Key points: Sustained hypoxic exposure increases ventilatory sensitivity to hypoxia as part of physiological acclimatisation. Oxygen‐sensitive signals are transduced in animal cells by post‐translational hydroxylation of transcription factors termed hypoxia‐inducible factors (HIFs). Mice heterozygous for the principal ‘oxygen‐sensing’ HIF hydroxylase PHD2 (prolyl hydroxylase domain 2) show enhanced ventilatory sensitivity to hypoxia. To analyse the underlying mechanisms, functional (hypoxic ventilatory responses, HVRs) and anatomical (cellular proliferation within carotid bodies) responses were studied in genetic models of inducible and constitutive inactivation of PHD2 and its principal hydroxylation substrates, HIF‐1α and HIF‐2α. Inducible PHD2 inactivation enhanced HVR, similar to constitutive inactivation; both responses were almost entirely compensated for by specific inactivation of HIF‐2α. Inducible inactivation of HIF‐2α, but not HIF‐1α, strikingly reduced ventilatory acclimatisation to hypoxia and associated carotid body cell proliferation. These findings demonstrate a key role for PHD2 and HIF‐2α in ventilatory control and carotid body biology. Ventilatory sensitivity to hypoxia increases in response to continued hypoxic exposure as part of acute acclimatisation. Although this process is incompletely understood, insights have been gained through studies of the hypoxia‐inducible factor (HIF) hydroxylase system. Genetic studies implicate these pathways widely in the integrated physiology of hypoxia, through effects on developmental or adaptive processes. In keeping with this, mice that are heterozygous for the principal HIF prolyl hydroxylase, PHD2, show enhanced ventilatory sensitivity to hypoxia and carotid body hyperplasia. Here we have sought to understand this process better through comparative analysis of inducible and constitutive inactivation of PHD2 and its principal targets HIF‐1α and HIF‐2α. We demonstrate that general inducible inactivation of PHD2 in tamoxifen‐treated Phd2^f/f;Rosa26^+/CreERT2 mice, like constitutive, heterozygous PHD2 deficiency, enhances hypoxic ventilatory responses (HVRs: 7.2 ± 0.6 vs. 4.4 ± 0.4 ml min⁻¹ g⁻¹ in controls, P < 0.01). The ventilatory phenotypes associated with both inducible and constitutive inactivation of PHD2 were strongly compensated for by concomitant inactivation of HIF‐2α, but not HIF‐1α. Furthermore, inducible inactivation of HIF‐2α strikingly impaired ventilatory acclimatisation to chronic hypoxia (HVRs: 4.1 ± 0.5 vs. 8.6 ± 0.5 ml min⁻¹ g⁻¹ in controls, P < 0.0001), as well as carotid body cell proliferation (400 ± 81 vs. 2630 ± 390 bromodeoxyuridine‐positive cells mm⁻² in controls, P < 0.0001). The findings demonstrate the importance of the PHD2/HIF‐2α enzyme–substrate couple in modulating ventilatory sensitivity to hypoxia.

Key points: Arterial chemoreceptors measure blood oxygen and are involved in the control of both breathing and the cardiovascular system. Oxygen is mostly used by cells in their mitochondria to generate energy. In this study we have investigated the effects of oxygen starvation (hypoxia) on the mitochondria of specialised oxygen sensing cells from arterial chemoreceptors. Our data confirm that the mitochondria of these oxygen sensing cells are unusually sensitive to modest hypoxia. This effect seems to stem from a reduced affinity of the oxygen utilising enzyme cytochrome oxidase for oxygen. These results are consistent with a functional adaptation of the mitochondria of oxygen sensing cells that may enable them to play a direct role in the oxygen sensing process itself. Abstract  The mechanism of oxygen sensing in arterial chemoreceptors is unknown but has often been linked to mitochondrial function. A common criticism of this hypothesis is that mitochondrial function is insensitive to physiological levels of hypoxia. Here we investigate the effects of hypoxia (down to 0.5% O₂) on mitochondrial function in neonatal rat type‐1 cells. The oxygen sensitivity of mitochondrial [NADH] was assessed by monitoring autofluorescence and increased in hypoxia with a P₅₀ of 15 mm Hg (1 mm Hg = 133.3 Pa) in normal Tyrode or 46 mm Hg in Ca²⁺‐free Tyrode. Hypoxia also depolarised mitochondrial membrane potential (ψ_m, measured using rhodamine 123) with a P₅₀ of 3.1, 3.3 and 2.8 mm Hg in normal Tyrode, Ca²⁺‐free Tyrode and Tyrode containing the Ca²⁺ channel antagonist Ni²⁺, respectively. In the presence of oligomycin and low carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP; 75 nm) ψ_m is maintained by electron transport working against an artificial proton leak. Under these conditions hypoxia depolarised ψ_m/inhibited electron transport with a P₅₀ of 5.4 mm Hg. The effects of hypoxia upon cytochrome oxidase activity were investigated using rotenone, myxothiazol, antimycin A, oligomycin, ascorbate and the electron donor tetramethyl‐p‐phenylenediamine. Under these conditions ψ_m is maintained by complex IV activity alone. Hypoxia inhibited cytochrome oxidase activity (depolarised ψ_m) with a P₅₀ of 2.6 mm Hg. In contrast hypoxia had little or no effect upon NADH (P₅₀= 0.3 mm Hg), electron transport or cytochrome oxidase activity in sympathetic neurons. In summary, type‐1 cell mitochondria display extraordinary oxygen sensitivity commensurate with a role in oxygen sensing. The reasons for this highly unusual behaviour are as yet unexplained.

