The voltage-gated Cl(-) channel (CLC) family comprises cell surface Cl(-) channels and intracellular Cl(-)/H(+) exchangers. CLCs in organelle membranes are thought to assist acidification by providing a passive chloride conductance that electrically counterbalances H(+) accumulation. Following recent descriptions of Cl(-)/H(+) exchange activity in endosomal CLCs we have re-evaluated their role. We expressed human CLC-5 in HEK293 cells, recorded currents under a range of Cl(-) and H(+) gradients by whole-cell patch clamp, and examined the contribution of CLC-5 to endosomal acidification using a targeted pH-sensitive fluorescent protein. We found that CLC-5 only conducted outward currents, corresponding to Cl(-) flux into the cytoplasm and H(+) from the cytoplasm. Inward currents were never observed, despite the range of intracellular and extracellular Cl(-) concentrations and pH used. Endosomal acidification in HEK293 cells was prevented by 25 microm bafilomycin-A1, an inhibitor of vacuolar-type H(+)-ATPase (v-ATPase), which actively pumps H(+) into the endosomal lumen. Overexpression of CLC-5 in HEK293 cells conferred an additional bafilomycin-insensitive component to endosomal acidification. This effect was abolished by making mutations in CLC-5 that remove H(+) transport, which result in either no current (E268A) or bidirectional Cl(-) flux (E211A). Endosomal acidification in a proximal tubule cell line was partially sensitive to inhibition of v-ATPase by bafilomycin-A1. Furthermore, in the presence of bafilomycin-A1, acidification was significantly reduced and nearly fully ablated by partial and near-complete knockdown of endogenous CLC-5 by siRNA. These data suggest that CLC-5 is directly involved in endosomal acidification by exchanging endosomal Cl(-) for H(+).

Cone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2gnat2:EGFP) zebrafish.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

We describe the first clinical isolate of Bordetella petrii from a patient with mandibular osteomyelitis. The only previously documented isolation of B. petrii occurred after the initial culture of a single strain from an environmental source.

Strains of type emm89 Streptococcus pyogenes have recently increased in frequency as a cause of human infections in several countries in Europe and North America. This increase has been molecular epidemiologically linked with the emergence of a new genetically distinct clone, designated clade 3. We sought to extend our understanding of this epidemic behavior by the genetic characterization of type emm89 strains responsible in recent years for an increased frequency of infections in Scotland.

Laundering of textiles is a significant source of waterborne microfiber pollution, and solutions are now being sought to mitigate this issue including improvements in clothing technology and integration of filtration systems into washing machines. Vented tumble dryers are a potential source of airborne microfiber pollution, as their built-in lint filtration systems have been found to be inefficient with significant quantities of textile microfibers being released to the external environment through their exhaust air ducts. The present study is the first to evaluate the impact of condenser dryers, finding that they are significant contributors to waterborne microfiber pollution from the lint filter (if users clean this with water), the condenser and the condensed water. Microfiber release from drying of real consumer loads in condenser and vented tumble dryers was compared, finding that real loads release surprisingly high levels of microfibers (total 341.5 ± 126.0 ppm for those dried in a condenser dryer and 256.0 ± 74.2 ppm for those dried in a vented dryer), similar in quantity to microfibers produced during the first highly-shedding drying cycle of a new T-shirt load (total 321.4 ± 11.2 ppm) in a condenser dryer. Vented dryers were found to be significant contributors to waterborne microfiber pollution if consumers clean the lint filter with water in accordance with some published appliance usage instructions, as most (86.1 ± 5.5% for the real consumer loads tested) of the microfibers generated during vented tumble drying were collected on the lint filter. Therefore, tumble dryers are a significant source of waterborne and (for vented dryers) airborne microfiber pollution. While reducing the pore size of tumble dryer lint filters and instructing consumers to dispose of fibers collected on lint filters as municipal solid waste could help reduce the issue, more sophisticated engineering solutions will likely be required to achieve a more comprehensive solution.

