Control of cell state transitions.

Understanding cell state transitions and purposefully controlling them is a longstanding challenge in biology. Here we present cell state transition assessment and regulation (cSTAR), an approach for mapping cell states, modelling transitions between them and predicting targeted interventions to convert cell fate decisions. cSTAR uses omics data as input, classifies cell states, and develops a workflow that transforms the input data into mechanistic models that identify a core signalling network, which controls cell fate transitions by influencing whole-cell networks. By integrating signalling and phenotypic data, cSTAR models how cells manoeuvre in Waddington's landscape1 and make decisions about which cell fate to adopt. Notably, cSTAR devises interventions to control the movement of cells in Waddington's landscape. Testing cSTAR in a cellular model of differentiation and proliferation shows a high correlation between quantitative predictions and experimental data. Applying cSTAR to different types of perturbation and omics datasets, including single-cell data, demonstrates its flexibility and scalability and provides new biological insights. The ability of cSTAR to identify targeted perturbations that interconvert cell fates will enable designer approaches for manipulating cellular development pathways and mechanistically underpinned therapeutic interventions.

Changes in cellular Ca2+ and Na+ regulation during the progression towards heart failure.

In adapting to disease and loss of tissue, the heart shows great phenotypic plasticity that involves changes to its structure, composition and electrophysiology. Together with parallel whole body cardiovascular adaptations, the initial decline in cardiac function resulting from the insult is compensated. However, in the long term, the heart muscle begins to fail and patients with this condition have a very poor prognosis, with many dying from disturbances of rhythm. The surviving myocytes of these hearts gain Na+ , which is positively inotropic because of alterations to Ca2+ fluxes mediated by the Na+ /Ca2+ exchange, but compromises Ca2+ -dependent energy metabolism in mitochondria. Uptake of Ca2+ into the sarcoplasmic reticulum (SR) is reduced because of diminished function of SR Ca2+ ATPases. The result of increased Ca2+ influx and reduced SR Ca2+ uptake is an increase in the diastolic cytosolic Ca2+ concentration, which promotes spontaneous SR Ca2+ release and induces delayed afterdepolarisations. Action potential duration prolongs because of increased late Na+ current and changes in expression and function of other ion channels and transporters increasing the probability of the formation of early afterdepolarisations. There is a reduction in T-tubule density and so the normal spatial arrangements required for efficient excitation-contraction coupling are compromised and lead to temporal delays in Ca2+ release from the SR. Therefore, the structural and electrophysiological responses that occur to provide compensation do so at the expense of (1) increasing the likelihood of arrhythmogenesis; (2) activating hypertrophic, apoptotic and Ca2+ signalling pathways; and (3) decreasing the efficiency of SR Ca2+ release.

Retrospective cohort analysis of weight changes during the COVID-19 pandemic in a pediatric asthma population.

OBJECTIVE: To investigate the BMI trajectories of children attending a tertiary asthma clinic during the COVID-19 pandemic. METHODS: Data were collected retrospectively on children and young people with asthma who attended the Royal Hospital for Children and Young People (RHCYP) before March 2020 (pre-COVID-19) and after August 2021 (the lifting of national restrictions). MAIN OUTCOME MEASURES: Changes in weight, height, and BMI Z score measured between 13/03/2019 and 13/03/2020 (timepoint 1) and then again during the period 01/08/2021 to 01/10/2022 (timepoint 2); changes in lung function parameters (FEV1) between the timepoints; proportion of study sample classed as obese and overweight at both timepoints; interaction analyses according to deprivation indices (SIMD decile), the use of high dose inhaled corticosteroid (ICS) therapy, and the presence of atopy. RESULTS: Eighty-nine children aged 5-18 years were studied. Weight and height Z scores significantly increased between timepoint 1 and 2 [weight Z score: +0.19 (0.08, +0.30), height Z score: +0.15 (+0.07, +0.23)], such that no significant change was observed in the BMI Z score [+ 0.07 (-0.05, +0.20)] or BMI centile [+0.5 (-3.1, +4.1)]. There was also no change in FEV1%predicted [-0.1 (-3.8, +3.6)] between the timepoints. CONCLUSIONS: No changes in BMI were observed in children with asthma before and after COVID-19 lockdowns. Improved linear growth was noted, implying an improvement in the overall physical health of our study cohort. This may suggest improved asthma control, which may reflect avoidance of viral triggers and/or improved adherence to treatment.