Objective: The aim of this review was to collate all available evidence on the impact of point-of-care C reactive protein (CRP) testing on patient-relevant outcomes in children and adults in ambulatory care.Design: This was a systematic review to identify controlled studies assessing the impact of point-of-care CRP in patients presenting to ambulatory care services. Ovid Medline, Embase, Cochrane Database of Systematic Reviews, Cochrane CENTRAL, DARE, Science Citation Index were searched from inception to March 2017.Eligibility criteria for selecting studies: Controlled studies assessing the impact of point-of-care CRP in patients presenting to ambulatory care services, measuring a change in clinical care, including but not limited to antibiotic prescribing rate, reconsultation, clinical recovery, patient satisfaction, referral and additional tests. No language restrictions were applied.Data extraction: Data were extracted on setting, date of study, a description of the intervention and control group, patient characteristics and results. Methodological quality of selected studies and assessment of potential bias was assessed independently by two authors using the Cochrane Risk of Bias tool.Results: 11 randomised controlled trials and 8 non-randomised controlled studies met the inclusion criteria, reporting on 16 064 patients. All included studies had a high risk of performance and selection bias. Compared with usual care, point-of-care CRP reduces immediate antibiotic prescribing (pooled risk ratio 0.81; 95% CI 0.71 to 0.92), however, at considerable heterogeneity (I²=72%). This effect increased when guidance on antibiotic prescribing relative to the CRP level was provided (risk ratios of 0.68; 95% CI 0.63 to 0.74 in adults and 0.56; 95% CI 0.33 to 0.95 in children). We found no significant effect of point-of-care CRP testing on patient satisfaction, clinical recovery, reconsultation, further testing and hospital admission.Conclusions: Performing a point-of-care CRP test in ambulatory care accompanied by clinical guidance on interpretation reduces the immediate antibiotic prescribing in both adults and children. As yet, available evidence does not suggest an effect on other patient outcomes or healthcare processes.PROSPERO registration number: CRD42016035426; Results.

In rat arterial chemoreceptors, background potassium channels play an important role in maintaining resting membrane potential and promoting depolarization and excitation in response to hypoxia or acidosis. It has been suggested that these channels are a heterodimer of TASK-1 and TASK-3 based on their similarity to heterologously expressed TASK-1/3 fusion proteins. In this study, we sought to confirm the identity of these channels through germline ablation of Task-1 (Kcnk3) and Task-3 (Kcnk9) in mice. Background K-channels were abundant in carotid body type-1 cells from wild-type mice and comparable to those previously described in rat type-1 cells with a main conductance state of 33 pS. This channel was absent from both Task-1^−/− and Task-3^−/− cells. In its place we observed a larger (38 pS) K⁺-channel in Task-1^−/− cells and a smaller (18 pS) K⁺-channel in Task-3^−/− cells. None of these channels were observed in Task-1^−/−/Task-3^−/− double knock-out mice. We therefore conclude that the predominant background K-channel in wild-type mice is a TASK-1/TASK-3 heterodimer, whereas that in Task-1^−/− mice is TASK-3 and, conversely, that in Task-3^−/− mice is TASK-1. All three forms of TASK channel in type-1 cells were inhibited by hypoxia, cyanide and the uncoupler FCCP, but the greatest sensitivity was seen in TASK-1 and TASK-1/TASK-3 channels. In summary, the background K-channel in type-1 cells is predominantly a TASK-1/TASK-3 heterodimer. Although both TASK-1 and TASK-3 are able to couple to the oxygen and metabolism sensing pathways present in type-1 cells, channels containing TASK-1 appear to be more sensitive.

