Triangularia manubriata sp. Nov.: A Novel Fungal Species Belonging to the Family Podosporaceae Isolated from Soil in Korea.

The fungal strain designated as KNUF-21-020, belonging to the genus Triangularia, was isolated from a soil sample collected in the Chungnam province, Korea. Phylogenetic analyses based on the concatenated nucleotide sequences of internal transcribed spacer regions and partial sequences of large subunit rRNA, beta-tubulin, and RNA polymerase II subunit genes revealed that the strain was grouped in a clade with Triangularia species. However, it occupied a distinct phylogenetic position. We also observed morphological differences between strain KNUF-21-020 and closely related species. Here, we provided detailed descriptions, illustrations, and discussions regarding the morphological and phylogenetic analyses of the closely related species to support the novelty of this isolated species. The phylogenetic analyses and morphological observations indicate that the strain KNUF-21-020 represents a novel species in the genus Triangularia (family: Podosporaceae). We have designated this species as Triangularia manubriata sp. nov.

Ultrasmall Mn-doped iron oxide nanoparticles with dual hepatobiliary and renal clearances for T1 MR liver imaging.

Although magnetic nanoparticles demonstrate significant potential as magnetic resonance imaging (MRI) contrast agents, their negative contrasts, liver accumulation, and limited excretion hinder their application. Herein, we developed ultrasmall Mn-doped iron oxide nanoparticles (UMIOs) with distinct advantages as T1 MRI contrast agents. Exceptionally small particle sizes (ca. 2 nm) and magnetization values (5 emu gMn+Fe-1) of UMIOs provided optimal T1 contrast effects with an ideally low r2/r1 value of ∼1. Furthermore, the use of Mn as a dopant facilitated hepatocyte uptake of the particles, allowing liver imaging. In animal studies, UMIOs exhibited significantly enhanced contrasts for sequential T1 imaging of blood vessels and the liver, distinguishing them from conventional magnetic nanoparticles. UMIOs were systematically cleared via dual hepatobiliary and renal excretion pathways, highlighting their safety profile. These characteristics imply substantial potential of UMIOs as T1 contrast agents for the accurate diagnosis of liver diseases.

Demyelination and Na+ Channel Redistribution Underlie Auditory and Vestibular Dysfunction in PMP22-Null Mice.

Altered expression of peripheral myelin protein 22 (PMP22) results in demyelinating peripheral neuropathy. PMP22 exhibits a highly restricted tissue distribution with marked expression in the myelinating Schwann cells of peripheral nerves. Auditory and vestibular Schwann cells and the afferent neurons also express PMP22, suggesting a unique role in hearing and balancing. Indeed, neuropathic patients diagnosed with PMP22-linked hereditary neuropathies often present with auditory and balance deficits, an understudied clinical complication. To investigate the mechanism by which abnormal expression of PMP22 may cause auditory and vestibular deficits, we studied gene-targeted PMP22-null mice. PMP22-null mice exhibit an unsteady gait, have difficulty maintaining balance, and live for only ∼3-5 weeks relative to unaffected littermates. Histological analysis of the inner ear revealed reduced auditory and vestibular afferent nerve myelination and profound Na+ channel redistribution without PMP22. Yet, Na+ current density was unaltered, in stark contrast to increased K+ current density. Atypical postsynaptic densities and a range of neuronal abnormalities in the organ of Corti were also identified. Analyses of auditory brainstem responses (ABRs) and vestibular sensory-evoked potential (VsEP) revealed that PMP22-null mice had auditory and vestibular hypofunction. These results demonstrate that PMP22 is required for hearing and balance, and the protein is indispensable for the formation and maintenance of myelin in the peripheral arm of the eighth nerve. Our findings indicate that myelin abnormalities and altered signal propagation in the peripheral arm of the auditory nerve are likely causes of auditory deficits in patients with PMP22-linked neuropathies.

The Piezo channel is a mechano-sensitive complex component in the mammalian inner ear hair cell.

