High correlated color temperature artificial lighting impairs retinal pigment epithelium integrity and chloride ion transport: A potential mechanism for choroidal thinning.

Among the environmental factors contributing to myopia, the role of correlated color temperature (CCT) of ambient light emerges as a key element warranting in-depth investigation. The choroid, a highly vascularized and dynamic structure, often undergoes thinning during the progression of myopia, though the precise mechanism remains elusive. The retinal pigment epithelium (RPE), the outermost layer of the retina, plays a pivotal role in regulating the transport of ion and fluid between the subretinal space and the choroid. A hypothesis suggests that variations in choroidal thickness (ChT) may be modulated by transepithelial fluid movement across the RPE. Our experimental results demonstrate that high CCT illumination significantly compromised the integrity of tight junctions in the RPE and disrupted chloride ion transport. This functional impairment of the RPE may lead to a reduction in fluid transfer across the RPE, consequently resulting in choroidal thinning and potentially accelerating axial elongation. Our findings provide support for the crucial role of the RPE in regulating ChT. Furthermore, we emphasize the potential hazards posed by high CCT artificial illumination on the RPE, the choroid, and refractive development, underscoring the importance of developing eye-friendly artificial light sources to aid in the prevention and control of myopia.

Ionic plasmon-polariton in application to neurosignaling.

The diffusive ion current is insufficient to explain the fast saltatory conduction observed in myelinated axons and in pain-sensing C fibers in the human nervous system, where the stimulus signal exhibits a velocity two orders of magnitude greater than the upper limit of ion diffusion velocity, even when the diffusion is accelerated by myelin, as in the discrete cable model including the Hodgkin-Huxley mechanism. The agreement with observations has been achieved in a wave-type model of stimulus signal kinetics via synchronized ion local density oscillations propagating as a wave in axons periodically corrugated by myelin segments in myelinated axons, or by periodically distributed rafts with clusters of Na^{+} channels in C fibers. The resulting so-called plasmon-polariton model for saltatory conduction reveals also the specific role of myelin, which is different from what was previously thought. This can be important for identifying a new target for the future treatment of demyelination diseases.

Ion transport in muscle acetylcholine receptor maintained by conserved salt bridges between the pore and lipid membrane.

Pores through ion channels rapidly transport small inorganic ions along their electrochemical gradients. Here, applying single-channel electrophysiology and mutagenesis to the archetypal muscle nicotinic acetylcholine receptor (AChR) channel, we show that a conserved pore-peripheral salt bridge partners with those in the other subunits to regulate ion transport. Disrupting the salt bridges in all five receptor subunits greatly decreases the amplitude of the unitary current and increases its fluctuations. However, disrupting individual salt bridges has unequal effects that depend on the structural status of the other salt bridges. The AChR ε- and δ-subunits are structurally unique in harboring a putative palmitoylation site near each salt bridge and bordering the lipid membrane. The effects of disrupting the palmitoylation sites mirror those of disrupting the salt bridges, but the effect of disrupting either of these structures depends on the structural status of the other. Thus, rapid ion transport through the AChR channel is maintained by functionally interdependent salt bridges linking the pore to the lipid membrane.

Integrative physiology and transcriptome reveal salt-tolerance differences between two licorice species: Ion transport, Casparian strip formation and flavonoids biosynthesis.

