, and an average sleep-diary derived sleep efficiency of 70.57%. There were 16 sub-categories and 4 categories of characteristics. The 16 sub-categories could be grouped under: 1) beliefs regarding the nature and treatment of insomnia, 2) behavioral responses to insomnia, 3) cognitive-emotional and physiological arousal, and 4) emotional experiences associated with insomnia. Significant difference between primary insomniacs and good sleepers was found on 9 out of 14 items of the quantitative scale we developed based on the qualitative study results. Seven items remained significant after Bonferroni correction (p < .003), including 1) puzzlement about cause, 2) realistic sleep expectation, 3) constant search for treatment, 4) nighttime negative emotions and physiological symptoms, 5) heightened vigilance, 6) association of sleep with suffering, and 7) sleep problem not understood by others. Conclusion: The present study serves as the first to use both qualitative and quantitative approaches to identify the subjective experience of Hong Kong Chinese insomnia patients. Questionnaire study confirmed that the identified experience was also found in a separate chronic insomnia sample. It sheds light on tailoring CBT-I for the local population with chronic insomnia. Further research on the efficacy and acceptance of a tailor-made local CBT-I program is needed.published_or_final_versionPsychiatryMasterMaster of Philosoph

Modulation of the Unfolded Protein Response by the Severe Acute Respiratory Syndrome Coronavirus Spike Protein

Perturbation of the function of endoplasmic reticulum (ER) causes stress leading to the activation of cell signaling pathways known as the unfolded protein response (UPR). Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) uses ER as a site for synthesis and processing of viral proteins. In this report, we demonstrate that infection with SARS-CoV induces the UPR in cultured cells. A comparison with M, E, and NSP6 proteins indicates that SARS-CoV spike (S) protein sufficiently induces transcriptional activation of several UPR effectors, including glucose-regulated protein 78 (GRP78), GRP94, and C/EBP homologous protein. A substantial amount of S protein accumulates in the ER. The expression of S protein exerts different effects on the three major signaling pathways of the UPR. Particularly, it induces GRP78/94 through PKR-like ER kinase but has no influence on activating transcription factor 6 or X box-binding protein 1. Taken together, our findings suggest that SARS-CoV S protein specifically modulates the UPR to facilitate viral replication

MOESM4 of Identification of Chinese medicine syndromes in persistent insomnia associated with major depressive disorder: a latent tree analysis

Additional file 2. Research protocol

MOESM3 of Identification of Chinese medicine syndromes in persistent insomnia associated with major depressive disorder: a latent tree analysis

Additional file 1. Research ethic approval

Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC

Severe acute respiratory syndrome coronavirus (SARS-CoV) is capable of inducing a storm of proinflammatory cytokines. In this study, we show that the SARS-CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). SARS-CoV and its ORF3a protein were found to be potent activators of pro-IL-1beta gene transcription and protein maturation, the 2 signals required for activation of the NLRP3 inflammasome.This work was supported by the Hong Kong Health and Medical Research Fund (13121032, 14130822, and HKM-15- M01), Hong Kong Research Grants Council (T11-707/15-R, C7011-15R, and 17124415), Government of Spain (BIO2013- 42869-R), U.S. National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (2P01AI060699- 06A1), and European Commission (Innovative Medicines Initiative Grant Agreement 115760). C.C.-R. received a fellowship from Fundacion La Caixa

A Comparative Proteomic Analysis of Erinacine A’s Inhibition of Gastric Cancer Cell Viability and Invasiveness

Background / Aims: Erinacine A, isolated from the ethanol extract of the Hericium erinaceus mycelium, has been demonstrated as a new alternative anticancer medicine. Drawing upon current research, this study presents an investigation of the molecular mechanism of erinacine A inhibition associated with gastric cancer cell growth. Methods: Cell viability was determined by Annexin V–FITC/propidium iodide staining and migration using a Boyden chamber assay to determine the effects of erinacine A treatment on the proliferation capacity and invasiveness of gastric cancer cells. A proteomic assay provided information that was used to identify the differentially-expressed proteins following erinacine A treatment, as well as the mechanism of its targets in the apoptotic induction of erinacine A. Results: Our results demonstrate that erinacine A treatment of TSGH 9201 cells increased cytotoxicity and the generation of reactive oxygen species (ROS), as well as decreased the invasiveness. Treatment of TSGH 9201 cells with erinacine A resulted in the activation of caspases and the expression of TRAIL. Erinacine A induction of apoptosis was accompanied by sustained phosphorylation of FAK/AKT/p70S6K and the PAK1 pathways, as well as the generation of ROS. Furthermore, the induction of apoptosis and anti-invasion properties by erinacine A could involve the differential expression of the 14-3-3 sigma protein (1433S) and microtubule-associated tumor suppressor candidate 2 (MTUS2), with the activation of the FAK/AKT/p70S6K and PAK1 signaling pathways. Conclusions: These results lead us to speculate that erinacine A may generate an apoptotic cascade in TSGH 9201 cells by activating the FAK/AKT/p70S6K/PAK1 pathway and upregulating proteins 1433S and MTUS2, providing a new mechanism underlying the anti-cancer effects of erinacine A in human gastric cancer cells