Mechanisms of GABAA and Glycine Receptor Analgesia in the Spinal Dorsal Horn: In Vitro Models as Translational Platforms for Drug Discovery

Available analgesics do not always provide adequate pain relief and are often associated with a significant side-effect profile. Following reports of a loss of inhibitory signalling in the spinal dorsal horn (DH) network in persistent pain conditions, γ-amino butyric acid type A (GABAA) and glycine receptors have been identified as promising targets. The present study assesses two in vitro models for their suitability for screening novel analgesics targeting GABAA and glycine receptors. Firstly, the embryonic cultured spinal DH cell model and secondly an acute rat spinal cord slice model. Immunofluorescence characterisation of the spinal DH culture illustrated that this model displays many similarities with the in vivo spinal DH. Immunofluorescence and RT-PCR demonstrated the presence of GABAA and glycine receptor subunits in the spinal DH culture. Calcium imaging and extracellular multi-electrode array (MEA) recording techniques were utilised to study the effect of GABAA, GABAB and glycine receptor drugs on the spinal DH culture network activity. All drugs tested significantly modulated the culture’s spontaneous firing. A further study assessed whether lentivirus and Accell siRNA mediated glycine receptor α subunit gene silencing modulates calcium responses in the DH culture model. The lentiviruses had low transfection efficiencies and caused cell death, however Accell siRNA transfection was successful and significantly decreased baseline spontaneous activity compared to untreated cultures. Single electrode and MEA extracellular recordings were performed with the acute spinal cord slice model. GABAA, GABAB and glycine receptor drugs modulated 4-aminopyridine-induced hyperexcitability in the substantia gelatinosa lamina of the slices. The MEA recordings illustrated that 4-aminopyridine-induced activity manifested more prominently in the DH than the ventral horn (VH) and that the DH network activity was highly synchronous. Taken together, these findings demonstrate that these in vitro models provide suitable platforms to test novel analgesics targeting GABAA and glycine receptors in the spinal DH network

Neisseria meningitidis Opc Invasin Binds to the Sulphated Tyrosines of Activated Vitronectin to Attach to and Invade Human Brain Endothelial Cells

The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y56 and Y59. Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43–68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain 45RGD47 was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may enable the bacterium to harness the benefits of the physiological processes in which the host effector molecule participates

Seeing a drummer’s performance modulates the subjective experience of groove when listening to popular music drum patterns

Spontaneous rhythmical movements, like foot-tapping and head-bobbing, often emerge when people listen to music, promoting the sensation of being in the ‘groove’: a psychological construct that additionally incorporates positive affect. Here we report the first study to investigate if seeing the music maker modulates this subjective experience of groove. Across trials participants (n = 36) listened to high and low groove drum beats, while concurrently observing a task-irrelevant point-light display (PLD) of the drummer. The PLD was either fully-corresponding with the audio or was incompatible across three other visual display conditions: a static PLD, a corresponding but asynchronous PLD (0.5s time shifted); or a non-corresponding PLD (e.g. low groove audio paired with high groove PLD). Participants rated: (a) their desire to move; and (b) the perceived groove in response to the auditory beats only, using 8-point Likert scales. In both measurements there were significant main effects of groove level and of visual display. Ratings were higher for high compared to low groove audio, and for the fully-corresponding condition compared to the other three visual conditions. The participants’ desire to move was also rated higher in the static condition compared to the non-corresponding condition, while the two-way interaction was also significant: ratings were higher for the high compared to low groove audio in the fully-corresponding, static and asynchronous conditions, but not in the non-corresponding condition. These findings identify the importance of seeing as well as hearing the musician for an enhanced listening experience, which necessitates a multimodal account of music perception