N-cadherin stabilises neural identity by dampening anti-neural signals.

A switch from E- to N-cadherin regulates the transition from pluripotency to neural identity, but the mechanism by which cadherins regulate differentiation was previously unknown. Here, we show that the acquisition of N-cadherin stabilises neural identity by dampening anti-neural signals. We use quantitative image analysis to show that N-cadherin promotes neural differentiation independently of its effects on cell cohesiveness. We reveal that cadherin switching diminishes the level of nuclear ß-catenin, and that N-cadherin also dampens FGF activity and consequently stabilises neural fate. Finally, we compare the timing of cadherin switching and differentiation in vivo and in vitro, and find that this process becomes dysregulated during in vitro differentiation. We propose that N-cadherin helps to propagate a stable neural identity throughout the emerging neuroepithelium, and that dysregulation of this process contributes to asynchronous differentiation in culture.

Integrins Increase Sarcoplasmic Reticulum Activity for Excitation-Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes.

Engagement of the sarcoplasmic reticulum (SR) Ca2+ stores for excitation-contraction (EC)-coupling is a fundamental feature of cardiac muscle cells. Extracellular matrix (ECM) proteins that form the extracellular scaffolding supporting cardiac contractile activity are thought to play an integral role in the modulation of EC-coupling. At baseline, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show poor utilisation of SR Ca2+ stores, leading to inefficient EC-coupling, like developing or human CMs in cardiac diseases such as heart failure. We hypothesised that integrin ligand-receptor interactions between ECM proteins and CMs recruit the SR to Ca2+ cycling during EC-coupling. hiPSC-CM monolayers were cultured on fibronectin-coated glass before 24 h treatment with fibril-forming peptides containing the integrin-binding tripeptide sequence arginine-glycine-aspartic acid (2 mM). Micropipette application of 40 mM caffeine in standard or Na+/Ca2+-free Tyrode's solutions was used to assess the Ca2+ removal mechanisms. Microelectrode recordings were conducted to analyse action potentials in current-clamp. Confocal images of labelled hiPSC-CMs were analysed to investigate hiPSC-CM morphology and ultrastructural arrangements in Ca2+ release units. This study demonstrates that peptides containing the integrin-binding sequence arginine-glycine-aspartic acid (1) abbreviate hiPSC-CM Ca2+ transient and action potential duration, (2) increase co-localisation between L-type Ca2+ channels and ryanodine receptors involved in EC-coupling, and (3) increase the rate of SR-mediated Ca2+ cycling. We conclude that integrin-binding peptides induce recruitment of the SR for Ca2+ cycling in EC-coupling through functional and structural improvements and demonstrate the importance of the ECM in modulating cardiomyocyte function in physiology.

Combined targeting of MEK and the glucocorticoid receptor for the treatment of RAS-mutant multiple myeloma.

BACKGROUND: Multiple myeloma (MM) remains incurable despite recent therapeutic advances. RAS mutations are frequently associated with relapsed/refractory disease. Efforts to target the mitogen-activated protein kinase (MAPK) pathway with the MEK inhibitor, trametinib (Tra) have been limited by toxicities and the development of resistance. Dexamethasone (Dex) is a corticosteroid commonly used in clinical practice, to enhance efficacy of anti-myeloma therapy. Therefore, we hypothesised that the combination of Tra and Dex would yield synergistic activity in RAS-mutant MM. METHODS: The response of human MM cell lines to drug treatment was analysed using cell proliferation assays, Western blotting, Annexin V and propidium iodide staining by flow cytometry and reverse phase protein arrays. The efficacy of trametinib and dexamethasone treatment in the MM.1S xenograft model was assessed by measuring tumor volume over time. RESULTS: The Tra/Dex combination demonstrated synergistic cytotoxicity in KRASG12A mutant lines MM.1S and RPMI-8226. The induction of apoptosis was associated with decreased MCL-1 expression and increased BIM expression. Reverse phase proteomic arrays revealed suppression of FAK, PYK2, FLT3, NDRG1 and 4EBP1 phosphorylation with the Tra/Dex combination. Notably, NDRG1 expression was associated with the synergistic response to Tra/Dex. MM cells were sensitive to PDK1 inhibition and IGF1-induced signalling partially protected from Tra/Dex treatment, highlighting the importance of this pathway. In the MM.1S tumor xenograft model, only the combination of Tra/Dex resulted in a significant inhibition of tumor growth. CONCLUSIONS: Overall Tra/Dex demonstrates antiproliferative activity in RAS-mutant MM cell lines associated with suppression of pro-survival PDK1 signalling and engagement of apoptotic pathways. Our data support further investigation of this combination in RAS-mutant MM.

