Detecting normal and cancer skin cells via glycosylation and adhesion signatures: A path to enhanced microfluidic phenotyping.

Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive properties. Our study investigates the adhesion of skin cells to specific lectins, particularly focusing on advancements in lectin-based biosensors with diagnostic potential. We explore whether we can successfully capture normal skin (melanocytes and keratinocytes) and melanoma (WM35, WM115, WM266-4) cells in a low-shear flow environment by coating surfaces with lectins. Specifically, we coated surfaces with Dolichos biflorus (DBA) and Maackia Amurensis (MAL) lectins, which were used to detect and capture specific skin cells from the flow of cell mixture. Alterations in glycan expression (confirmed by fluorescent microscopy) demonstrated that DBA binds predominantly to normal skin cells, while MAL interacts strongly with melanoma cells. Assessing adhesion under static and dynamic low-shear stress conditions (up to 30 mPa) underscores the reliability of DBA and MAL as markers for discriminating specific cell type. Melanocytes and keratinocytes adhere to DBA-coated surfaces, while melanoma cells prefer MAL-coated surfaces. A comprehensive analysis encompassing cell shape, cytoskeleton, and focal adhesions shows the independence of our approach from the inherent characteristics of cells, thus demonstrating its robustness. Our results carry practical implications for lectin-biosensor designs, emphasizing the significance of glycan-based discrimination of pathologically altered cells. Combined with microfluidics, it demonstrates the value of cell adhesion as a discriminant of cancer-related changes, with potential applications spanning diagnostics, therapeutic interventions, and advanced biomedical technologies.

Plasma Treatment of PDMS for Microcontact Printing (µCP) of Lectins Decreases Silicone Transfer and Increases the Adhesion of Bladder Cancer Cells.

The present study investigates silicone transfer occurring during microcontact printing (µCP) of lectins with polydimethylsiloxane (PDMS) stamps and its impact on the adhesion of cells. Static adhesion assays and single-cell force spectroscopy (SCFS) are used to compare adhesion of nonmalignant (HCV29) and cancer (HT1376) bladder cells, respectively, to high-affinity lectin layers (PHA-L and WGA, respectively) prepared by physical adsorption and µCP. The chemical composition of the µCP lectin patterns was monitored by time-of-flight secondary ion mass spectrometry (ToF-SIMS). We show that the amount of transferred silicone in the µCP process depends on the preprocessing of the PDMS stamps. It is revealed that silicone contamination within the patterned lectin layers inhibits the adhesion of bladder cells, and the work of adhesion is lower for µCP lectins than for drop-cast lectins. The binding capacity of microcontact printed lectins was larger when the PDMS stamps were treated with UV ozone plasma as compared to sonication in ethanol and deionized water. ToF-SIMS data show that ozone-based treatment of PDMS stamps used for µCP of lectin reduces the silicone contamination in the imprinting protocol regardless of stamp geometry (flat vs microstructured). The role of other possible contributors, such as the lectin conformation and organization of lectin layers, is also discussed.

Bladder Cancer Cells Interaction with Lectin-Coated Surfaces under Static and Flow Conditions.

Aberrant expression of glycans, i.e., oligosaccharide moiety covalently attached to proteins or lipids, is characteristic of various cancers, including urothelial ones. The binding of lectins to glycans is classified as molecular recognition, which makes lectins a strong tool for understanding their role in developing diseases. Here, we present a quantitative approach to tracing glycan-lectin interactions in cells, from the initial to the steady phase of adhesion. The cell adhesion was measured between urothelial cell lines (non-malignant HCV29 and carcinoma HT1376 and T24 cells) and lectin-coated surfaces. Depending on the timescale, single-cell force spectroscopy, and adhesion assays conducted in static and flow conditions were applied. The obtained results reveal that the adhesion of urothelial cells to two specific lectins, i.e., phytohemagglutinin-L and wheat germ agglutinin, was specific and selective. Thus, these lectins can be applied to selectively capture, identify, and differentiate between cancer types in a label-free manner. These results open up the possibility of designing lectin-based biosensors for diagnostic or prognostic purposes and developing strategies for drug delivery that could target cancer-associated glycans.

Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties.

Therapies that restore dystrophin expression are presumed to correct Duchenne muscular dystrophy (DMD), with antisense-mediated exon skipping being the leading approach. Here we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. Dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed 2 weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed, with dystrophin uniformly distributed at the sarcolemma of corrected myofibers at all doses. Serum microRNA biomarkers (i.e., miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase levels were restored toward wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e., Young's modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression and that both serum microRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in DMD.

Changes in nanomechanical properties of single neuroblastoma cells as a model for oxygen and glucose deprivation (OGD).

Although complex, the biological processes underlying ischemic stroke are better known than those related to biomechanical alterations of single cells. Mechanisms of biomechanical changes and their relations to the molecular processes are crucial for understanding the function and dysfunction of the brain. In our study, we applied atomic force microscopy (AFM) to quantify the alterations in biomechanical properties in neuroblastoma SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) and reoxygenation (RO). Obtained results reveal several characteristics. Cell viability remained at the same level, regardless of the OGD and RO conditions, but, in parallel, the metabolic activity of cells decreased with OGD duration. 24 h RO did not recover the metabolic activity fully. Cells subjected to OGD appeared softer than control cells. Cell softening was strongly present in cells after 1 h of OGD and with longer OGD duration, and in RO conditions, cells recovered their mechanical properties. Changes in the nanomechanical properties of cells were attributed to the remodelling of actin filaments, which was related to cofilin-based regulation and impaired metabolic activity of cells. The presented study shows the importance of nanomechanics in research on ischemic-related pathological processes such as stroke.

miR-218 affects the ECM composition and cell biomechanical properties of glioblastoma cells.

Glioblastoma (GBM) is the most common malignant brain tumour. GBM cells have the ability to infiltrate into the surrounding brain tissue, which results in a significant decrease in the patient's survival rate. Infiltration is a consequence of the low adhesion and high migration of the tumour cells, two features being associated with the highly remodelled extracellular matrix (ECM). In this study, we report that ECM composition is partially regulated at the post-transcriptional level by miRNA. Particularly, we show that miR-218, a well-known miRNA suppressor, is involved in the direct regulation of ECM components, tenascin-C (TN-C) and syndecan-2 (SDC-2). We demonstrated that the overexpression of miR-218 reduces the mRNA and protein expression levels of TN-C and SDC-2, and subsequently influences biomechanical properties of GBM cells. Atomic force microscopy (AFM) and real-time migration analysis revealed that miR-218 overexpression impairs the migration potential and enhances the adhesive properties of cells. AFM analysis followed by F-actin staining demonstrated that the expression level of miR-218 has an impact on cell stiffness and cytoskeletal reorganization. Global gene expression analysis showed deregulation of a number of genes involved in tumour cell motility and adhesion or ECM remodelling upon miR-218 treatment, suggesting further indirect interactions between the cells and ECM. The results demonstrated a direct impact of miR-218 reduction in GBM tumours on the qualitative ECM content, leading to changes in the rigidity of the ECM and GBM cells being conducive to increased invasiveness of GBM.

Rheological properties of skeletal muscles in a Duchenne muscular dystrophy murine model before and after autologous cell therapy.

Duchenne muscular dystrophy (DMD) is still an incurable muscle degenerative disease; thus, numerous studies focused on novel therapeutic approaches. However, a simple assay of muscle function restoration remains needed. Herein, we used an oscillatory shear rheometer to evaluate changes in rheological properties of mouse muscles (tibialis anterior, TA) and their restoration upon autologous cell therapy by comparing the viscoelastic properties of normal, diseased and treated muscles. Amplitude sweep tests of muscle samples were performed under 20% compression over a range of shear strain between 0.01 and 2% and frequency of 1 rad/s. The samples were tested in plane-plane geometry and horizontal myofiber alignment. Typical linear viscoelastic region (LVER) patterns were found for each muscle type. For healthy muscles, a broad LVER between shear deformations (γ) of 0.013-0.62% was observed. The LVER of DMD mdx/SCID muscles was found at 0.14% to 0.46% shear deformation, and no shear dependence of storage (G') and loss (G") moduli at γ range changing from 0.034% to 0.26% was found for transplanted tissues. G'LVER and G"LVER moduli of healthy muscles were significantly higher than G'LVER and G"LVER of dystrophic tissues. Additionally, muscle resistance assessment by rheometer indicated that muscles transplanted with stem cells restored elastic properties to levels close to those of healthy muscles. Interestingly, histological staining and rheological data indicate that the loss factor is strongly related to structural changes of examined muscles.