BACKGROUND: Glycyrrhiza inflata Bat. and Glycyrrhiza uralensis Fisch. are both original plants of 'Gan Cao' in the Chinese Pharmacopoeia, and G. uralensis is currently the mainstream variety of licorice and has a long history of use in traditional Chinese medicine. Both of these species have shown some degree of tolerance to salinity, G. inflata exhibits higher salt tolerance than G. uralensis and can grow on saline meadow soils and crusty saline soils. However, the regulatory mechanism responsible for the differences in salt tolerance between different licorice species is unclear. Due to land area-related limitations, the excavation and cultivation of licorice varieties in saline-alkaline areas that both exhibit tolerance to salt and contain highly efficient active substances are needed. The systematic identification of the key genes and pathways associated with the differences in salt tolerance between these two licorice species will be beneficial for cultivating high-quality salt-tolerant licorice G. uralensis plant varieties and for the long-term development of the licorice industry. In this research, the differences in growth response indicators, ion accumulation, and transcription expression between the two licorice species were analyzed. RESULTS: This research included a comprehensive comparison of growth response indicators, including biomass, malondialdehyde (MDA) levels, and total flavonoids content, between two distinct licorice species and an analysis of their ion content and transcriptome expression. In contrast to the result found for G. uralensis, the salt treatment of G. inflata ensured the stable accumulation of biomass and total flavonoids at 0.5 d, 15 d, and 30 d and the restriction of Na+ to the roots while allowing for more K+ and Ca2+ accumulation. Notably, despite the increase in the Na+ concentration in the roots, the MDA concentration remained low. Transcriptome analysis revealed that the regulatory effects of growth and ion transport on the two licorice species were strongly correlated with the following pathways and relevant DEGs: the TCA cycle, the pentose phosphate pathway, and the photosynthetic carbon fixation pathway involved in carbon metabolism; Casparian strip formation (lignin oxidation and translocation, suberin formation) in response to Na+; K+ and Ca2+ translocation, organic solute synthesis (arginine, polyamines, GABA) in response to osmotic stresses; and the biosynthesis of the nonenzymatic antioxidants carotenoids and flavonoids in response to antioxidant stress. Furthermore, the differential expression of the DEGs related to ABA signaling in hormone transduction and the regulation of transcription factors such as the HSF and GRAS families may be associated with the remarkable salt tolerance of G. inflata. CONCLUSION: Compared with G. uralensis, G. inflata exhibits greater salt tolerance, which is primarily attributable to factors related to carbon metabolism, endodermal barrier formation and development, K+ and Ca2+ transport, biosynthesis of carotenoids and flavonoids, and regulation of signal transduction pathways and salt-responsive transcription factors. The formation of the Casparian strip, especially the transport and oxidation of lignin precursors, is likely the primary reason for the markedly higher amount of Na+ in the roots of G. inflata than in those of G. uralensis. The tendency of G. inflata to maintain low MDA levels in its roots under such conditions is closely related to the biosynthesis of flavonoids and carotenoids and the maintenance of the osmotic balance in roots by the absorption of more K+ and Ca2+ to meet growth needs. These findings may provide new insights for developing and cultivating G. uralensis plant species selected for cultivation in saline environments or soils managed through agronomic practices that involve the use of water with a high salt content.

Advances in TRPV6 inhibitors for tumors by targeted therapies: Macromolecular proteins, synthetic small molecule compounds, and natural compounds.

TRPV6, a Ca2+-selective member of the transient receptor potential vanilloid (TRPV) family, plays a key role in extracellular calcium transport, calcium ion reuptake, and maintenance of a local low calcium environment. An increasing number of studies have shown that TRPV6 is involved in the regulation of various diseases. Notably, overexpression of TRPV6 is closely related to the occurrence of various cancers. Research confirmed that knocking down TRPV6 could effectively reduce the proliferation and invasiveness of tumors by mainly mediating the calcium signaling pathway. Hence, TRPV6 has become a promising new drug target for numerous tumor treatments. However, the development of TRPV6 inhibitors is still in the early stage, and the existing TRPV6 inhibitors have poor selectivity and off-target effects. In this review, we focus on summarizing and describing the structure characters, and mechanisms of existing TRPV6 inhibitors to provide new ideas and directions for the development of novel TRPV6 inhibitors.

Inorganic polyphosphate and ion transport across biological membranes.

Inorganic polyphosphate (polyP) is widely recognized for playing important roles and processes involved in energy and phosphate storage, regulation of gene expression, and calcium signaling. The less well-known role of polyP is as a direct mediator of ion transport across biological membranes. Here, we will briefly summarize current knowledge of the molecular mechanisms of how polyP can be involved in membrane ion transport. We discuss three types of mechanisms that might involve polyP: (1) formation of non-protein channel complex that includes calcium, polyP, and polyhydroxybutyrate (PHB); (2) modulation of the channel activity of PHBlated protein channels; and (3) direct effects of polyP on the function of the voltage-gated ion channels in the process that do not involve PHB.