Reversal of proliferation deficits caused by chromosome 16p13.11 microduplication through targeting NFκB signaling: an integrated study of patient-derived neuronal precursor cells, cerebral organoids and in vivo brain imaging.

The molecular basis of how chromosome 16p13.11 microduplication leads to major psychiatric disorders is unknown. Here we have undertaken brain imaging of patients carrying microduplications in chromosome 16p13.11 and unaffected family controls, in parallel with iPS cell-derived cerebral organoid studies of the same patients. Patient MRI revealed reduced cortical volume, and corresponding iPSC studies showed neural precursor cell (NPC) proliferation abnormalities and reduced organoid size, with the NPCs therein displaying altered planes of cell division. Transcriptomic analyses of NPCs uncovered a deficit in the NFκB p65 pathway, confirmed by proteomics. Moreover, both pharmacological and genetic correction of this deficit rescued the proliferation abnormality. Thus, chromosome 16p13.11 microduplication disturbs the normal programme of NPC proliferation to reduce cortical thickness due to a correctable deficit in the NFκB signalling pathway. This is the first study demonstrating a biologically relevant, potentially ameliorable, signalling pathway underlying chromosome 16p13.11 microduplication syndrome in patient-derived neuronal precursor cells.

Mer-mediated eosinophil efferocytosis regulates resolution of allergic airway inflammation.

BACKGROUND: Eosinophils play a central role in propagation of allergic diseases, including asthma. Both recruitment and retention of eosinophils regulate pulmonary eosinophilia, but the question of whether alterations in apoptotic cell clearance by phagocytes contributes directly to resolution of allergic airway inflammation remains unexplored. OBJECTIVES: In this study we investigated the role of the receptor tyrosine kinase Mer in mediating apoptotic eosinophil clearance and allergic airway inflammation resolution in vivo to establish whether apoptotic cell clearance directly affects the resolution of allergic airway inflammation. METHODS: Alveolar and bone marrow macrophages were used to study Mer-mediated phagocytosis of apoptotic eosinophils. Allergic airway inflammation resolution was modeled in mice by using ovalbumin. Fluorescently labeled apoptotic cells were administered intratracheally or eosinophil apoptosis was driven by administration of dexamethasone to determine apoptotic cell clearance in vivo. RESULTS: Inhibition or absence of Mer impaired phagocytosis of apoptotic human and mouse eosinophils by macrophages. Mer-deficient mice showed delayed resolution of ovalbumin-induced allergic airway inflammation, together with increased airway responsiveness to aerosolized methacholine, increased bronchoalveolar lavage fluid protein levels, altered cytokine production, and an excess of uncleared dying eosinophils after dexamethasone treatment. Alveolar macrophage phagocytosis was significantly Mer dependent, with the absence of Mer attenuating apoptotic cell clearance in vivo to enhance inflammation in response to apoptotic cells. CONCLUSIONS: We demonstrate that Mer-mediated apoptotic cell clearance by phagocytes contributes to resolution of allergic airway inflammation, suggesting that augmenting apoptotic cell clearance is a potential therapeutic strategy for treating allergic airway inflammation.

Ammonite habitat revealed via isotopic composition and comparisons with co-occurring benthic and planktonic organisms.

