Outcomes of very high C-reactive protein levels in paediatric practice.

Siba Prosad Paul, Alifa De Aguiar, Joanna Barnden, Anna Cannon and Megan Eaton discuss the implications of high levels of the inflammatory marker, drawing on local audit.

Mutational Analysis of the Putative High-Affinity Propofol Binding Site in Human ß3 Homomeric GABAA Receptors.

Propofol is a sedative and anesthetic agent that can both activate GABA(A) receptors and potentiate receptor activation elicited by submaximal concentrations of the transmitter. A recent modeling study of the ß3 homomeric GABA(A) receptor postulated a high-affinity propofol binding site in a hydrophobic pocket in the middle of a triangular cleft lined by the M1 and M2 membrane-spanning domains of one subunit and the M2 domain of the neighboring subunit. The goal of the present study was to gain functional evidence for the involvement of this pocket in the actions of propofol. Human ß3 and α1ß3 receptors were expressed in Xenopus oocytes, and the effects of substitutions of selected residues were probed on channel activation by propofol and pentobarbital. The data demonstrate the vital role of the ß3(Y143), ß3(F221), ß3(Q224), and ß3(T266) residues in the actions of propofol but not pentobarbital in ß3 receptors. The effects of ß3(Y143W) and ß3(Q224W) on activation by propofol are likely steric because propofol analogs with less bulky ortho substituents activated both wild-type and mutant receptors. The T266W mutation removed activation by propofol in ß3 homomeric receptors; however, this mutation alone or in combination with a homologous mutation (I271W) in the α1 subunit had almost no effect on activation properties in α1ß3 heteromeric receptors. We hypothesize that heteromeric α1ß3 receptors can be activated by propofol interactions with ß3-ß3, α1-ß3, and ß3-α1 interfaces, but the exact locations of the binding site and/or nature of interactions vary in different classes of interfaces.

γ-aminobutyric acid type A α4, ß2, and δ subunits assemble to produce more than one functionally distinct receptor type.

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, ß1-3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4ßδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4ß2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct ß2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, ß2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4ßδ receptors may assemble in a similar configuration in neurons and oocytes.

The benzodiazepine diazepam potentiates responses of α1ß2γ2L γ-aminobutyric acid type A receptors activated by either γ-aminobutyric acid or allosteric agonists.

BACKGROUND: The γ-aminobutyric acid (GABA) type A receptor is a target for several anesthetics, anticonvulsants, anxiolytics, and sedatives. Neurosteroids, barbiturates, and etomidate both potentiate responses to GABA and allosterically activate the receptor. We examined the ability of a benzodiazepine, diazepam, to potentiate responses to allosteric agonists. METHODS: The GABA type A receptors were expressed in human embryonic kidney 293 cells and studied using whole-cell and single-channel patch clamp. The receptors were activated by the orthosteric agonist GABA and allosteric agonists pentobarbital, etomidate, and alfaxalone. RESULTS: Diazepam is equally potent at enhancing responses to orthosteric and allosteric agonists. Diazepam EC50s were 25 ± 4, 26 ± 6, 33 ± 6, and 26 ± 3 nm for receptors activated by GABA, pentobarbital, etomidate, and alfaxalone, respectively (mean ± SD, 5-6 cells at each condition). Mutations to the benzodiazepine-binding site (α1(H101C), γ2(R144C), γ2(R197C)) reduced or removed potentiation for all agonists, and an inverse agonist at the benzodiazepine site reduced responses to all agonists. Single-channel data elicited by GABA demonstrate that in the presence of 1 µm diazepam the prevalence of the longest open-time component is increased from 13 ± 7 (mean ± SD, n = 5 patches) to 27 ± 8% (n = 3 patches) and the rate of channel closing is decreased from 129 ± 28 s(-1) to 47 ± 6 s(-1) (mean ± SD) CONCLUSIONS: We conclude that benzodiazepines do not act by enhancing affinity of the orthosteric site for GABA but rather by increasing channel gating efficacy. The results also demonstrate the presence of interactions between allosteric activators and potentiators, raising a possibility of effects on dosage requirements or changes in side effects.