The inner ear is the hub where hair cells (HCs) transduce sound, gravity, and head acceleration stimuli to the brain. Hearing and balance rely on mechanosensation, the fastest sensory signals transmitted to the brain. The mechanoelectrical transducer (MET) channel is the entryway for the sound-balance-brain interface, but the channel-complex composition is not entirely known. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as MET-complex components. The Pz channels, expressed in HC stereocilia, and cell lines are co-localized and co-assembled with MET complex partners. Mice expressing non-functional Pz1 and Pz2 at the ROSA26 locus have impaired auditory and vestibular traits that can only be explained if the Pzs are integral to the MET complex. We suggest that Pz subunits constitute part of the MET complex and that interactions with other MET complex components yield functional MET units to generate HC MET currents.

Immune Activation Modulation via Magnetically Localized Bacteria Based Micro/Bio Robot (BBMBR).

Understanding tumor's microenvironment is one of the key factors in the cancer therapy. Especially, from the perspective of immunotherapy, immune desert or cold tumor is referred as significantly downregulated T cell in-filtration due to lack of immune surveillance in the tumor microenvironment. There are many studies are dedicated to convert cold tumor to hot tumor for enhancing the efficacy of immunotherapy. In this study, we suggested selective immune activation system through the spatiotemporal control of the bacteria as an immune boosting agent. To this end, we have developed bacteria-based micro/bio robot system (BBMBR) by attaching bacteria with magnetic nanoparticles (MNP) so that the localization can be controlled through the magnetic field. The biomanufacturing results showed that BBMBR includes 6.6 ± 1.54 MNP attached and the presence ratio of bacteria-MNP out of total bacteria population reached 75.2 ± 3.37%. Spatial controllability experiments have shown that rotational and translation localization has been controlled as intended. The function of the immune modulation system through BBMBR was confirmed through experiments that magnetically driven BBMBR localization induced localized immune activation. M1-phenotype differentiation of macrophage cells were quantified CD80 staining, and overall immune response level was evaluated through IL-6 measurements. As the distance from the activation point increased, the population of M1 differentiated macrophages decreased, and when the movement of BBMBR was magnetically restricted, overall immune activation was found to be regulated downward. Proposed BBMBR and immune modulation framework could introduce a powerful new paradigm in cancer treatment by improving the localization controllability of immune-boosting agent and the spatial immune activation strategies.

Genetic and pharmacologic alterations of claudin9 levels suffice to induce functional and mature inner hair cells.

Hearing loss is the most common form of sensory deficit. It occurs predominantly due to hair cell (HC) loss. Mammalian HCs are terminally differentiated by birth, making HC loss incurable. Here, we show the pharmacogenetic downregulation of Cldn9, a tight junction protein, generates robust supernumerary inner HCs (IHCs) in mice. The putative ectopic IHCs have functional and synaptic features akin to typical IHCs and were surprisingly and remarkably preserved for at least fifteen months >50% of the mouse's life cycle. In vivo, Cldn9 knockdown using shRNA on postnatal days (P) P1-7 yielded analogous functional putative ectopic IHCs that were equally durably conserved. The findings suggest that Cldn9 levels coordinate embryonic and postnatal HC differentiation, making it a viable target for altering IHC development pre- and post-terminal differentiation.

The Piezo channel is central to the mechano-sensitive channel complex in the mammalian inner ear.

The inner ear is the hub where hair cells transduce sound, gravity, and head acceleration stimuli carried by neural codes to the brain. Of all the senses, hearing and balance, which rely on mechanosensation, are the fastest sensory signals transmitted to the central nervous system. The mechanoelectrical transducer (MET) channel in hair cells is the entryway for the sound-balance-brain interface, but the channel's composition has eluded biologists due to its complexity. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as central components of the MET complex. The Pz channel subunits are expressed in hair-cell stereocilia, are co-localized and co-assembled, and are essential components of the MET complex in vitro and in situ, including integration with the transmembrane channel (Tmc1/2) protein. Mice expressing non-functional Pz1 and Pz2, but not functional Pz1 at the ROSA26 locus under the control of hair-cell promoters, have impaired auditory and vestibular traits that can only be explained if Pz channel multimers are integral to the MET complex. We affirm that Pz protein subunits constitute MET channels and that functional interactions with components of the MET complex yield current properties resembling hair-cell MET currents. Our results demonstrate Pz is a MET channel component central to interacting with MET complex proteins. Results account for the MET channel pore and complex.

Colorimetric mercury detection with enhanced sensitivity using magnetic-Au hybrid nanoparticles.