Ammonites are among the best-known fossils of the Phanerozoic, yet their habitat is poorly understood. Three common ammonite families (Baculitidae, Scaphitidae, and Sphenodiscidae) co-occur with well-preserved planktonic and benthic organisms at the type locality of the upper Maastrichtian Owl Creek Formation, offering an excellent opportunity to constrain their depth habitats through isotopic comparisons among taxa. Based on sedimentary evidence and the micro- and macrofauna at this site, we infer that the 9-m-thick sequence was deposited at a paleodepth of 70-150 m. Taxa present throughout the sequence include a diverse assemblage of ammonites, bivalves, and gastropods, abundant benthic foraminifera, and rare planktonic foraminifera. No stratigraphic trends are observed in the isotopic data of any taxon, and thus all of the data from each taxon are considered as replicates. Oxygen isotope-based temperature estimates from the baculites and scaphites overlap with those of the benthos and are distinct from those of the plankton. In contrast, sphenodiscid temperature estimates span a range that includes estimates of the planktonic foraminifera and of the warmer half of the benthic values. These results suggest baculites and scaphites lived close to the seafloor, whereas sphenodiscids sometimes inhabited the upper water column and/or lived closer to shore. In fact, the rarity and poorer preservation of the sphenodiscids relative to the baculites and scaphites suggests that the sphenodiscid shells may have only reached the Owl Creek locality by drifting seaward after death.

Effect of ovariectomy on intracellular Ca2+ regulation in guinea pig cardiomyocytes.

This study addressed the hypothesis that long-term deficiency of ovarian hormones after ovariectomy (OVx) alters cellular Ca2+-handling mechanisms in the heart, resulting in the formation of a proarrhythmic substrate. It also tested whether estrogen supplementation to OVx animals reverses any alterations to cardiac Ca2+ handling and rescues proarrhythmic behavior. OVx or sham operations were performed on female guinea pigs using appropriate anesthetic and analgesic regimes. Pellets containing 17ß-estradiol (1 mg, 60-day release) were placed subcutaneously in selected OVx animals (OVx + E). Cardiac myocytes were enzymatically isolated, and electrophysiological measurements were conducted with a switch-clamp system. In fluo-4-loaded cells, Ca2+ transients were 20% larger, and fractional sarcoplasmic reticulum (SR) Ca2+ release was 7% greater in the OVx group compared with the sham group. Peak L-type Ca2+ current was 16% larger in OVx myocytes with channel inactivation shifting to more positive membrane potentials, creating a larger "window" current. SR Ca2+ stores were 22% greater in the OVx group, and these cells showed a higher frequency of Ca2+ sparks and waves and shorter wave-free intervals. OVx myocytes showed higher frequencies of early afterdepolarizations, and a greater percentage of these cells showed delayed afterdepolarizations after exposure to isoprenaline compared with sham myocytes. The altered Ca2+ regulation occurring in the OVx group was not observed in the OVx + E group. These findings suggest that long-term deprivation of ovarian hormones in guinea pigs lead to changes in myocyte Ca2+-handling mechanisms that are considered proarrhythmogenic. 17ß-Estradiol replacement prevented these adverse effects.NEW & NOTEWORTHY Ovariectomized guinea pig cardiomyocytes have higher frequencies of Ca2+ waves, and isoprenaline-challenged cells display more early afterdepolarizations, delayed afterdepolarizations, and extra beats compared with sham myocytes. These alterations to Ca2+ regulation were not observed in myocytes from ovariectomized guinea pigs supplemented with 17ß-estradiol, suggesting that ovarian hormone deficiency modifies cardiac Ca2+ regulation, potentially creating proarrhythmic substrates.

Age- and strain-related aberrant Ca2+ release is associated with sudden cardiac death in the ACTC E99K mouse model of hypertrophic cardiomyopathy.