The organisation of nurse staffing in intensive care units: a qualitative study

© 2022 The Authors. Journal of Nursing Management published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License. https://creativecommons.org/licenses/by/4.0/Aims: To examine the organisation of the nursing workforce in intensive care units and identify factors that influence how the workforce operates. Background: Pre-pandemic UK survey data show that up to 60% of intensive care units did not meet locally agreed staffing numbers and 40% of ICUs were closing beds at least once a week because of workforce shortages, specifically nursing. Nurse staffing in intensive care is based on the assumption that sicker patients need more nursing resource than those recovering from critical illness. These standards are based on historical working, and expert professional consensus, deemed the weakest form of evidence. Methods: Focus groups with intensive care health care professionals (n= 52 participants) and individual interviews with critical care network leads and policy leads (n= 14 participants) in England between December 2019 and July 2020. Data were analysed using framework analysis. Findings: Three themes were identified: the constraining or enabling nature of intensive care and hospital structures; whole team processes to mitigate nurse staffing shortfalls; and the impact of nurse staffing on patient, staff and intensive care flow outcomes. Staff made decisions about staffing throughout a shift and were influenced by a combination of factors illuminated in the three themes. Conclusions: Whilst nurse: patient ratios were clearly used to set the nursing establishment, it was clear that rostering and allocation/re-allocation during a shift took into account many other factors, such as patient and family nursing needs, staff wellbeing, intensive care layout and the experience, and availability, of other members of the multi-professional team. This has important implications for future planning for intensive care nurse staffing and highlights important factors to be accounted for in future research studies. Implications for Nursing Management in order to safeguard patient and staff safety, factors such as the ICU layout need to be considered in staffing decisions and the local business case for nurse staffing needs to reflect these factors. Patient safety in intensive care may not be best served by a blanket ‘ratio’ approach to nurse staffing, intended to apply uniformly across health services.Peer reviewe

No role for estrogen receptor 1 gene intron 1 Pvu II and exon 4 C325G polymorphisms in migraine susceptibility

BACKGROUND: We have previously reported an association between the estrogen receptor 1 (ESR1) gene exon 8 G594A polymorphism and migraine susceptibility in two independent Australian cohorts. In this paper we report results of analysis of two further single nucleotide polymorphisms (SNPs) in the ESR1 gene in the same study group, the T/C Pvu II SNP in intron 1 and the C325G SNP in exon 4, as well as results of linkage disequilibrium (LD) analysis on these markers. METHODS: We investigated these variants by case-control association analysis in a cohort of 240 migraineurs and 240 matched controls. The SNPs were genotyped using specific restriction enzyme assays. Results were analysed using contingency table methods incorporating the chi-squared statistic. LD results are presented as D' statistics with associated P values. RESULTS: We found no evidence for association of the Pvu II T/C polymorphism and the C325G polymorphism and migraine susceptibility and no evidence for LD between these two SNPs and the previously implicated exon 8 G594A marker. CONCLUSION: We have found no role for the polymorphisms in intron 1 and exon 4 with migraine susceptibility. To further investigate our previously implicated exon 8 marker, we suggest the need for studies with a high density of polymorphisms be undertaken, with particular focus on markers in LD with the exon 8 marker

IFN-γ amplifies NFκB-dependent Neisseria meningitidis invasion of epithelial cells via specific upregulation of CEA-related cell adhesion molecule 1

Temporal relationship between viral and bacterial infections has been observed, and may arise via the action of virus-induced inflammatory cytokines. These, by upregulating epithelial receptors targeted by bacteria, may encourage greater bacterial infiltration. In this study, human epithelial cells exposed to interferon-gamma but not tumour necrosis factor-alpha or interleukin 1-beta supported increased meningococcal adhesion and invasion. The increase was related to Opa but not Opc or pili adhesin expression. De novo synthesis of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a major Opa receptor, occurred in epithelial cells exposed to the cytokine, or when infected with Opa-expressing bacteria. Cell line-dependent differences in invasion that were observed could be correlated with CEACAM expression levels. There was also evidence for Opa/pili synergism leading to high levels of monolayer infiltration by capsulate bacteria. The use of nuclear factor-kappa B (NFκB) inhibitors, diferuloylmethane (curcumin) and SN50, abrogated bacterial infiltration of both untreated and interferon-gamma-treated cells. The studies demonstrate the importance of CEACAMs as mediators of increased cellular invasion under conditions of inflammation and bring to light the potential role of NFκB pathway in Opa-mediated invasion by meningococci. The data imply that cell-surface remodelling by virally induced cytokines could be one factor that increases host susceptibility to bacterial infection