Single-molecule force spectroscopy reveals structural differences of heparan sulfate chains during binding to vitronectin.

The syndecans represent an ongoing research field focused on their regulatory roles in normal and pathological conditions. The role of syndecans in cancer progression is well documented, implicating their importance in diagnosis and even proposing various potential cancer treatments. Thus, the characterization of the unbinding properties at the single-molecule level will appeal to their use as targets for therapeutics. In our study, syndecan-1 and syndecan-4 were measured during the interaction with the vitronectin HEP II binding site. Our findings show that syndecans are calcium ion dependent molecules that reveal distinct, unbinding properties indicating the alterations in the structure of heparan sulfate (HS) chains, possibly in the chain sequence or sulfation pattern. In this way, we suppose that HS chain affinity to extracellular matrix proteins may govern cancer invasion by altering the syndecans' ability to interact with cancer-related receptors present in the tumor microenvironment, thereby promoting the activation of various signaling cascades regulating tumor cell behavior.

Traction force microscopy - Measuring the forces exerted by cells.

Cells generate mechanical forces (traction forces, TFs) while interacting with the extracellular matrix or neighbouring cells. Forces are generated by both cells and extracellular matrix (ECM) and transmitted within the cell-ECM or cell-cell contacts involving focal adhesions or adherens junctions. Within more than two decades, substantial progress has been achieved in techniques that measure TFs. One of the techniques is traction force microscopy (TFM). This review discusses the TFM and its advances in measuring TFs exerted by cells (single cells and multicellular systems) at cell-ECM and cell-cell junctional intracellular interfaces. The answers to how cells sense, adapt and respond to mechanical forces unravel their role in controlling and regulating cell behaviour in normal and pathological conditions.

On the Determination of Mechanical Properties of Aqueous Microgels-Towards High-Throughput Characterization.

Aqueous microgels are distinct entities of soft matter with mechanical signatures that can be different from their macroscopic counterparts due to confinement effects in the preparation, inherently made to consist of more than one domain (Janus particles) or further processing by coating and change in the extent of crosslinking of the core. Motivated by the importance of the mechanical properties of such microgels from a fundamental point, but also related to numerous applications, we provide a perspective on the experimental strategies currently available and emerging tools being explored. Albeit all techniques in principle exploit enforcing stress and observing strain, the realization differs from directly, as, e.g., by atomic force microscope, to less evident in a fluid field combined with imaging by a high-speed camera in high-throughput strategies. Moreover, the accompanying analysis strategies also reflect such differences, and the level of detail that would be preferred for a comprehensive understanding of the microgel mechanical properties are not always implemented. Overall, the perspective is that current technologies have the capacity to provide detailed, nanoscopic mechanical characterization of microgels over an extended size range, to the high-throughput approaches providing distributions over the mechanical signatures, a feature not readily accessible by atomic force microscopy and micropipette aspiration.

Indenting soft samples (hydrogels and cells) with cantilevers possessing various shapes of probing tip.