Patients with hypertrophic cardiomyopathy, particularly young adults, can die from arrhythmia, but the mechanism underlying abnormal rhythm formation remains unknown. C57Bl6 × CBA/Ca mice carrying a cardiac actin ( ACTC) E99K (Glu99Lys) mutation reproduce many aspects of human hypertrophic cardiomyopathy, including increased myofilament Ca2+ sensitivity and sudden death in a proportion (up to 40%) of young (28-40 day old) animals. We studied the hearts of transgenic (TG; ACTC E99K) mice and their non-TG (NTG) littermates when they were in their vulnerable period (28-40 days old) and when they were adult (8-12 wk old). Ventricular myocytes were isolated from the hearts of TG and NTG mice at these two time points. We also examined the hearts of mice that died suddenly (SCD). SCD animals had approximately four times more collagen compared with age-matched NTG mice, yet myocyte cell size was normal. Young TG mice had double the collagen content of NTG mice. Contraction and Ca2+ transients were greater in cells from young TG mice compared with their NTG littermates but not in cells from adult mice (TG or NTG). Cells from young TG mice had a greater propensity for Ca2+ waves than NTG littermates, and, despite similar sarcoplasmic reticulum Ca2+ content, a proportion of these cells had larger Ca2+ spark mass. We found that the probability of SCD in young TG mice was increased when the mutation was expressed in animals with a CBA/Ca2+ background and almost eliminated in mice bred on a C57Bl6 background. The latter TG mice had normal cellular Ca2+ homeostasis. NEW & NOTEWORTHY Mice with the actin Glu99Lys hypertrophic cardiomyopathy mutation ( ACTC E99K) are prone to sudden cardiac death around 40 days, associated with increased Ca2+ transients, spark mass, and fibrosis. However, adult survivors have normal Ca2+ transients and spark density accompanied by hypertrophy. Penetrance of the sudden cardiac death phenotype depends on the genetic background of the mouse. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/calcium-regulation-in-e99k-mouse-heart/ .

Functional consequences of co-expressing connexin40 or connexin45 with connexin43 on intercellular electrical coupling.

The functional characteristics of the co-expression of connexin43, connexin40, and connexin45 proteins in human myocardium are thought to play an important role in governing normal propagation of the cardiac electrical impulse and in generating the myocardial substrate for some arrhythmias and conduction disturbances. A rat liver epithelial cell line, that endogenously expresses connexin43, was used to induce also expression of connexin40 or connexin45 after stable transfection using an inducible ecdysone system. Electrical coupling was estimated from measurement of the input resistance of transfected cells using an intracellular microelectrode to inject current and record changes to membrane potential. However, varied expression of the transfected connexin40 or connexin45 did not change electrical coupling, although connexin43/40 co-expression led to better coupling than connexin43/45 co-expression. Quantification of endogenous connexin43 expression, at both mRNA and protein levels, showed that it was altered in a manner dependent on the transfected connexin isotype. The data using rat liver epithelial cells indicate an increased electrical coupling upon expression of connexin40 and connexin43 but decreased coupling with connexin45 and connexin43 co-expression.

The mechanism of flecainide action in CPVT does not involve a direct effect on RyR2.

RATIONALE: Flecainide, a class 1c antiarrhythmic, has emerged as an effective therapy in preventing arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) refractory to ß-adrenergic receptor blockade. It has been proposed that the clinical efficacy of flecainide in CPVT is because of the combined actions of direct blockade of ryanodine receptors (RyR2) and Na(+) channel inhibition. However, there is presently no direct evidence to support the notion that flecainide blocks RyR2 Ca(2+) flux in the physiologically relevant (luminal-to-cytoplasmic) direction. The mechanism of flecainide action remains controversial. OBJECTIVE: To examine, in detail, the effect of flecainide on the human RyR2 channel and to establish whether the direct blockade of physiologically relevant RyR2 ion flow by the drug contributes to its therapeutic efficacy in the clinical management of CPVT. METHODS AND RESULTS: Using single-channel analysis, we show that, even at supraphysiological concentrations, flecainide did not inhibit the physiologically relevant, luminal-to-cytosolic flux of cations through the channel. Moreover, flecainide did not alter RyR2 channel gating and had negligible effect on the mechanisms responsible for the sarcoplasmic reticulum charge-compensating counter current. Using permeabilized cardiac myocytes to eliminate any contribution of plasmalemmal Na(+) channels to the observed actions of the drug at the cellular level, flecainide did not inhibit RyR2-dependent sarcoplasmic reticulum Ca(2+) release. CONCLUSIONS: The principal action of flecainide in CPVT is not via a direct interaction with RyR2. Our data support a model of flecainide action in which Na(+)-dependent modulation of intracellular Ca(2+) handling attenuates RyR2 dysfunction in CPVT.