Due to the neural toxicity of mercury, there is a need for the development of on-site detection systems for Hg2+ monitoring. To this end, a new colorimetric mercury detection probe, Fe3O4@SiO2@Au (magnetic-Au; Mag-Au) hybrid nanoparticles, has been developed. The Au on the surface of Mag-Au is an indicator of Hg2+, which forms an AuHg alloy (amalgam) on their surface (Mag-Au@Hg), with excellent peroxidase-like activity. The oxidation of 3,3',5,5'-tetramethylbenzidine by Mag-Au@Hg resulted in a color change of the indicator solution, which was enhanced with increasing Hg2+ concentration. Mag-Au can be used to detect Hg2+ at nanomolar concentrations. Additionally, magnetic separation can be used to easily purify and concentrate the Mag-Au@Hg from samples, and thus avoid interference from unwanted residues or colored samples. The feasibility of Mag-Au for Hg2+ detection was tested with an artificial urine solution and it can be used to detect Hg2+ in various real samples, such as river water, seawater, food, and biological samples.

Enhancing catalytic efficiency of carbon dots by modulating their Mn doping and chemical structure with metal salts.

Nanozymes are emerging materials in various fields owing to their advantages over natural enzymes, such as controllable and facile synthesis, tunability in catalytic activities, cost-effectiveness, and high stability under stringent conditions. In this study, the effect of metal salts on the formation and catalytic activity of carbon dots (CDs), a promising nanozyme, is demonstrated. By introducing Mn sources that possess different counter anions, the chemical structure and composition of the CDs produced are affected, thereby influencing their enzymatic activities. The synergistic catalytic effect of the Mn and N-doped CDs (Mn&N-CDs) is induced by effective metal doping in the carbogenic domain and a high proportion of graphitic and pyridinic N. This highly enhanced catalytic effect of Mn&N-CDs allows them to respond sensitively to the interference factors of enzymatic reactions. Consequently, ascorbic acid, which is an essential nutrient for maintaining our health and is a reactive oxygen scavenger, can be successfully monitored using color change by forming oxidized 3,3',5,5'-tetramethylbenzidine with H2O2 and Mn&N-CDs. This study provides a basic understanding of the formation of CDs and how their catalytic properties can be controlled by the addition of different metal sources, thereby providing guidelines for the development of CDs for industrial applications.

Mag-spinner: a next-generation Facile, Affordable, Simple, and porTable (FAST) magnetic separation system.

Mag-spinner, a system in which magnets are combined with a spinner system, is a new type of magnetic separation system for the preprocessing of biological and medical samples. Interference by undesired components restricts the detection accuracy and efficiency. Thus, the development of appropriate separation techniques is required for better detection of the desired targets, to enrich the target analytes and remove the undesired components. The strong response of iron oxide nanoclusters can successfully capture the targets quickly and with high efficiency. As a result, cancer cells can be effectively separated from blood using the developed mag-spinner system. Indeed, this system satisfies the requirements for desirable separation systems, namely (i) fast sorting rates, (ii) high separation efficiency, (iii) the ability to process native biological fluids, (iv) simple operating procedures, (v) low cost, (vi) operational convenience, and (vii) portability. Therefore, this system is widely applicable to sample preparation without limitations on place, cost, and equipment.

Zn-assisted modification of the chemical structure of N-doped carbon dots and their enhanced quantum yield and photostability.

This article presents the Zn-assisted synthesis of N-doped carbon dots (N-CDs) with an enhanced quantum yield (QY) and photostability. There have been intensive studies to improve or tune the optical properties of carbon dots (CDs) to meet the demand for luminescent materials in various fields, including energy conversion, photocatalysis, bioimaging, and phototherapy. For these applications, the photostability of the CDs is also a critical factor, but the related studies are relatively less common. The Zn-assisted N-CDs (denoted as Zn:N-CDs) obtained by the addition of Zn(OAc)2 to the precursors during the synthesis of N-CDs not only exhibited an enhanced quantum yield but also improved photostability compared to those of N-CDs. A comprehensive study of the chemical composition of Zn:N-CD and N-CD using X-ray photoelectron spectroscopy indicated a correlation between their chemical structure and photostability. Zn(OAc)2, which acts as a catalytic reagent, induced the modification of chemical structures at the edges of carbogenic sp2 domains, without being doped in N-CD, and the heteroatom-carbon bonds in Zn:N-CD seemed to be more resistant to light compared to those in N-CDs. The increased QY and photostability of Zn:N-CDs make them more suitable as an optical probe and they could be used in fingerprint identification. With Zn:N-CDs, the microstructure of fingerprints was confirmed clearly for a long duration effectively.

