The attenuation of NF-κB/p65 nuclear translocation by DYN 1–17 and the N-terminal fragments in LPS-stimulated THP-1 cells.

<p>The LPS-stimulated THP-1 cells were treated with the N-terminal fragments of DYN 1–17 and U50,488H at 1 μM and 10 nM for an hour. The treated cells were fixed with paraformaldehyde (3.7%) and immunolabelled with primary anti-NF-κB/p65 monoclonal antibody and visualized using Alexa Fluor 555^® secondary antibody. DAPI staining was used to identify the nuclei. The nuclear translocation of NF-κB/p65 in each treatment group was assessed using the Image Xpress screening system. Non-stimulated THP-1 cells (NS) served as negative control. The NF-κB/p65 translocation percentage in each treatment group was normalised and expressed relative to LPS-stimulated control group. Data shown are the means ± S.E.M. of at least three independent experiments performed in triplicates, *p ≤ 0.05.</p

NF-κB/p65 nuclear localisation in differentiated THP-1 cells.

<p>Cells were fixed, permeabilised, stained with anti-NF-κB/p65 rabbit monoclonal antibody and visualised with Alexa Fluor^® 555 goat anti-rabbit IgG (green). The nuclei were counterstained with DAPI (blue). Microscopy images <b>A.</b> Inactive NF-κB/p65 proteins localisation in the cytoplasm of the non-stimulated THP-1 cells (top row) and <b>B.</b> Translocated NF-κB/p65 proteins into the nuclei of THP-1 cells following LPS stimulation (bottom row). Images were acquired for each fluorescence channel, using suitable TRITC and DAPI filters and a 63× objective. Scale bar: 10μm</p

Effect of IMD-0354 on the NF-κB/p65 nuclear translocation in LPS-stimulated THP-1 cells.

<p>The LPS-stimulated THP-1 cells were treated with IMD-0354 (10 μM) and DMSO (0.1% v/v, as diluent control). The treated cells were fixed with paraformaldehyde (3.7%) and immunolabelled with primary anti-NF-κB/p65 rabbit monoclonal antibody and visualized using Alexa Fluor 555^® secondary antibody. DAPI staining was used to identify the nuclei. The nuclear translocation of NF-κB/p65 in each treatment group was assessed using the Image Xpress system. Non-stimulated THP-1 cells (NS), receiving similar treatment served as negative control. The percentage of NF-κB/p65 nuclear translocation in each treatment group was normalised and expressed relative to LPS-stimulated control group. Data represents means ± S.E.M. of at least three independent experiments performed in triplicates, *p ≤ 0.05.</p

The expression of KOP receptor on LPS-stimulated THP-1 cells.

<p>Cells were fixed, permeabilised, immunolabelled with KOP receptor monoclonal antibody and visualised with Alexa Fluor 647^® goat anti-mouse IgG (red). The nuclei were counterstained with DAPI (blue). Images were acquired for each fluorescence channel, using suitable FITC and DAPI filters with 20 × objective. Scale bar: 25μm</p

The modulation of DYN 1–17 and the N-terminal fragments on the release of IL-1β and TNF-α in LPS-stimulated THP-1 cells.

<p>The LPS-stimulated THP-1 cells were treated with the N-terminal fragments of DYN 1–17 and U50,488H at 0.1 μM, 1 nM and 10 pM for 24 hr. The culture supernatants were collected and IL-1β and TNF-α release was measured using IL-1β and TNF-α AlphaLISA kit, respectively. The AlphaLISA signal was read using an Enspire-Alpha 2390 Multilabel Plate Reader. Non-stimulated THP-1 cells (NS) served as negative control. The release of IL-1β and TNF-α in each treatment group was normalised and expressed relative to LPS-stimulated control group. Data shown are the means ± S.E.M. of at least three independent experiments performed in triplicates. Statistical significance as denoted by * and # represent the IL-1β and TNF-α release between peptide-treated (solid-line) and LPS-stimulated control group or ML-190-treated group (dotted-line), respectively, p ≤ 0.05.</p

Effect of ML-190 on the attenuation of NF-κB/p65 nuclear translocation by DYN 1–17 and the N-terminal fragments in LPS-stimulated THP-1 cells.

<p>The LPS-stimulated THP-1 cells were pre-treated with ML-190, a KOP receptor antagonist (1 μM, 1 hour) followed by co-treatment of the DYN 1–17 and the N-terminal fragments (10 nM) or U50,488H (10 nM) with ML-190 (1 μM, 1 hour). The treated cells were fixed with paraformaldehyde (3.7%) and immunolabelled with primary anti-NF-κB/p65 rabbit monoclonal antibody and visualized using Alexa Fluor 555^® secondary antibody. DAPI staining was used to identify the nuclei. The nuclear translocation of NF-κB/p65 in each treatment group was assessed using the Image Xpress system. Non-stimulated THP-1 cells (NS) served as negative control. The percentage of NF-κB/p65 nuclear translocation in each treatment group was normalised and expressed relative to LPS-stimulated control group (contains DMSO 0.1% as diluent control). Data shown are the means ± S.E.M. of at least three independent experiments performed in triplicates, *p ≤ 0.05.</p