A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation.

Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic "compensation" and congestive "decompensation." Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae.

Continental warming preceding the Palaeocene-Eocene thermal maximum.

Marine and continental records show an abrupt negative shift in carbon isotope values at ∼55.8 Myr ago. This carbon isotope excursion (CIE) is consistent with the release of a massive amount of isotopically light carbon into the atmosphere and was associated with a dramatic rise in global temperatures termed the Palaeocene-Eocene thermal maximum (PETM). Greenhouse gases released during the CIE, probably including methane, have often been considered the main cause of PETM warming. However, some evidence from the marine record suggests that warming directly preceded the CIE, raising the possibility that the CIE and PETM may have been linked to earlier warming with different origins. Yet pre-CIE warming is still uncertain. Disentangling the sequence of events before and during the CIE and PETM is important for understanding the causes of, and Earth system responses to, abrupt climate change. Here we show that continental warming of about 5 °C preceded the CIE in the Bighorn Basin, Wyoming. Our evidence, based on oxygen isotopes in mammal teeth (which reflect temperature-sensitive fractionation processes) and other proxies, reveals a marked temperature increase directly below the CIE, and again in the CIE. Pre-CIE warming is also supported by a negative amplification of δ(13)C values in soil carbonates below the CIE. Our results suggest that at least two sources of warming-the earlier of which is unlikely to have been methane-contributed to the PETM.

Myosin regulatory light chain (RLC) phosphorylation change as a modulator of cardiac muscle contraction in disease.

Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.

Lung clearance index (LCI2.5) changes after initiation of Elexacaftor/Tezacaftor/Ivacaftor in children with cystic fibrosis aged between 6 and 11 years: The "real-world" differs from trial data.

BACKGROUND: Elexacaftor in combination with Tezacaftor and Ivacaftor (ETI) became licensed in the United Kingdom in early 2022 for children aged 6-11 years with cystic fibrosis (CF) and an eligible mutation. Many in this age group have excellent prior lung health making quantitative measurement of benefit challenging. Clinical trials purport that lung clearance index (LCI2.5) measurement is most suitable for this purpose. OBJECTIVES: This study aimed to understand the clinical utility of LCI2.5 in detecting change after commencing ETI in the real world. PATIENT SELECTION/METHODS: Baseline anthropometric data were collected along with spirometry (forced expiratory volume in 1 s [FEV1], forced vital capacityFV and LCI2.5 measures in children aged 6-11 years with CF before starting ETI. Measures were repeated after a mean (range) of 8.2 (7-14) months of ETI treatment. The primary endpoint was a change in LCI2.5, with secondary endpoints including change in FEV1 and change in body mass index (BMI) also reported. RESULTS: Twelve children were studied (seven male, mean age 9.5 years at baseline). Our study population had a mean (SD) LCI2.5 of 7.01 (1.14) and FEV1 of 96 (13) %predicted at baseline. Mean (95% confidence interval) changes in LCI2.5 [-0.7 (-1.4, 0), p = .06] and BMI [+0.7 (+0.1, +1.3), p = .03] were observed, along with changes in FEV1 of +3.1 (-1.9, +8.1) %predicted. CONCLUSIONS: Real-world changes in LCI2.5 (-0.7) are different to those reported in clinical trials (-2.29). Lower baseline LCI2.5 as a result of prior modulator exposure, high baseline lung health, and new LCI2.5 software analyses all contribute to lower LCI2.5 values being recorded in the real world of children with CF.

Changes in physiological, functional and structural markers of cystic fibrosis lung disease with treatment of a pulmonary exacerbation.