Intensity-controlled (relative to VO2max) treadmill exercise training in adult rats results in the activation and ensuing differentiation of endogenous c-kit(pos) cardiac stem/progenitor cells (eCSCs) into newly formed cardiomyocytes and capillaries. Whether these training-induced adaptations persist following detraining is undetermined. Twelve male Wistar rats (~230 g) were exercised at 80-85% of their VO2max for 30 min day(-1), 4 days week(-1) for 4 weeks (TR; n = 6), followed by 4 weeks of detraining (DTR; n = 6). Twelve untrained rats acted as controls (CTRL). Exercise training significantly enhanced VO2max (11.34 mL kg(-1) min(-1)) and wet heart weight (29%) above CTRL (P < 0.05). Echocardiography revealed that exercise training increased LV mass (~32%), posterior and septal wall thickness (~15%), ejection fraction and fractional shortening (~10%) compared to CTRL (P < 0.05). Cardiomyocyte diameter (17.9 ± 0.1 μm vs. 14.9 ± 0.6 μm), newly formed (BrdU(pos)/Ki67(pos)) cardiomyocytes (7.2 ± 1.3%/1.9 ± 0.7% vs. 0.2 ± 0.1%/0.1 ± 0.1%), total cardiomyocyte number (45.6 ± 0.6 × 10(6) vs. 42.5 ± 0.4 × 10(6)), c-kit(pos) eCSC number (884 ± 112 per 10(6) cardiomyocytes vs. 482 ± 132 per 10(6) cardiomyocytes), and capillary density (4123 ± 227 per mm(2) vs. 2117 ± 118 per mm(2)) were significantly greater in the LV of trained animals (P < 0.05) than CTRL. Detraining removed the stimulus for c-kit(pos) eCSC activation (640 ± 98 per 10(6) cardiomyocytes) and resultant cardiomyocyte hyperplasia (0.4 ± 0.3% BrdU(pos)/0.2 ± 0.2% Ki67(pos) cardiomyocytes). Capillary density (3673 ± 374 per mm(2)) and total myocyte number (44.7 ± 0.5 × 10(6)) remained elevated following detraining, but cardiomyocyte hypertrophy (15.0 ± 0.4 μm) was lost, resulting in a reduction of anatomical (wall thickness ~4%; LV mass ~10% and cardiac mass ~8%, above CTRL) and functional (EF & FS ~2% above CTRL) parameters gained through exercise training. These findings demonstrate that cardiac adaptations, produced by 4 weeks of intensity-controlled exercise training are lost after a similar period of detraining.

An overdose of Isoproterenol (ISO) causes acute cardiomyocyte (CM) dropout and activates the resident cardiac c-kitpos stem/progenitor cells (CSCs) generating a burst of new CM formation that replaces those lost to ISO. Recently, unsuccessful attempts to reproduce these findings using c-kitCre knock-in (KI) mouse models were reported. We tested whether c-kit haploinsufficiency in c-kitCreKI mice was the cause of the discrepant results in response to ISO. Male C57BL/6J wild-type (wt) mice and c-kitCreKI mice were given a single dose of ISO (200 and/or 400 mg/Kg s.c.). CM formation was measured with different doses and duration of BrdU or EdU. We compared the myogenic and regenerative potential of the c-kitCreCSCs with wtCSCs. Acute ISO overdose causes LV dysfunction with dose-dependent CM death by necrosis and apoptosis, whose intensity follows a basal-apical and epicardium to sub-endocardium gradient, with the most severe damage confined to the apical sub-endocardium. The damage triggers significant new CM formation mainly in the apical sub-endocardial layer. c-kit haploinsufficiency caused by c-kitCreKIs severely affects CSCs myogenic potential. c-kitCreKI mice post-ISO fail to respond with CSC activation and show reduced CM formation and suffer chronic cardiac dysfunction. Transplantation of wtCSCs rescued the defective regenerative cardiac phenotype of c-kitCreKI mice. Furthermore, BAC-mediated transgenesis of a single c-kit gene copy normalized the functional diploid c-kit content of c-kitCreKI CSCs and fully restored their regenerative competence. Overall, these data show that c-kit haploinsufficiency impairs the endogenous cardioregenerative response after injury affecting CSC activation and CM replacement. Repopulation of c-kit haploinsufficient myocardial tissue with wtCSCs as well c-kit gene deficit correction of haploinsufficient CSCs restores CM replacement and functional cardiac repair. Thus, adult neo-cardiomyogenesis depends on and requires a diploid level of c-kit.

Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a β-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1-2% of total c-kitpos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Iron nitride (Fe3N) and iron carbide (Fe3C) nanoparticles can be prepared via sol-gel synthesis. While sol-gel methods are simple, it can be difficult to control the crystalline composition, i.e., to achieve a Rietveld-pure product. In a previous in situ synchrotron study of the sol-gel synthesis of Fe3N/Fe3C, we showed that the reaction proceeds as follows: Fe3O4 → FeOx → Fe3N → Fe3C. There was considerable overlap between the different phases, but we were unable to ascertain whether this was due to the experimental setup (side-on heating of a quartz capillary which could lead to thermal gradients) or whether individual particle reactions proceed at different rates. In this paper, we use in situ wide- and small-angle X-ray scattering (wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS)) to demonstrate that the overlapping phases are indeed due to variable reaction rates. While the initial oxide nanoparticles have a small range of diameters, the size range expands considerably and very rapidly during the oxide-nitride transition. This has implications for the isolation of Rietveld-pure Fe3N, and in an extensive laboratory study, we were indeed unable to isolate phase-pure Fe3N. However, we made the surprising discovery that Rietveld-pure Fe3C nanoparticles can be produced at 500 °C with a sufficient furnace dwell time. This is considerably lower than the previous reports of the sol-gel synthesis of Fe3C nanoparticles.

Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery of intracellular calcium signalling in cardiomyocytes. While a range of optical super-resolution microscopy techniques have revealed the nanoscale structure of these clusters, the three-dimensional (3D) nanoscale topologies of the clusters have remained mostly unresolved. In this paper, we demonstrate the exploitation of molecular-scale resolution in enhanced expansion microscopy (EExM) along with various 2D and 3D visualization strategies to observe the topological complexities, geometries and molecular sub-domains within the RyR clusters. Notably, we observed sub-domains containing RyR-binding protein junctophilin-2 (JPH2) occupying the central regions of RyR clusters in the deeper interior of the myocytes (including dyads), while the poles were typically devoid of JPH2, lending to a looser RyR arrangement. By contrast, peripheral RyR clusters exhibited variable co-clustering patterns and ratios between RyR and JPH2. EExM images of dyadic RyR clusters in right ventricular (RV) myocytes isolated from rats with monocrotaline-induced RV failure revealed hallmarks of RyR cluster fragmentation accompanied by breaches in the JPH2 sub-domains. Frayed RyR patterns observed adjacent to these constitute new evidence that the destabilization of the RyR arrays inside the JPH2 sub-domains may seed the primordial foci of dyad remodelling observed in heart failure. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

A common feature of glasses is the "boson peak", observed as an excess in the heat capacity over the crystal or as an additional peak in the terahertz vibrational spectrum. The microscopic origins of this peak are not well understood; the emergence of locally ordered structures has been put forward as a possible candidate. Here, we show that depolarised Raman scattering in liquids consisting of highly symmetric molecules can be used to isolate the boson peak, allowing its detailed observation from the liquid into the glass. The boson peak in the vibrational spectrum matches the excess heat capacity. As the boson peak intensifies on cooling, wide-angle x-ray scattering shows the simultaneous appearance of a pre-peak due to molecular clusters consisting of circa 20 molecules. Atomistic molecular dynamics simulations indicate that these are caused by over-coordinated molecules. These findings represent an essential step toward our understanding of the physics of vitrification.

The mental health of healthcare workers (HCWs) is critical to their long-term well-being and future disaster preparedness. Goal 1 of this study was to identify rates of mental health problems experienced by HCWs. Goal 2 was to test a model of risk stemming from pandemic-related stressors and vulnerability factors. This cross-sectional study included HCWs (N ​= ​2,246 [1,573 clinical providers; 673 non-clinical staff]) in the Rocky Mountain West who voluntarily completed an online survey in April/May 2020. Respondents completed measures for traumatic stress symptoms, depression, anxiety, alcohol use, and sleep. Logistic regressions stratified by professional role (clinical versus non-clinical) were specified to predict clinical screening cutoff (positive/negative) as a function of five pandemic-related stressors (immunocompromised self; immunocompromised household member; care provision to infected patients; clinical management role; positive cases). Results showed that more than half of HCWs surveyed (52.5%) screened positive (above cutoff) for traumatic stress, depression, or anxiety, with ~20% reporting problematic alcohol use, and variable insufficient sleep from ~10% off shift to ~50% on shift. Clinical employees with an immunocompromised household member had increased odds of screening positive for a mental health problem. Non-clinical HCWs who were immunocompromised were at elevated risk for screening positive a mental health problem. Being female, minority status, and younger increased odds for mental health problems. Implications include alleviating a portion of the mental health burden of HCWs involved in response to the SARS-CoV-2 pandemic by considering policies to protect immunocompromised HCWs and their families (e.g., vaccine priorities, telework options).

Receptor tyrosine kinase inhibitors improve cancer survival but their cardiotoxicity requires investigation. We investigated these inhibitors' effects on human cardiac progenitor cells in vitro and rat heart in vivo. We applied imatinib, sunitinib or sorafenib to human cardiac progenitor cells, assessing cell viability, proliferation, stemness, differentiation, growth factor production and second messengers. Alongside, sunitinib effects were assessed in vivo. Inhibitors decreased (p < 0.05) cell viability, at levels equivalent to 'peak' (24 h; imatinib: 91.5 ± 0.9%; sunitinib: 83.9 ± 1.8%; sorafenib: 75.0 ± 1.6%) and 'trough' (7 days; imatinib: 62.3 ± 6.2%; sunitinib: 86.2 ± 3.5%) clinical plasma levels, compared to control (100% viability). Reduced (p < 0.05) cell cycle activity was seen with imatinib (29.3 ± 4.3% cells in S/G2/M-phases; 50.3 ± 5.1% in control). Expression of PECAM-1, Nkx2.5, Wnt2, linked with cell differentiation, were decreased (p < 0.05) 2, 2 and 6-fold, respectively. Expression of HGF, p38 and Akt1 in cells was reduced (p < 0.05) by sunitinib. Second messenger (p38 and Akt1) blockade affected progenitor cell phenotype, reducing c-kit and growth factor (HGF, EGF) expression. Sunitinib for 9 days (40 mg/kg, i.p.) in adult rats reduced (p < 0.05) cardiac ejection fraction (68 ± 2% vs. baseline (83 ± 1%) and control (84 ± 4%)) and reduced progenitor cell numbers. Receptor tyrosine kinase inhibitors reduce cardiac progenitor cell survival, proliferation, differentiation and reparative growth factor expression.