Cisplatin Neurotoxicity Targets Specific Subpopulations and K+ Channels in Tyrosine-Hydroxylase Positive Dorsal Root Ganglia Neurons.

Among the features of cisplatin chemotherapy-induced peripheral neuropathy are chronic pain and innocuous mechanical hypersensitivity. The complete etiology of the latter remains unknown. Here, we show that cisplatin targets a heterogeneous population of tyrosine hydroxylase-positive (TH+) primary afferent dorsal root ganglion neurons (DRGNs) in mice, determined using single-cell transcriptome and electrophysiological analyses. TH+ DRGNs regulate innocuous mechanical sensation through C-low threshold mechanoreceptors. A differential assessment of wild-type and vitamin E deficient TH+ DRGNs revealed heterogeneity and specific functional phenotypes. The TH+ DRGNs comprise; fast-adapting eliciting one action potential (AP; 1-AP), moderately-adapting (≥2-APs), in responses to square-pulse current injection, and spontaneously active (SA). Cisplatin increased the input resistance and AP frequency but reduced the temporal coding feature of 1-AP and ≥2-APs neurons. By contrast, cisplatin has no measurable effect on the SA neurons. Vitamin E reduced the cisplatin-mediated increased excitability but did not improve the TH+ neuron temporal coding properties. Cisplatin mediates its effect by targeting outward K+ current, likely carried through K2P18.1 (Kcnk18), discovered through the differential transcriptome studies and heterologous expression. Studies show a potential new cellular target for chemotherapy-induced peripheral neuropathy and implicate the possible neuroprotective effects of vitamin E in cisplatin chemotherapy.

Hyperthermia Effect of Nanoclusters Governed by Interparticle Crystalline Structures.

Magnetic nanoparticles have an important role as heat generators in magnetic fluid hyperthermia, a type of next-generation cancer treatment. Despite various trials to improve the heat generation capability of magnetic nanoparticles, iron oxide nanoparticles are the only approved heat generators for clinical applications, which require a large injection dose due to their low hyperthermia efficiency. In this study, iron oxide nanoclusters (NCs) with a highly enhanced hyperthermia effect and adjustable size were synthesized through a facile and simple solvothermal method. Among the samples, the NCs with a size of 25 nm showed the highest hyperthermia efficiency. Differently sized NCs exhibit inconsistent interparticle crystalline alignments, which affect their magnetic properties (e.g., coercivity and saturation magnetization). As a result, the optimal NCs exhibited a significantly enhanced heat generation efficiency compared with that of isolated iron oxide nanoparticles (ca. 7 nm), and their hyperthermia effect on skin cancer cells was confirmed.

Highly Enhanced Enzymatic Activity of Mn-Induced Carbon Dots and Their Application as Colorimetric Sensor Probes.

Nanomaterial-based enzyme mimetics (nanozymes) have attracted significant interest because of their lower cost and higher stability compared to natural enzymes. In this study, we focused on improving the enzymatic properties of metal induced N-doped carbon dots (N-CDs), which are nanozymes of interest, and their applications for sensory systems. For this purpose, Mn(acetate)2 was introduced during the synthetic step of N-doped carbon dots, and its influence on the enzymatic properties of Mn-induced N-CDs (Mn:N-CDs) was investigated. Their chemical structure was analyzed through infrared spectroscopy and X-ray photoelectron spectrometry; the results suggest that Mn ions lead to the variation in the population of chemical bonding in Mn:N-CDs, whereas these ions were not incorporated into N-CD frameworks. This structural change improved the enzymatic properties of Mn:N-CDs with respect to those of N-CDs when the color change of a 3,3',5,5'-tetramethylbenzidine/H2O2 solution was examined in the presence of Mn:N-CDs and N-CDs. Based on this enhanced enzymatic property, a simple colorimetric system with Mn:N-CDs was used for the detection of γ-aminobutyric acid, which is an indicator of brain-related disease. Therefore, we believe that Mn:N-CDs will be an excellent enzymatic probe for the colorimetric sensor system.