Chloride/Multiple Anion Exchanger SLC26A Family: Systemic Roles of SLC26A4 in Various Organs.

Solute carrier family 26 member 4 (SLC26A4) is a member of the SLC26A transporter family and is expressed in various tissues, including the airway epithelium, kidney, thyroid, and tumors. It transports various ions, including bicarbonate, chloride, iodine, and oxalate. As a multiple-ion transporter, SLC26A4 is involved in the maintenance of hearing function, renal function, blood pressure, and hormone and pH regulation. In this review, we have summarized the various functions of SLC26A4 in multiple tissues and organs. Moreover, the relationships between SLC26A4 and other channels, such as cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and sodium chloride cotransporter, are highlighted. Although the modulation of SLC26A4 is critical for recovery from malfunctions of various organs, development of specific inducers or agonists of SLC26A4 remains challenging. This review contributes to providing a better understanding of the role of SLC26A4 and development of therapeutic approaches for the SLC26A4-associated hearing loss and SLC26A4-related dysfunction of various organs.

Coupling of Electric and Flow Fields to Enhance Ion Transport for Energy-Efficient Electrochemical Tap-Water Softening.

Electrochemical-induced precipitation is a sustainable approach for tap-water softening, but the hardness removal performance and energy efficiency are vastly limited by the ultraslow ion transport and the superlow local HCO3-/Ca2+ ratio compared to the industrial scenarios. To tackle the challenges, we herein report an energy-efficient electrochemical tap-water softening strategy by utilizing an integrated cathode-anode-cathode (CAC) reactor in which the direction of the electric field is reversed to that of the flow field in the upstream cell, while the same in the downstream cell. As a result, the transport of ions, especially HCO3-, is significantly accelerated in the downstream cell under a flow field. The local HCO3-/Ca2+ ratio is increased by 1.5 times, as revealed by the finite element numerical simulation and in situ imaging. In addition, a continuous flow electrochemical system with an integrated CAC reactor is operated for 240 h to soften tap water. Experiments show that a much lower cell voltage (9.24 V decreased) and energy consumption (28% decreased) are obtained. The proposed ion-transport enhancement strategy by coupled electric and flow fields provides a new perspective on developing electrochemical technologies to meet the flexible and economic demand for tap-water softening.

Chloride ions in health and disease.

Chloride is a key anion involved in cellular physiology by regulating its homeostasis and rheostatic processes. Changes in cellular Cl- concentration result in differential regulation of cellular functions such as transcription and translation, post-translation modifications, cell cycle and proliferation, cell volume, and pH levels. In intracellular compartments, Cl- modulates the function of lysosomes, mitochondria, endosomes, phagosomes, the nucleus, and the endoplasmic reticulum. In extracellular fluid (ECF), Cl- is present in blood/plasma and interstitial fluid compartments. A reduction in Cl- levels in ECF can result in cell volume contraction. Cl- is the key physiological anion and is a principal compensatory ion for the movement of the major cations such as Na+, K+, and Ca2+. Over the past 25 years, we have increased our understanding of cellular signaling mediated by Cl-, which has helped in understanding the molecular and metabolic changes observed in pathologies with altered Cl- levels. Here, we review the concentration of Cl- in various organs and cellular compartments, ion channels responsible for its transportation, and recent information on its physiological roles.

K+-Driven Cl-/HCO3- Exchange Mediated by Slc4a8 and Slc4a10.

Slc4a genes encode various types of transporters, including Na+-HCO3- cotransporters, Cl-/HCO3- exchangers, or Na+-driven Cl-/HCO3- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl-/HCO3- exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl-/HCO3- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl-/HCO3- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl- and HCO3--dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3- ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.

The 2-oxoglutarate/malate carrier extends the family of mitochondrial carriers capable of fatty acid and 2,4-dinitrophenol-activated proton transport.