The identification of cancer-related changes in cells and tissues based on the measurements of elastic properties using atomic force microscopy (AFM) seems to be approaching clinical application. Several limiting aspects have already been discussed; however, still, no data have shown how specific AFM probe geometries are related to the biomechanical evaluation of cancer cells. Here, we analyze and compare the nanomechanical results of mechanically homogenous polyacrylamide gels and heterogeneous bladder cancer cells measured using AFM probes of various tip geometry, including symmetric and non-symmetric pyramids and a sphere. Our observations show large modulus variability aligned with both types of AFM probes used and with the internal structure of the cells. Altogether, these results demonstrate that it is possible to differentiate between compliant and rigid samples of kPa elasticity; however, simultaneously, they highlight the strong need for standardized protocols for AFM-based elasticity measurements if applied in clinical practice including the use of a single type of AFM cantilever.

Probing the recognition specificity of αVß1 integrin and syndecan-4 using force spectroscopy.

The knowledge on how cells interact with microenvironment is particularly important in understanding the interaction of cancer cells with surrounding stroma, which affects cell migration, adhesion, and metastasis. The main cell surface receptors responsible for the interaction with extracellular matrix (ECM) are integrins, however, they are not the only ones. Integrins are accompanied to other molecules such as syndecans. The role of the latter has not yet been fully established. In our study, we would like to answer the question of whether integrins and syndecans, possessing similar functions, share also similar unbinding properties. By using single molecule force spectroscopy (SMFS), we conducted measurements of the unbinding properties of αVß1 and syndecan-4 in the interaction with vitronectin (VN), which, as each ECM protein, possesses two binding sites specific to integrins and syndecans. The unbinding force and the kinetic off rate constant derived from SMFS describe the stability of single molecular complex. Obtained data show one barrier transition for each complex. The proposed model shows that the unbinding of αVß1 from VN proceeds before the unbinding of SDC-4. However, despite different unbinding kinetics, the access to both receptors is needed for cell growth and proliferation.

Sequential binary protein patterning on surface domains of thermo-responsive polymer blends cast by horizontal-dipping.

We characterize an approach enabling dual protein positioning over broad polymer areas based on subsequent selective adsorption of two fluorescently labelled lectins, Concanavalin A (Con A) and Lentil Lectin (LcH), on self-assembled gradient patterns of thermoresponsive poly(N-isopropyl acrylamide) (PNIPAM) and polystyrene (PS) polymers blend, prepared by horizontal dipping technique. The film morphologies of gradient samples prior dual selective protein adsorption are mapped with scanning microscopy (AFM) and secondary ion mass spectrometry (ToF-SIMS), whereas adsorbed proteins are imaged with fluorescence microscope. ToF-SIMS analysis reveals surface composition consisting of PNIPAM-rich domains in PS-rich matrix. The two-step protein adsorption experiment results in selective adsorption of Con A and LcH to PNIPAM- and PS-rich phases, respectively. Integral geometry approach is used to compare quantitatively morphology of polymer patterns varied in domain size due to horizontal dipping casting. Minkowski measures are also used to compare quantitatively fluorescence micrographs of protein patches with SIMS images of original isotropic polymer patterns. It confirms that PNIPAM domains size increases with increasing speed. Further, Minkowski analysis unveiled that adsorbed proteins cover about 60-70% of polymer surface. What is more fluorescence micrographs acknowledge both no lectins contamination and no adsorption to interphase areas. Additionally, protein displacement effect is observed.

AFM-based nanomechanical characterization of bronchoscopic samples in asthma patients.

Asthma is not a single disease, but recently, it is considered as a syndrome characterized through various clinical presentations and different etiopathologies. Large degree of the disease heterogeneity manifests in distinct characteristics that translate into variability of properties at single cell and molecular levels. Here, we conducted measurements of mechanical properties of bronchial tissue samples collected from patients suffering from asthma. The results obtained from different applied protocols for sample preparation may indicate that deep freezing and storage in liquid nitrogen, followed by consecutive unfreezing of tissue samples, preserve tissue mechanical properties as indicated by a parameter referred here as a tissue relative stiffness index. Tissue relative stiffness index quantifies both the degree of heterogeneity and deformability of tissue samples regarding healthy one. These studies demonstrate that the freezing protocol, optimized towards asthma tissue, can facilitate atomic force microscopy use what, together with recent findings on standardization of elasticity measurements, enables the measurements of large group of samples with minimized influence of errors stemming from the applied methodology of tissue stiffness determination.