Key points: Arterial hypoxaemia leads to a rapid increase in ventilation. If the hypoxaemia is sustained, a further increase in ventilation develops over hours to days in a process termed ventilatory acclimatisation. Studies in transgenic mice implicate the hypoxia‐inducible factor (HIF) pathway in the latter process. The aim of this study was to investigate the role of HIF prolyl hydroxylase (PHD) enzymes in ventilatory acclimatisation. We find that PHD2^+/−, but not PHD1^−/− or PHD3^−/−, mice mimic chronic hypoxia in exhibiting exaggerated ventilatory responses to acute hypoxia. This was associated with carotid body overgrowth. However, use of a PHD inhibitor (PHI) induced both hypoxic ventilatory sensitivity and carotid body proliferation only marginally despite strongly inducing erythropoiesis. Taken together, these findings implicate HIF/PHD2 in ventilatory control and carotid body biology but highlight the difficulty of translation from genetic models to pharmacological intervention. Abstract  Oxygen‐dependent prolyl hydroxylation of hypoxia‐inducible factor (HIF) by a set of closely related prolyl hydroxylase domain enzymes (PHD1, 2 and 3) regulates a range of transcriptional responses to hypoxia. This raises important questions about the role of these oxygen‐sensing enzymes in integrative physiology. We investigated the effect of both genetic deficiency and pharmacological inhibition on the change in ventilation in response to acute hypoxic stimulation in mice. Mice exposed to chronic hypoxia for 7 days manifest an exaggerated hypoxic ventilatory response (HVR) (10.8 ± 0.3 versus 4.1 ± 0.7 ml min⁻¹ g⁻¹ in controls; P < 0.01). HVR was similarly exaggerated in PHD2^+/− animals compared to littermate controls (8.4 ± 0.7 versus 5.0 ± 0.8 ml min⁻¹ g⁻¹; P < 0.01). Carotid body volume increased (0.0025 ± 0.00017 in PHD2^+/− animals versus 0.0015 ± 0.00019 mm³ in controls; P < 0.01). In contrast, HVR in PHD1^−/− and PHD3^−/− mice was similar to littermate controls. Acute exposure to a small molecule PHD inhibitor (PHI) (2‐(1‐chloro‐4‐hydroxyisoquinoline‐3‐carboxamido) acetic acid) did not mimic the ventilatory response to hypoxia. Further, 7 day administration of the PHI induced only modest increases in HVR and carotid body cell proliferation, despite marked stimulation of erythropoiesis. This was in contrast with chronic hypoxia, which elicited both exaggerated HVR and cellular proliferation. The findings demonstrate that PHD enzymes modulate ventilatory sensitivity to hypoxia and identify PHD2 as the most important enzyme in this response. They also reveal differences between genetic inactivation of PHDs, responses to hypoxia and responses to a pharmacological inhibitor, demonstrating the need for caution in predicting the effects of therapeutic modulation of the HIF hydroxylase system on different physiological responses.

Objective Since 2008, the Oxford Diagnostic Horizon Scan Programme has been identifying and summarising evidence on new and emerging diagnostic technologies relevant to primary care. We used these reports to determine the sequence and timing of evidence for new point-of-care diagnostic tests and to identify common evidence gaps in this process. Design Systematic overview of diagnostic horizon scan reports. Primary outcome measures We obtained the primary studies referenced in each horizon scan report (n=40) and extracted details of the study size, clinical setting and design characteristics. In particular, we assessed whether each study evaluated test accuracy, test impact or cost-effectiveness. The evidence for each point-of-care test was mapped against the Horvath framework for diagnostic test evaluation. Results We extracted data from 500 primary studies. Most diagnostic technologies underwent clinical performance (ie, ability to detect a clinical condition) assessment (71.2%), with very few progressing to comparative clinical effectiveness (10.0%) and a cost-effectiveness evaluation (8.6%), even in the more established and frequently reported clinical domains, such as cardiovascular disease. The median time to complete an evaluation cycle was 9 years (IQR 5.5–12.5 years). The sequence of evidence generation was typically haphazard and some diagnostic tests appear to be implemented in routine care without completing essential evaluation stages such as clinical effectiveness. Conclusions Evidence generation for new point-of-care diagnostic tests is slow and tends to focus on accuracy, and overlooks other test attributes such as impact, implementation and cost-effectiveness. Evaluation of this dynamic cycle and feeding back data from clinical effectiveness to refine analytical and clinical performance are key to improve the efficiency of point-of-care diagnostic test development and impact on clinically relevant outcomes. While the ‘road map’ for the steps needed to generate evidence are reasonably well delineated, we provide evidence on the complexity, length and variability of the actual process that many diagnostic technologies undergo.