AIMS: Metabolic reprogramming in cancer cells has been linked to mitochondrial dysfunction. The mitochondrial 2-oxoglutarate/malate carrier (OGC) has been suggested as a potential target for preventing cancer progression. Although OGC is involved in the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. We aimed to investigate whether OGC may contribute to the alteration of mitochondrial inner membrane potential by transporting protons. METHODS: The expression of OGC in mouse tissues and cancer cells was investigated by PCR and Western blot analysis. The proton transport function of recombinant murine OGC was evaluated by measuring the membrane conductance (Gm) of planar lipid bilayers. OGC-mediated substrate transport was measured in proteoliposomes using 14C-malate. RESULTS: OGC increases proton Gm only in the presence of natural (long-chain fatty acids, FA) or chemical (2,4-dinitrophenol) protonophores. The increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of the FA. OGC substrates and inhibitors compete with FA for the same protein binding site. Arginine 90 was identified as a critical amino acid for the binding of FA, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism. CONCLUSION: OGC extends the family of mitochondrial transporters with dual function: (i) metabolite transport and (ii) proton transport facilitated in the presence of protonophores. Elucidating the contribution of OGC to uncoupling may be essential for the design of targeted drugs for the treatment of cancer and other metabolic diseases.

Symplasmic and transmembrane zinc transport is modulated by cadmium in the Cd/Zn hyperaccumulator Sedum alfredii.

This study investigated the influence of Cd (25 µM) on Zn accumulation in a hyperaccumulating (HE) and a non-hyperaccumulating (NHE) ecotype of Sedum alfredii Hance at short-term supply of replete (Zn5, 5 µM) and excess (Zn400, 400 µM) Zn. Cd inhibited Zn accumulation in both ecotypes, especially under Zn400, in organs with active metal sequestration, i.e. roots of NHE and shoots of HE. Direct biochemical Cd/Zn competition at the metal-protein interaction and changes in transporter gene expression contributed to the observed accumulation patterns in the roots. Specifically, in HE, Cd stimulated SaZIP4 and SaPCR2 under Zn5, but downregulated SaIRT1 and SaZIP4 under Zn400. However, Cd downregulated related transporter genes, except for SaNRAMP1, in NHE, irrespective of Zn. Cadmium stimulated casparian strip (CSs) development in NHE, as part of the defense response, while it had a subtle effect on the (CS) in HE. Moreover, Cd delayed the initiation of the suberin lamellae (SL) in HE, but stimulated SL deposition in NHE under both Zn5 or Zn400. Changes in suberization were mainly ascribed to suberin-biosynthesis-related genes and hormonal signaling. Altogether, Cd regulated Zn accumulation mainly via symplasmic and transmembrane transport in HE, while Cd inhibited both symplasmic and apoplasmic Zn transport in NHE.

Long-term outcome of uniport vs. multiport video-assisted thoracoscopic lobectomy for lung cancer.

This study aims to compare the perioperative outcomes and long-term survival of U-VATS lobectomy for NSCLC with multiportal VATS (M-VATS, involving two ports or more) lobectomy. A total of 339 patients who underwent intentional VATS lobectomy for lung cancer between 2012 and 2017 were included in the analysis. Perioperative outcomes and long-term survival were evaluated. Propensity score matching was utilized to minimize baseline characteristic differences between the two groups. Out of the total cases, 17 (5.01%) were converted to open thoracotomy. The conversion rates were 4.96% (7/141) in the U-VATS group and 5.05% (10/198) in the M-VATS group. A total of 322 consecutive patients underwent VATS lobectomy and mediastinal lymphadenectomy. After propensity matching, 106 pairs were obtained, consisting of 83 males and 129 females. Intraoperative bleeding volume, number of retrieved lymph nodes, explored nodal stations, drainage time and volume, and postoperative hospital stay were similar between the two groups. Both groups exhibited comparable morbidity and mortality rates. From the multivariable analysis, there was no significant difference observed in terms of overall survival (OS) and disease-free survival (DFS) between the two patient cohorts. U-VATS demonstrated comparable perioperative outcomes and long-term efficacy to M-VATS. However, further confirmation of these findings is required.

The crossing of two unwound transmembrane regions that is the hallmark of the NhaA structural fold is critical for antiporter activity.