The family of CLC proteins comprises both Cl(-) channels and Cl(-)/H(+) exchange transporters with varying degrees of voltage dependence. The human CLC-5 is an electrogenic voltage-dependent 2Cl(-)/1H(+) exchanger that gives rise to strongly outwardly rectifying currents when expressed. We conducted whole-cell recordings from HEK293 cells transiently transfected with either wild-type CLC-5 or a permeation-deficient mutant, E268A. With E268A CLC-5 we recorded transient voltage-dependent currents that represent the gating currents associated with CLC-5 activation and had kinetics that could be described by voltage-dependent forward and reverse transition rates. In extracellular solutions rich in Cl(-) or Br(-), CLC-5 exhibited a gating charge of 1.3, but this was reduced to 0.9 in solutions comprising the impermeant anions aspartate, methanesulfonate, sulfate, or HEPES. Extracellular ion depletion by local perfusion with isotonic mannitol failed to reduce the gating charge further. Lowering intracellular pH from 7.4 to 5.4 did not shift the voltage-dependence of the gating currents, but reducing and increasing intracellular Cl(-) shifted the charge-voltage relationship to more negative and positive potentials, respectively. Our data suggest that voltage sensing is an intrinsic property of the CLC-5 protein and that permeant anions, particularly Cl(-), modulate a voltage-dependent transition to an activated state from which Cl(-)/H(+) exchange can occur.

Functional complexity of the eukaryotic mitochondrial proteome is augmented by independent gene acquisition from bacteria since its endosymbiotic origins. Mammalian homologs of many ancestral mitochondrial proteins have uncharacterized catalytic activities. Recent forward genetic approaches attributed functions to proteins in established metabolic pathways, thereby limiting the possibility of identifying novel biology relevant to human disease. We undertook a bottom-up biochemistry approach to discern evolutionarily conserved mitochondrial proteins with catalytic potential.

Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis, which are typically transmitted via respiratory droplets, are leading causes of invasive diseases, including bacteraemic pneumonia and meningitis, and of secondary infections subsequent to post-viral respiratory disease. The aim of this study was to investigate the incidence of invasive disease due to these pathogens during the early months of the COVID-19 pandemic.

The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.

Abdominal aortic aneurysm (AAA) is a common cause of morbidity and mortality and has a significant heritability. We carried out a genome-wide association discovery study of 1866 patients with AAA and 5435 controls and replication of promising signals (lead SNP with a p value < 1 × 10(-5)) in 2871 additional cases and 32,687 controls and performed further follow-up in 1491 AAA and 11,060 controls. In the discovery study, nine loci demonstrated association with AAA (p < 1 × 10(-5)). In the replication sample, the lead SNP at one of these loci, rs1466535, located within intron 1 of low-density-lipoprotein receptor-related protein 1 (LRP1) demonstrated significant association (p = 0.0042). We confirmed the association of rs1466535 and AAA in our follow-up study (p = 0.035). In a combined analysis (6228 AAA and 49182 controls), rs1466535 had a consistent effect size and direction in all sample sets (combined p = 4.52 × 10(-10), odds ratio 1.15 [1.10-1.21]). No associations were seen for either rs1466535 or the 12q13.3 locus in independent association studies of coronary artery disease, blood pressure, diabetes, or hyperlipidaemia, suggesting that this locus is specific to AAA. Gene-expression studies demonstrated a trend toward increased LRP1 expression for the rs1466535 CC genotype in arterial tissues; there was a significant (p = 0.029) 1.19-fold (1.04-1.36) increase in LRP1 expression in CC homozygotes compared to TT homozygotes in aortic adventitia. Functional studies demonstrated that rs1466535 might alter a SREBP-1 binding site and influence enhancer activity at the locus. In conclusion, this study has identified a biologically plausible genetic variant associated specifically with AAA, and we suggest that this variant has a possible functional role in LRP1 expression.