Effect of heteroatoms on the optical properties and enzymatic activity of N-doped carbon dots.

Carbon dots (CDs) are attractive nanomaterials because of their facile synthesis, biocompatibility, superior physicochemical properties, and low cost of their precursors. Recent advances in CDs have particularly relied on the modulation of their properties by heteroatom doping (e.g., nitrogen). Although nitrogen-doped CDs (N-CDs) have attracted considerable attention owing to their different properties compared to those of the original CDs, the effects of the heteroatom content and types of bonding on the properties of N-doped CDs remain underexplored. In this work, we prepared N-CDs with controlled nitrogen contents, and fully examined their optical properties, enzymatic activity, and toxicity. We demonstrate that (i) the type of carbon-heteroatom bonding (i.e., carbon-oxygen and carbon-nitrogen bonds) can be altered by changing the ratio of carbon to heteroatom sources, and (ii) both the heteroatom content and the heteroatom-bonding character significantly influence the properties of the doped CDs. Notably, N-CDs exhibited higher quantum yields and peroxidase-like activities than the non-doped CDs. Furthermore, the negatively charged N-CDs exhibited negligible cytotoxicity. Such comprehensive investigations on the physicochemical properties of N-CDs are expected to guide the design of N-CDs for targeted applications.

Population Structure and Genetic Diversity in Korean Cowpea Germplasm Based on SNP Markers.

Cowpea is one of the most essential legume crops providing inexpensive dietary protein and nutrients. The aim of this study was to understand the genetic diversity and population structure of global and Korean cowpea germplasms. A total of 384 cowpea accessions from 21 countries were genotyped with the Cowpea iSelect Consortium Array containing 51,128 single-nucleotide polymorphisms (SNPs). After SNP filtering, a genetic diversity study was carried out using 35,116 SNPs within 376 cowpea accessions, including 229 Korean accessions. Based on structure and principal component analysis, a total of 376 global accessions were divided into four major populations. Accessions in group 1 were from Asia and Europe, those in groups 2 and 4 were from Korea, and those in group 3 were from West Africa. In addition, 229 Korean accessions were divided into three major populations (Q1, Jeonra province; Q2, Gangwon province; Q3, a mixture of provinces). Additionally, the neighbor-joining tree indicated similar results. Further genetic diversity analysis within the global and Korean population groups indicated low heterozygosity, a low polymorphism information content, and a high inbreeding coefficient in the Korean cowpea accessions. The population structure analysis will provide useful knowledge to support the genetic potential of the cowpea breeding program, especially in Korea.

A magnetic resonance tuning sensor for the MRI detection of biological targets.

Sensors that detect specific molecules of interest in a living organism can be useful tools for studying biological functions and diseases. Here, we provide a protocol for the construction of nanosensors that can noninvasively detect biologically important targets with magnetic resonance imaging (MRI). The key operating principle of these sensors is magnetic resonance tuning (MRET), a distance-dependent phenomenon occurring between a superparamagnetic quencher and a paramagnetic enhancer. The change in distance between the two magnetic components modulates the longitudinal (T1) relaxivity of the enhancer. In this MRET sensor, distance variation is achieved by interactive linkers that undergo binding, cleavage, or folding/unfolding upon their interaction with target molecules. By the modular incorporation of suitable linkers, the MRET sensor can be applied to a wide range of targets. We showcase three examples of MRET sensors for enzymes, nucleic acid sequences, and pH. This protocol comprises three stages: (i) chemical synthesis and surface modification of the quencher, (ii) conjugation with interactive linkers and enhancers, and (iii) MRI sensing of biological targets. The entire procedure takes up to 3 d.

Cross-Linked Fluorescent Supramolecular Nanoparticles for Intradermal Controlled Release of Antifungal Drug-A Therapeutic Approach for Onychomycosis.