Cell pH and Na+ homeostasis requires Na+/H+ antiporters. The crystal structure of NhaA, the main Escherichia coli Na+/H+ antiporter, revealed a unique NhaA structural fold shared by prokaryotic and eukaryotic membrane proteins. Out of the 12 NhaA transmembrane segments (TMs), TMs III-V and X-XII are topologically inverted repeats with unwound TMs IV and XI forming the X shape characterizing the NhaA fold. We show that intramolecular cross-linking under oxidizing conditions of a NhaA mutant with two Cys replacements across the crossing (D133C-T340C) inhibits antiporter activity and impairs NhaA-dependent cell growth in high-salts. The affinity purified D133C-T340C protein binds Li+ (the Na+ surrogate substrate of NhaA) under reducing conditions. The cross-linking traps the antiporter in an outward-facing conformation, blocking the antiport cycle. As many secondary transporters are found to share the NhaA fold, including some involved in human diseases, our data have importance for both basic and clinical research.

Single shot detection of alterations across multiple ionic currents from assimilation of cell membrane dynamics.

The dysfunction of ion channels is a causative factor in a variety of neurological diseases, thereby defining the implicated channels as key drug targets. The detection of functional changes in multiple specific ionic currents currently presents a challenge, particularly when the neurological causes are either a priori unknown, or are unexpected. Traditional patch clamp electrophysiology is a powerful tool in this regard but is low throughput. Here, we introduce a single-shot method for detecting alterations amongst a range of ion channel types from subtle changes in membrane voltage in response to a short chaotically driven current clamp protocol. We used data assimilation to estimate the parameters of individual ion channels and from these we reconstructed ionic currents which exhibit significantly lower error than the parameter estimates. Such reconstructed currents thereby become sensitive predictors of functional alterations in biological ion channels. The technique correctly predicted which ionic current was altered, and by approximately how much, following pharmacological blockade of BK, SK, A-type K+ and HCN channels in hippocampal CA1 neurons. We anticipate this assay technique could aid in the detection of functional changes in specific ionic currents during drug screening, as well as in research targeting ion channel dysfunction.

Pulley Effect in the Capture of DNA Translocation through Solid-State Nanopores.

Nanopores are powerful single-molecule sensors for analyzing biomolecules such as DNA and proteins. Understanding the dynamics of DNA capture and translocation through nanopores is essential for optimizing their performance. In this study, we examine the effects of applied voltage and pore diameter on current blockage, translocation time, collision, and capture location by translocating λ-DNA through 5.7 and 16 nm solid-state nanopores. Ionic current changes are used to infer DNA conformations during translocation. We find that translocation time increases with pore diameter, which can be attributed to the decrease of the stall force. Linear and exponential decreases of collision frequency with voltage are observed in the 16 and 5.7 nm pores, respectively, indicating a free energy barrier in the small pore. Moreover, the results reveal a voltage-dependent bias in the capture location toward the DNA ends, which is explained by a "pulley effect" deforming the DNA as it approaches the pore. This study provides insights into the physics governing DNA capture and translocation, which can be useful for promoting single-file translocation to enhance nanopore sensing.

The molecular sociology of NHERF1 PDZ proteins controlling renal hormone-regulated phosphate transport.

Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control extracellular phosphate levels by regulating renal NPT2A-mediated phosphate transport by a process requiring the PDZ scaffold protein NHERF1. NHERF1 possesses two PDZ domains, PDZ1 and PDZ2, with identical core-binding GYGF motifs explicitly recognizing distinct binding partners that play different and specific roles in hormone-regulated phosphate transport. The interaction of PDZ1 and the carboxy-terminal PDZ-binding motif of NPT2A (C-TRL) is required for basal phosphate transport. PDZ2 is a regulatory domain that scaffolds multiple biological targets, including kinases and phosphatases involved in FGF23 and PTH signaling. FGF23 and PTH trigger disassembly of the NHERF1-NPT2A complex through reversible hormone-stimulated phosphorylation with ensuing NPT2A sequestration, down-regulation, and cessation of phosphate absorption. In the absence of NHERF1-NPT2A interaction, inhibition of FGF23 or PTH signaling results in disordered phosphate homeostasis and phosphate wasting. Additional studies are crucial to elucidate how NHERF1 spatiotemporally coordinates cellular partners to regulate extracellular phosphate levels.