Agonist-specific conformational changes in the α1-γ2 subunit interface of the GABA A receptor.

The GABA(A) receptor undergoes conformational changes upon the binding of agonist that lead to the opening of the channel gate and a flow of small anions across the cell membrane. Besides the transmitter GABA, allosteric ligands such as the general anesthetics pentobarbital and etomidate can activate the receptor. Here, we have investigated the agonist specificity of structural changes in the extracellular domain of the receptor. We used the substituted cysteine accessibility method and focused on the γ2(S195C) site (loop F). We show that modification of the site with (2-sulfonatoethyl)methanethiosulfonate (MTSES) results in an enhanced response to GABA, indicating accessibility of the resting receptor to the modifying agent. Coapplication of GABA or muscimol, but not of gabazine, with MTSES prevented the effect, suggesting that GABA and muscimol elicit a conformational change that reduces access to the γ2(S195C) site. Exposure of the receptors to MTSES in the presence of the allosteric activators pentobarbital and etomidate resulted in an enhanced current response indicating accessibility and labeling of the γ2(S195C) site. However, comparison of the rates of modification indicated that labeling in the presence of etomidate was significantly faster than that in the presence of pentobarbital or gabazine or in resting receptors. We infer from the data that the structure of the α1-γ2 subunit interface undergoes agonist-specific conformational changes.

Value and Barriers to Use of the SIMPL Tool for Resident Feedback.

OBJECTIVE: The System for Improving and Measuring Procedural Learning (SIMPL) is a smart-phone application used to provide residents with an evaluation of operative autonomy and feedback. This study investigated the perceived benefits and barriers to app use. DESIGN: A database of previously performed SIMPL evaluations was analyzed to identify high, low, and never users. Potential predisposing factors to use were explored. A survey investigating key areas of value and barriers to use for the SIMPL application was sent to resident and faculty users. Respondents were asked to self-identify how often they used the app. The perceived benefits and barriers were correlated with the level of usage. Qualitative analysis of free text responses was used to determine strategies to increase usage. SETTING: General surgery training programs who are members of the Procedural Learning and Safety Collaborative. PARTICIPANTS: Surgical residents and faculty. RESULTS: At least 1 SIMPL evaluation was created for 411 residents and 524 faculty. Thirty percent of both faculty and residents were high-frequency users. Thirty percent of faculty were never users. One hundred eighty-eight residents and 207 faculty (response rate 46%) completed the survey. High-frequency resident users were more likely to perceive a benefit for both numerical evaluations (76% vs 30%) and dictated feedback (92% vs 30%). Faculty and residents commonly blamed each other for not creating and completing evaluations regularly (87% of residents, 81% of faculty). Suggested strategies to increase usage included reminders and integration with existing data systems. CONTRIBUTIONS: Frequent users perceive value from the application, particularly from dictated feedback and see a positive impact on feedback in their programs. Faculty engagement represents a major barrier to adoption. Mechanisms which automatically remind residents to initiate an evaluation will help improve utilization but programs must work to enhance faculty willingness to respond and dictate feedback.

Relationship of procedural numbers with meaningful procedural autonomy in general surgery residents.