The existing approaches to onychomycosis demonstrate limited success since the commonly used oral administration and topical cream only achieve temporary effective drug concentration at the fungal infection sites. An ideal therapeutic approach for onychomycosis should have (i) the ability to introduce antifungal drugs directly to the infected sites; (ii) finite intradermal sustainable release to maintain effective drug levels over prolonged time; (iii) a reporter system for monitoring maintenance of drug level; and (iv) minimum level of inflammatory responses at or around the fungal infection sites. To meet these expectations, we introduced ketoconazole-encapsulated cross-linked fluorescent supramolecular nanoparticles (KTZ⊂c-FSMNPs) as an intradermal controlled release solution for treating onychomycosis. A two-step synthetic approach was adopted to prepare a variety of KTZ⊂c-FSMNPs. Initial characterization revealed that 4800 nm KTZ⊂c-FSMNPs exhibited high KTZ encapsulation efficiency/capacity, optimal fluorescent property, and sustained KTZ release profile. Subsequently, 4800 nm KTZ⊂c-FSMNPs were chosen for in vivo studies using a mouse model, wherein the KTZ⊂c-FSMNPs were deposited intradermally via tattoo. The results obtained from (i) in vivo fluorescence imaging, (ii) high-performance liquid chromatography quantification of residual KTZ, (iii) matrix-assisted laser desorption/ionization mass spectrometry imaging mapping of KTZ distribution in intradermal regions around the tattoo site, and (iv) histology for assessment of local inflammatory responses and biocompatibility, suggest that 4800 nm KTZ⊂c-FSMNPs can serve as an effective treatment for onychomycosis.

Distance-dependent magnetic resonance tuning as a versatile MRI sensing platform for biological targets.

Nanoscale distance-dependent phenomena, such as Förster resonance energy transfer, are important interactions for use in sensing and imaging, but their versatility for bioimaging can be limited by undesirable photon interactions with the surrounding biological matrix, especially in in vivo systems. Here, we report a new type of magnetism-based nanoscale distance-dependent phenomenon that can quantitatively and reversibly sense and image intra-/intermolecular interactions of biologically important targets. We introduce distance-dependent magnetic resonance tuning (MRET), which occurs between a paramagnetic 'enhancer' and a superparamagnetic 'quencher', where the T1 magnetic resonance imaging (MRI) signal is tuned ON or OFF depending on the separation distance between the quencher and the enhancer. With MRET, we demonstrate the principle of an MRI-based ruler for nanometre-scale distance measurement and the successful detection of both molecular interactions (for example, cleavage, binding, folding and unfolding) and biological targets in in vitro and in vivo systems. MRET can serve as a novel sensing principle to augment the exploration of a wide range of biological systems.

Cross-Linked Fluorescent Supramolecular Nanoparticles as Finite Tattoo Pigments with Controllable Intradermal Retention Times.

Tattooing has been utilized by the medical community for precisely demarcating anatomic landmarks. This practice is especially important for identifying biopsy sites of nonmelanoma skin cancer (NMSC) due to the long interval (i.e., up to 3 months) between the initial diagnostic biopsy and surgical treatment. Commercially available tattoo pigments possess several issues, which include causing poor cosmesis, being mistaken for a melanocytic lesion, requiring additional removal procedures when no longer desired, and potentially inducing inflammatory responses. The ideal tattoo pigment for labeling of skin biopsy sites for NMSC requires (i) invisibility under ambient light, (ii) fluorescence under a selective light source, (iii) a finite intradermal retention time (ca. 3 months), and (iv) biocompatibility. Herein, we introduce cross-linked fluorescent supramolecular nanoparticles (c-FSNPs) as a "finite tattoo" pigment, with optimized photophysical properties and intradermal retention time to achieve successful in vivo finite tattooing. Fluorescent supramolecular nanoparticles encapsulate a fluorescent conjugated polymer, poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV), into a core via a supramolecular synthetic approach. FSNPs which possess fluorescent properties superior to those of the free MPS-PPV are obtained through a combinatorial screening process. Covalent cross-linking of FSNPs results in micrometer-sized c-FSNPs, which exhibit a size-dependent intradermal retention. The 1456 nm sized c-FSNPs display an ideal intradermal retention time (ca. 3 months) for NMSC lesion labeling, as observed in an in vivo tattoo study. In addition, the c-FSNPs induce undetectable inflammatory responses after tattooing. We believe that the c-FSNPs can serve as a "finite tattoo" pigment to label potential malignant NMSC lesions.