Additional file 1: of Rapid-Onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD): exome sequencing of trios, monozygotic twins and tumours

Low-coverage proportions of candidate genes in replication exomes. (XLSX 43 kb

Datasheet2_Communication and access to healthcare: Experiences of Aboriginal and Torres Strait Islander people managing pain in Queensland, Australia.pdf

BackgroundPain management requires a multidisciplinary approach and a collaborative relationship between patient-provider in which communication is crucial. This study examines the communication experiences of Aboriginal and Torres Strait Islander patients and Aboriginal and Torres Strait Islander Hospital Liaison Officers (ATSIHLOs), to improve understanding of how pain is managed in and through patient-health professional communication.MethodsThis qualitative study involved a purposive sample of patients attending three persistent pain clinics and ATSIHLOs working in two hospitals in Queensland, Australia. Focus groups and in-depth interviews explored the communication experiences of patients managing pain and ATSIHLOs supporting patients with pain. This study adopted a descriptive phenomenological methodology, as described by Colaizzi (1978). Relevant statements (patient and ATSIHLOs quotes) about the phenomenon were extracted from the transcripts to formulate meanings. The formulated meanings were subsequently sorted into thematic clusters and then integrated into themes. The themes were then incorporated into a concise description of the phenomenon of communication within pain management. Findings were validated by participants.ResultsA total of 21 Aboriginal and Torres Strait Islander participants were involved in this study. Exploration of the communication experiences of patients and ATSIHLOs revealed overlapping themes of important barriers to and enablers of communication that affected access to care while managing pain. Acknowledging historical and cultural factors were particularly important to build trust between patients and health professionals. Some patients reported feeling stigmatized for identifying as Aboriginal and Torres Strait Islander, while others were reluctant to disclose their background for fear of not having the same opportunity for treatment. Differences in the expression of pain and the difficulty to use standard pain measurement scales were identified. Communication was described as more than the content delivered, it is visual and emotional expressed through body language, voice intonation, language and the speed of the conversation.ConclusionCommunication can significantly affect access to pain management services. Aboriginal and Torres Strait Islander patients highlighted the burden of emotional pain caused by historical factors, negative stereotypes and the fear of discrimination. Pain management services and their health professionals need to acknowledge how these factors impact patients trust and care.</p

Datasheet1_Communication and access to healthcare: Experiences of Aboriginal and Torres Strait Islander people managing pain in Queensland, Australia.pdf

BackgroundPain management requires a multidisciplinary approach and a collaborative relationship between patient-provider in which communication is crucial. This study examines the communication experiences of Aboriginal and Torres Strait Islander patients and Aboriginal and Torres Strait Islander Hospital Liaison Officers (ATSIHLOs), to improve understanding of how pain is managed in and through patient-health professional communication.MethodsThis qualitative study involved a purposive sample of patients attending three persistent pain clinics and ATSIHLOs working in two hospitals in Queensland, Australia. Focus groups and in-depth interviews explored the communication experiences of patients managing pain and ATSIHLOs supporting patients with pain. This study adopted a descriptive phenomenological methodology, as described by Colaizzi (1978). Relevant statements (patient and ATSIHLOs quotes) about the phenomenon were extracted from the transcripts to formulate meanings. The formulated meanings were subsequently sorted into thematic clusters and then integrated into themes. The themes were then incorporated into a concise description of the phenomenon of communication within pain management. Findings were validated by participants.ResultsA total of 21 Aboriginal and Torres Strait Islander participants were involved in this study. Exploration of the communication experiences of patients and ATSIHLOs revealed overlapping themes of important barriers to and enablers of communication that affected access to care while managing pain. Acknowledging historical and cultural factors were particularly important to build trust between patients and health professionals. Some patients reported feeling stigmatized for identifying as Aboriginal and Torres Strait Islander, while others were reluctant to disclose their background for fear of not having the same opportunity for treatment. Differences in the expression of pain and the difficulty to use standard pain measurement scales were identified. Communication was described as more than the content delivered, it is visual and emotional expressed through body language, voice intonation, language and the speed of the conversation.ConclusionCommunication can significantly affect access to pain management services. Aboriginal and Torres Strait Islander patients highlighted the burden of emotional pain caused by historical factors, negative stereotypes and the fear of discrimination. Pain management services and their health professionals need to acknowledge how these factors impact patients trust and care.</p

Supplemental Material - Yarning about pain: Evaluating communication training for health professionals at persistent pain services in Queensland, Australia

Supplemental Material for Yarning about pain: Evaluating communication training for health professionals at persistent pain services in Queensland, Australia by Christina M Bernardes, Stuart Ekberg, Stephen Birch, Andrew Claus, Matthew Bryant, Renata Meuter, Jermaine Isua, Paul Gray, Joseph P Kluver, Eva Malacova, Corey Jones, Kushla Houkamau, Marayah Taylor, Ivan Lin and Gregory Pratt in British Journal of Pain</p