BACKGROUND: Concerns exist regarding the competency of general surgery graduates with performing core general surgery procedures. Current competence assessment incorporates minimal procedural numbers requirements. METHODS: Based on the Zwisch scale we evaluated the level of autonomy achieved by categorical PGY1-5 general surgery residents at 14 U.S. general surgery resident training programs between September 1, 2015 and December 31, 2016. With 5 of the most commonly performed core general surgery procedures, we correlated the level of autonomy achieved by each resident with the number of procedures they had performed before the evaluation period, with the intent of identifying specific target numbers that would correlate with the achievement of meaningful autonomy for each procedure with most residents. RESULTS: Whereas a definitive target number was identified for laparoscopic appendectomy (i.e. 25), for the other 4 procedures studied (i.e. laparoscopic cholecystectomy, 52; open inguinal hernia repair, 42; ventral hernia repair, 35; and partial colectomy, 60), target numbers identified were less definitive and/or were higher than many residents will experience during their surgical residency training. CONCLUSIONS: We conclude that procedural target numbers are generally not effective in predicting procedural competence and should not be used as the basis for determining residents' readiness for independent practice.

Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol.

BACKGROUND: Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the "+" of the ß subunit, in the ß-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the "-" side of the ß subunit, in the α-ß interface (or ß-ß interface, in the case of homomeric ß receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the ß(H267) residue lines a cavity that docks propofol with favorable interaction energy. METHOD: We used two-electrode voltage clamp to determine the functional effects of mutations to the "+" and "-" sides of the ß subunit on activation of the α1ß3 GABAA receptor by propofol. RESULTS: We found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-ß interface leads to strong reduction in gating efficacy for propofol. CONCLUSION: We conclude that α1ß3 GABAA receptors can be activated by propofol interactions with the ß-ß, α-ß, and ß-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.

Modulation of the human ρ1 GABAA receptor by inhibitory steroids.

RATIONALE: Modulators of the ρ1 GABAA receptor may be useful in the treatment of visual, sleep, and cognitive disorders. Neuroactive steroids and analogues have been shown to modulate ρ1 receptor function, but the molecular mechanisms are poorly understood. OBJECTIVES: We employed electrophysiology and voltage-clamp fluorometry to compare the actions of several neuroactive steroids and analogues on the human ρ1 GABAA receptor. RESULTS: Results confirmed that P294S and T298F mutations affect modulation by steroids. The P294S mutation abolished inhibition by (3α,5ß)-3-hydroxypregnan-20-one (3α5ßP) while the T298F mutation eliminated inhibition by 17ß-estradiol. Voltage-clamp fluorometry demonstrated that steroids differing in the presence of a charged group on C3 or nature of substituent on C17 uniquely modified fluorescence changes elicited by GABA in the extracellular domain. The I307Q mutation reversed the inhibitory effect of 3α5ßP but was without effect on modulation by (3α,5ß)-3-hydroxypregnan-20-one sulfate or 17ß-estradiol. The effect of 3α5ßP on the fluorescence change generated at Y241C was dependent on whether the steroid acted as an inhibitor or a potentiator. Further, the effect was limited to uncharged 5ß-reduced steroids containing an acetyl group on C17. CONCLUSIONS: The data demonstrate that steroids and analogues differ with respect to conformational changes elicited by these drugs as well as sensitivity to the effects of mutations. Steroids and analogues could be provisionally divided into three major groups based on their actions on the ρ1 GABAA receptor: 5ß-reduced uncharged steroids, sulfated and carboxylated steroids, and 17ß-estradiol. Further division among 5ß-reduced uncharged steroids was based on substituent at position C17.

11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors.

RATIONALE: While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified. OBJECTIVES: This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors. RESULTS: Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5ß-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C-H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1ß2γ2L subunits, modulated [(35)S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [(3)H]CW12 and [(3)H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and ß2 subunits or ß3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific. CONCLUSIONS: CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.

Energetic contributions to channel gating of residues in the muscle nicotinic receptor ß1 subunit.

In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the ß1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit.

Characteristics of concatemeric GABA(A) receptors containing α4/δ subunits expressed in Xenopus oocytes.

BACKGROUND AND PURPOSE: GABA(A) receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABA(A) receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH: We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +ß2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. KEY RESULTS: δ-ß2 -α4 +ß2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn(2+) . We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS: The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/ß2/γ2-containing receptors.