The Effect of Dextran Sulfate-as Model Glycosaminoglycan Analogue-on Membrane Lipids: DPPC, Cholesterol, and DPPC-Cholesterol Mixture. The Monolayer Study.

Glycosaminoglycans (GAGs) are essential components of the extracellular matrices (ECMs) located on the outer surface of cellular membranes. They belong to the group of polysaccharides involved in diverse biological processes acting on the surface and across natural lipid membranes. Recently, particular attention has been focused on possible role of GAGs in the amyloid deposits. The amyloid formation is related to a disorder in protein folding, causing that soluble-in normal conditions-peptides become deposited extracellularly as insoluble fibrils, impairing tissue structure and its function. One of the hypothesis holds that GAGs may inhibit amyloid formation by interacting with the lipid membrane by blocking the accumulation of protein aggregates on the membrane surface. Although the biophysical properties of GAGs are described rather well, little is known about the nature of association between these polysaccharides and components of natural cell membranes. Therefore, a study of GAGs influence on membrane lipids is of particular importance. The aim of the present work is to get insight into the effect of hydrophilic dextran sulfate (DS)-that can be considered as GAG analogue-on membrane lipids organization. This study was based on examining interactions between DS sodium salt of molecular weight equal to about 40 kDa (DS40), dissolved in water subphase, and a model membrane, mimicked as Langmuir monolayer, formed by representative natural membrane lipids: cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as well as their mixtures. Due to the fact that calcium ions in excess may accumulate in the lipid membrane, attracting high molecular weight molecules to their surface, the influence of calcium ions present in the subphase on the DS40 activity has also been examined. It has been found that negatively charged DS, forming a sublayer underneath the monolayer, barely interacts with membrane lipids; however, in the presence of calcium ions the electrostatic interactions between DS40 and lipid membrane are significantly enhanced, leading to the formation of network-like crystalline structures at the surface of model membrane, which can prevent incorporation and interaction with other extracellular molecules, e.g., proteins.

Fibroblasts change spreading capability and mechanical properties in a direct interaction with keratinocytes in conditions mimicking wound healing.

Keratinocytes are predominant in the uppermost layer of the skin, while fibroblasts dominate in the dermal layer. These cells interact with each other directly when fibroblasts migrate to a region of the wound where they induce keratinocytes proliferation through double paracrine signalling. Since a response from both keratinocytes and fibroblasts dominates during the inflammatory and proliferative phases, the exact knowledge how these two types of cells interact with each other is crucial for deeper understanding of mechanisms involved in the wound healing process. The aim of this study was to quantify alterations in mechanical properties of cells, i.e. fibroblasts and keratinocytes, in conditions mimicking direct cellular interactions observed in wound healing. Single cell elasticity was measured using atomic force microscope. To verify the influence of keratinocyte neighbors on fibroblasts elasticity (and vice versa), the effect of cellular confluency was studied in parallel. Our results enabled us to distinguish cellular density-related effects from intercellular interactions occurring between fibroblasts and keratinocytes. While the presence of keratinocytes affects fibroblasts spreading capability and mechanical properties, the keratinocytes remain unaffected by the fibroblasts. These results highlight the importance of the cellular deformability in understanding of the role of biomechanics in double paracrine signalling as fibroblast-keratinocyte interaction can change the potential of the wound healing.