Piezo1 and Its Function in Different Blood Cell Lineages.

Mechanosensation is a fundamental function through which cells sense mechanical stimuli by initiating intracellular ion currents. Ion channels play a pivotal role in this process by orchestrating a cascade of events leading to the activation of downstream signaling pathways in response to particular stimuli. Piezo1 is a cation channel that reacts with Ca2+ influx in response to pressure sensation evoked by tension on the cell lipid membrane, originating from cell-cell, cell-matrix, or hydrostatic pressure forces, such as laminar flow and shear stress. The application of such forces takes place in normal physiological processes of the cell, but also in the context of different diseases, where microenvironment stiffness or excessive/irregular hydrostatic pressure dysregulates the normal expression and/or activation of Piezo1. Since Piezo1 is expressed in several blood cell lineages and mutations of the channel have been associated with blood cell disorders, studies have focused on its role in the development and function of blood cells. Here, we review the function of Piezo1 in different blood cell lineages and related diseases, with a focus on megakaryocytes and platelets.

Fluoride Ion Binding and Translocation in the CLCF Fluoride/Proton Antiporter: Molecular Insights from Combined Quantum-Mechanical/Molecular-Mechanical Modeling.

CLCF fluoride/proton antiporters move fluoride ions out of bacterial cells, leading to fluoride resistance in these bacteria. However, many details about their operating mechanisms remain unclear. Here, we report a combined quantum-mechanical/molecular-mechanical (QM/MM) study of a CLCF homologue from Enterococci casseliflavus (Eca), in accord with the previously proposed windmill mechanism. Our multiscale modeling sheds light on two critical steps in the transport cycle: (i) the external gating residue E118 pushing a fluoride in the external binding site into the extracellular vestibule and (ii) an incoming fluoride reconquering the external binding site by forcing out E118. Both steps feature competitions for the external binding site between the negatively charged carboxylate of E118 and the fluoride. Remarkably, the displaced E118 by fluoride accepts a proton from the nearby R117, initiating the next transport cycle. We also demonstrate the importance of accurate quantum descriptions of fluoride solvation. Our results provide clues to the mysterious E318 residue near the central binding site, suggesting that the transport activities are unlikely to be disrupted by the glutamate interacting with a well-solvated fluoride at the central binding site. This differs significantly from the structurally similar CLC chloride/proton antiporters, where a fluoride trapped deep in the hydrophobic pore causes the transporter to be locked down. A free-energy barrier of 10-15 kcal/mol was estimated via umbrella sampling for a fluoride ion traveling through the pore to repopulate the external binding site.

Responsive Anionophores with AND Logic Multi-Stimuli Activation.

Artificial ion transport systems have emerged as an important class of compounds that promise applications in chemotherapeutics as anticancer agents or to treat channelopathies. Stimulus-responsive systems that offer spatiotemporally controlled activity for targeted applications remain rare. Here we utilize dynamic hydrogen bonding interactions of a 4,6-dihydroxy-isophthalamide core to generate a modular platform enabling access to stimuli-responsive ion transporters that can be activated in response to a wide variety of external stimuli, including light, redox, and enzymes, with excellent OFF-ON activation profiles. Alkylation of the two free hydroxyl groups with stimulus-responsive moieties locks the amide bonds through intramolecular hydrogen bonding and hence makes them unavailable for anion binding and transport. Triggering using a particular stimulus to cleave both cages reverses the hydrogen bonding arrangement, to generate a highly preorganized anion binding cavity for efficient transmembrane transport. Integration of two cages that are responsive to orthogonal stimuli enables multi-stimuli activation, where both stimuli are required to trigger transport in an AND logic process. Importantly, the strategy provides a facile method to post-functionalize the highly active transporter core with a variety of stimulus-responsive moieties for targeted activation with multiple triggers.