BACKGROUND: Clinical trials in cystic fibrosis (CF) have been hindered by the paucity of well characterised and clinically relevant outcome measures. AIM: To evaluate a range of conventional and novel biomarkers of CF lung disease in a multicentre setting as a contributing study in selecting outcome assays for a clinical trial of CFTR gene therapy. METHODS: A multicentre observational study of adult and paediatric patients with CF (>10 years) treated for a physician-defined exacerbation of CF pulmonary symptoms. Measurements were performed at commencement and immediately after a course of intravenous antibiotics. Disease activity was assessed using 46 assays across five key domains: symptoms, lung physiology, structural changes on CT, pulmonary and systemic inflammatory markers. RESULTS: Statistically significant improvements were seen in forced expiratory volume in 1 s (p<0.001, n=32), lung clearance index (p<0.01, n=32), symptoms (p<0.0001, n=37), CT scores for airway wall thickness (p<0.01, n=31), air trapping (p<0.01, n=30) and large mucus plugs (p=0.0001, n=31), serum C-reactive protein (p<0.0001, n=34), serum interleukin-6 (p<0.0001, n=33) and serum calprotectin (p<0.0001, n=31). DISCUSSION: We identify the key biomarkers of inflammation, imaging and physiology that alter alongside symptomatic improvement following treatment of an acute CF exacerbation. These data, in parallel with our study of biomarkers in patients with stable CF, provide important guidance in choosing optimal biomarkers for novel therapies. Further, they highlight that such acute therapy predominantly improves large airway parameters and systemic inflammation, but has less effect on airway inflammation.

Mechanical and energetic properties of papillary muscle from ACTC E99K transgenic mouse models of hypertrophic cardiomyopathy.

We compared the contractile performance of papillary muscle from a mouse model of hypertrophic cardiomyopathy [α-cardiac actin (ACTC) E99K mutation] with nontransgenic (non-TG) littermates. In isometric twitches, ACTC E99K papillary muscle produced three to four times greater force than non-TG muscle under the same conditions independent of stimulation frequency and temperature, whereas maximum isometric force in myofibrils from these muscles was not significantly different. ACTC E99K muscle relaxed slower than non-TG muscle in both papillary muscle (1.4×) and myofibrils (1.7×), whereas the rate of force development after stimulation was the same as non-TG muscle for both electrical stimulation in intact muscle and after a Ca²âº jump in myofibrils. The EC50 for Ca²âº activation of force in myofibrils was 0.39 ± 0.33 µmol/l in ACTC E99K myofibrils and 0.80 ± 0.11 µmol/l in non-TG myofibrils. There were no significant differences in the amplitude and time course of the Ca²âº transient in myocytes from ACTC E99K and non-TG mice. We conclude that hypercontractility is caused by higher myofibrillar Ca²âº sensitivity in ACTC E99K muscles. Measurement of the energy (work + heat) released in actively cycling heart muscle showed that for both genotypes, the amount of energy turnover increased with work done but with decreasing efficiency as energy turnover increased. Thus, ACTC E99K mouse heart muscle produced on average 3.3-fold more work than non-TG muscle, and the cost in terms of energy turnover was disproportionately higher than in non-TG muscles. Efficiency for ACTC E99K muscle was in the range of 11-16% and for non-TG muscle was 15-18%.

Consensus statement for inert gas washout measurement using multiple- and single- breath tests.

Inert gas washout tests, performed using the single- or multiple-breath washout technique, were first described over 60 years ago. As measures of ventilation distribution inhomogeneity, they offer complementary information to standard lung function tests, such as spirometry, as well as improved feasibility across wider age ranges and improved sensitivity in the detection of early lung damage. These benefits have led to a resurgence of interest in these techniques from manufacturers, clinicians and researchers, yet detailed guidelines for washout equipment specifications, test performance and analysis are lacking. This manuscript provides recommendations about these aspects, applicable to both the paediatric and adult testing environment, whilst outlining the important principles that are essential for the reader to understand. These recommendations are evidence based, where possible, but in many places represent expert opinion from a working group with a large collective experience in the techniques discussed. Finally, the important issues that remain unanswered are highlighted. By addressing these important issues and directing future research, the hope is to facilitate the incorporation of these promising tests into routine clinical practice.