Autologous Cell Therapy Approach for Duchenne Muscular Dystrophy using PiggyBac Transposons and Mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease currently without cure. We investigated the use of the PiggyBac transposon for full-length dystrophin expression in murine mesoangioblast (MABs) progenitor cells. DMD murine MABs were transfected with transposable expression vectors for full-length dystrophin and transplanted intramuscularly or intra-arterially into mdx/SCID mice. Intra-arterial delivery indicated that the MABs could migrate to regenerating muscles to mediate dystrophin expression. Intramuscular transplantation yielded dystrophin expression in 11%-44% of myofibers in murine muscles, which remained stable for the assessed period of 5 months. The satellite cells isolated from transplanted muscles comprised a fraction of MAB-derived cells, indicating that the transfected MABs may colonize the satellite stem cell niche. Transposon integration site mapping by whole-genome sequencing indicated that 70% of the integrations were intergenic, while none was observed in an exon. Muscle resistance assessment by atomic force microscopy indicated that 80% of fibers showed elasticity properties restored to those of wild-type muscles. As measured in vivo, transplanted muscles became more resistant to fatigue. This study thus provides a proof-of-principle that PiggyBac transposon vectors may mediate full-length dystrophin expression as well as functional amelioration of the dystrophic muscles within a potential autologous cell-based therapeutic approach of DMD.

Glass transition in temperature-responsive poly(butyl methacrylate) grafted polymer brushes. Impact of thickness and temperature on wetting, morphology, and cell growth.

Poly(n-butyl methacrylate) (PBMA) grafted polymer brushes attached to glass were fabricated in a three-step process involving surface initiated atom transfer radical polymerization. The surface properties of the coatings after subsequent fabrication steps were confirmed using ToF-SIMS and ellipsometry. Measurements of water contact angle and AFM revealed temperature-induced changes in the hydrophobicity and morphology of the coating. The glass transition temperatures (Tg) of the PBMA coatings with different thicknesses were determined from the AFM measurements. For the PBMA grafted brush coatings with thicknesses less than 62 nm, Tg increases sharply with increasing thickness. The PBMA grafted coatings of thickness equal to 86 nm and 43 nm as well as control glass substrates were used as substrates for culturing a urinary bladder cancer HTB-5 cell line. After 144 h of culturing, a well-developed monocellular layer may be observed on the PBMA coating of thickness equal to 86 nm. In turn, the cells incubated on thinner (43 nm) PBMA coatings as well as on a control glass sample only start to form a confluent layer.

Atomic force microscopy as a tool for assessing the cellular elasticity and adhesiveness to identify cancer cells and tissues.

From the first experiments of the atomic force microscopy (AFM) with biological samples, the range of its potential applications grows extensively. One of them is the use of AFM to characterize biophysical fingerprints of cancer progression in search of non-labelled biomarkers of the disease. The technique offers various functionalities, starting from surface imaging to detection of interaction forces, delivering quantitative parameters that can describe changes characteristic for various diseases, including cancer. In this review, the special emphasis was laid on these studies that compare the AFM-derived properties of reference and cancerous cells using all functionalities from cellular deformability measurements to quantification of the interaction forces at the single-molecule and single-cell levels. Despite the large effort and evidence of the microscope applicability to detect pathologically altered cells, there are still practical challenges remained to be solved before AFM can be implemented for routine cancer tracking and diagnosis. To-date, the AFM can be used to achieve a better understanding of cancer-related processes and mechanisms that could be further employed to design high-resolution clinical assays in a quantitative way.

Synthesis and Postpolymerization Modification of Thermoresponsive Coatings Based on Pentaerythritol Monomethacrylate: Surface Analysis, Wettability, and Protein Adsorption.

Properties of novel temperature-responsive hydroxyl-containing poly(pentaerythritol monomethacrylate) (PPM) coatings, polymerized from oligoperoxide grafted to glass surface premodified with (3-aminopropyl)triethoxysilane, are presented. Molecular composition, chemical state, thickness, and wettability are examined with time of flight-secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), ellipsometry, and contact angle measurements, respectively. Temperature-induced changes in hydrophobicity of grafted PPM brushes are revealed by water contact angle and ellipsometric measurements. Partial postpolymerization modification of hydroxyl groups (maximum a few percent), performed with acetyl chloride or pyromellitic acid chloride, is demonstrated to preserve thermal response of coatings. Adsorption of bovine serum albumin to PPM brushes, observed with fluorescence microscopy, is higher than on glass in contrast to similar hydroxyl-containing layers reported as nonfouling. Enhanced and temperature-controlled protein adsorption is obtained after postpolymerization modification with pyromellitic acid chloride.