Formalin‐Induced Short‐ and Long‐Term Modulation of Cav Currents Expressed in Xenopus Oocytes: An In Vitro Cellular Model for Formalin‐Induced Pain

Abstract: Xenopus oocytes expressing high voltage‐gated calcium channels (Ca_v) were exposed to formalin (0.5%, v/v, 5 min.) and the oocyte death and Ca_v currents were studied for up to 10 days. Ca_v channels were expressed with α₁β₁b and α₂δ sub‐units and the currents (I_Ba) were studied by voltage clamp. None of the oocytes was dead during the exposure to formalin. Oocyte death was significant between day 1 and day 5 after the exposure to formalin and was uniform among the oocytes expressing various Ca_v channels. Peak I_Ba of all Ca_v and A₁, the inactivating current component was decreased whereas the non‐inactivated R current was not affected by 5 min. exposure to formalin. On day 1 after the exposure to formalin, Ca_v1.2c currents were increased, 2.1 and 2.2 currents were decreased and 2.3 currents were unaltered. On day 5, both peak I_Ba and A₁ currents were increased. Ca_v1.2c, 2.2 and 2.3 currents were increased and Ca_v2.1 was unaltered on day 10 after the exposure to formalin. Protein kinase C (PKC) may be involved in formalin‐induced increase in Ca_v currents due to the (i) requirement for Ca_vβ₁b sub‐units; (ii) decreased phorbol‐12‐myristate,13‐acetate potentiation of Ca_v2.3 currents; (iii) absence of potentiation of Ca_v2.3 currents following down‐regulation of PKC; and (iv) absence of potentiation of Ca_v2.2 or 2.3 currents with Ser→Ala mutation of Ca_vα₁2.2 or 2.3 sub‐units. Increased Ca_v currents and PKC activation may coincide with changes observed in in vivo pain investigations, and oocytes incubated with formalin may serve as an in vitro model for some cellular mechanisms of pain.     

9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol, from Eulophia ochreata, inhibits inflammatory signalling mediated by Toll-like receptors

Background and purpose:

9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol (RSCL-0520) is a phenanthrene isolated from Eulophia ochreata, one of the Orchidaceae family, known by local tradition to exhibit medicinal properties. However, no anti-inflammatory activity or any molecular mechanisms involved have been reported or elucidated. Here, for the first time, we evaluate the anti-inflammatory properties of RSCL-0520 on responses induced by lipopolysaccharide (LPS) and mediated via Toll-like receptors (TLRs).

Experimental approach:

The in vitro anti-inflammatory activities of RSCL-0520 were investigated in LPS-stimulated monocytic cells, measuring activation of cytokine and inflammatory genes regulated by nuclear factor-κB (NF-κB). Tumour necrosis factor (TNF)-α levels in serum following LPS stimulation in mice and carrageenan-induced paw oedema in rats were used as in vivo models.

Key results:

Pretreatment with RSCL-0520 effectively inhibited LPS-induced, TLR4-mediated, NF-κB-activated inflammatory genes in vitro, and reduced both LPS-induced TNF-α release and carrageenan-induced paw oedema in rats. Treatment with RSCL-0520 reduced LPS-stimulated mRNA expression of TNF-α, COX-2, intercellular adhesion molecule-1, interleukin (IL)-8 and IL-1β, all regulated through NF-κB activation. RSCL-0520, however, did not interfere with any cellular processes in the absence of LPS.

Conclusions and implications:

RSCL-0520 blocked signals generated by TLR4 activation, as shown by down-regulation of NF-κB-regulated inflammatory cytokines. The inhibitory effect involved both MyD88-dependent and -independent signalling cascades. Our data elucidated the molecular mechanisms involved, and support the search for plant-derived TLR antagonists, as potential anti inflammatory agents.     

Alkali-free bioactive glasses for bone tissue engineering: a preliminary investigation

An alkali-free series of bioactive glasses has been designed and developed in the glass system CaO-MgO-SiO₂-P₂O₅-CaF₂ along the diopside (CaMgSi₂O₆)-fluorapatite (Ca₅(PO₄)₃F)-tricalcium phosphate (3CaO·P₂O₅) join. The silicate network in all the investigated glasses is predominantly coordinated in Q² (Si) units, while phosphorus tends to remain in an orthophosphate (Q⁰) environment. The in vitro bioactivity analysis of glasses has been made by immersion of glass powders in simulated body fluid (SBF) while chemical degradation has been studied in Tris-HCl in accordance with ISO-10993-14. Some of the investigated glasses exhibit hydroxyapatite formation on their surface within 1-12 h of their immersion in SBF solution. The sintering and crystallization kinetics of glasses has been investigated by differential thermal analysis and hot-stage microscopy, respectively while the crystalline phase evolution in resultant glass-ceramics has been studied in the temperature range of 800-900 °C using powder X-ray diffraction and scanning electron microscopy. The alkaline phosphatase activity and osteogenic differentiation for glasses have been studied in vitro on sintered glass powder compacts using rat bone marrow mesenchymal stem cells. The as-designed glasses are ideal candidates for their potential applications in bone tissue engineering in the form of bioactive glasses as well as glass/glass-ceramic scaffolds.     

N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity

The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.     

Haemorrhage control in pelvic fractures--a survey of surgical capabilities

Background

In the emergency management of patients with pelvic fractures, there is ongoing debate about the roles of angiography and open pelvic packing. It is agreed that some form of haemorrhage control is required for patients who are haemo-dynamically unstable despite resuscitation. We set out to determine whether on-call general and orthopaedic surgeons would feel able to perform emergency surgical procedures for these patients and whether vascular radiology was available to them.

Methods

Surveys were sent to all 221 general and orthopaedic surgeons in Wales. Questions included: sub-speciality interest, geographical region, whether there is a pelvic binder in their hospital, availability of interventional radiology, and whether surgeons would perform a range of procedures to control haemorrhage in the emergency setting.

Results

There were 141 responses to the survey, giving a 64% response rate. Only 18% reported that their unit had a formal rota for interventional radiology out of hours. 16% did not know. 96% of orthopaedic surgeons would perform external fixation, although only 49% would use a C-clamp. 90% of general surgeons would be able to pack the pelvis from within the abdominal compartment and 84% would be prepared to cross-clamp the aorta if the situation required. Despite being widely recommended in the literature as a method of haemorrhage control, our survey revealed only 45% would perform extra(pre)-peritoneal packing of the pelvis (58% of general surgeons; 34% of orthopaedic surgeons) and only 12% had received formal training in this procedure.

Conclusions

With appropriately targeted training it is likely that the care of patients with pelvic fractures can be significantly improved.