Chloride Gradient Is Involved in Ammonium Influx in Human Erythrocytes.

The ammonia/ammonium (NH3/NH4+, AM) concentration in human erythrocytes (RBCs) is significantly higher than in plasma. Two main possible mechanisms for AM transport, including simple and facilitated diffusion, are described; however, the driving force for AM transport is not yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a unique feature of RBCs to swell and lyse in isotonic NH4+ buffer. The kinetics of cell swelling and lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1 main ligand concentrations (Cl- and HCO3-). Additionally, the AM import kinetics was analyzed enzymatically and colorimetrically. In NH4+ buffer, RBCs concentration-dependently swelled and lysed when [NH4+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride concentration. The complete substitution of chloride with glutamate prevented NH4+-induced cell swelling and hemolysis, and the restoration of [Cl-] dose-dependently amplified the rates of RBC swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride anions in the buffer, significantly decreased NH4+ import. Our data indicate that the eAE1-mediated chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM transport in RBCs.

Guidelines in Practice: Team Communication.

The perioperative setting is a complex environment requiring interdisciplinary team collaboration to avoid adverse events. To protect the safety of patients and perioperative team members, communication among personnel should be clear and effective. The recently updated AORN "Guideline for team communication" provides perioperative nurses with recommendations on the topic. To promote effective communication in perioperative areas, all personnel should value and commit to a culture of safety. This article discusses recommendations for supporting a culture of safety, developing and implementing an effective hand-off process and surgical safety checklist, and developing education strategies for team communication. It also includes a scenario describing the implementation of a standardized, electronic surgical safety checklist in the OR. Perioperative nurses should review the guideline in its entirety and apply the recommendations for team communication in their working environments.

Identity Formation in Individuals between 16 and 25 Years Old with Borderline Personality Disorder.

Background/Objectives: Identity disruption is a key feature of borderline personality disorder (BPD), characterized by disturbances in self-image. This study aimed to use the Dimensions of Identity Development Scale (DIDS) in a population aged 16-25, to assess differences in identity status and correlations with BPD features as well as whether a correlation exists between the BPD features, the scores obtained on the DIDS and the scores of the different dimensions of this disorder. Methods: We analyzed data from 132 individuals: 44 with BPD using the Diagnostic Interview for Borderline-Revised (DIB-R). Statistical analyses included quantile regression to determine the differences in the DIDS after adjusting for confounding factors identified during group comparisons and Spearman correlation between the DIDS, the BPD features and the DIB-R. Results: Results indicated significantly lower DIDS scores in the BPD group, particularly in commitment making, exploration breadth (EB), identity with commitment (IM) and ruminative exploration (RE). After adjusting, only EB differs significantly between the two groups. All dimensions of the DIDS except for the exploration in depth (ED) are correlated with BPD features. Significant correlations could be demonstrated between cognitive dimension and ED, between the total DIDS and the number of suicide attempt (SA) and between the IM and the number of SA. Conclusions: Our clinical sample showed distinct identity formation compared to controls, with a lower EB associated with BPD. RE correlated with BPD, suggesting that the individuals engage in repetitive exploratory processes. SA was negatively associated with overall identity development and commitment, indicating impulsive behaviors in BPD intersect with identity struggles.

Glucometrics and device satisfaction in children and adolescents with type 1 diabetes using different treatment modalities: A multicenter real-world observational study.

AIMS: To analyze metabolic outcomes, diabetes impact and device satisfaction in children and adolescents with type 1 diabetes in Italy who used different treatment modalities for diabetes care in a real-life context. METHODS: In this multicenter, nationwide, cross-sectional study, 1464 participants were enrolled at a routine visit. The following treatment modalities were considered MDI + SMBG; MDI + CGM; Sensor Augmented Pump Therapy; predictive management of low glucose; Hybrid Closed Loop (HCL); Advanced Hybrid Closed Loop (AHCL). Health related quality of life was evaluated by the Italian version of the Diabetes Impact and Device Satisfaction Scale (DIDS) questionnaire. RESULTS: Patients treated with AID systems were more likely to have HbA1c ≤ 6.5 %, higher percentage of time with glucose levels between 70 and 180 mg/dL, lower percentage of time with glucose levels above 180 mg/dL, higher device satisfaction, and reduced impact of diabetes. All the therapeutic modalities with respect to MDI + CGM, except for MDI + SMBG, contributed to increase the device satisfaction. HCL and AHCL respect to MDI + CGM were associated with lower diabetes impact. CONCLUSION: Real-life use of automated insulin delivery systems is associated with reduced type 1 diabetes impact, increased device satisfaction, and achievement of glycemic goals.

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein.

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic ß loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

The Assessment of Dual-Cycle Identity Models Among Secondary School Students: The Hungarian Adaptation of DIDS and U-MICS.

The aim of the present study was to evaluate the factor structure and validity of the Hungarian versions of the Dimensions for Identity Development Scale (DIDS) and Utrecht-Management of Identity Commitments Scale (U-MICS). Both models assume that the iterative process of exploring and evolving commitments occurs in two distinct cycles. The sample for testing the factor structure of DIDS consisted of 808 adolescents (357 boys and 451 girls) aged between 14 and 21 years (Mage = 16.86; SD = 1.35). The sample for testing the factor structure of U-MICS consisted of 803 adolescents (353 boys and 450 girls) aged between 14 and 21 years (Mage = 16.88; SD = 1.34). Results indicated a five factor model of DIDS in the present sample. All the five dimensions correlated as hypothesized both internally and externally. In line with previous research, six clusters emerged based on the dimensions of DIDS, including ruminative moratorium. Regarding U-MICS, results indicated a three factor model in the present sample. All the three dimensions were internally and externally correlated as hypothesized regarding both ideological and interpersonal identity domains. With regard to the identity status cluster solution, five clusters emerged in both the educational and friendship domains. We found specific variation regarding identity clusters in the two identity domains. Our results support the use of these two measurements in Hungarian context. Further, our results confirm the divergent developmental dynamics of ideological and interpersonal identity domains.

The effect of hydrogen sulfide on the contractility of cerebral arterioles. A pilot study.

BACKGROUND AND AIMS: Endogenous gaseous substances, such as NO and CO have been found to be effective vasodilators earlier. H2S has been identified as an additional one, however, for that substance both vasodilatory and vasoconstrictor responses have been described in different vascular territories. Our aim was to examine the effect of hydrogen sulfide on the tone of cerebral arterioles and some aspects of its mechanism. METHODS: The work was performed on excised rat anterior cerebral artery segments in vitro (diameter range 150-250 µm), using a pressure myograph system. We used NaHS as exogenous H2S donor, propargylglycine (PAG) to abolish the endogenous synthesis of hydrogen sulfide and 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) to examine the potential role of Cl-/HCO3 - exchanger in the effects of H2S. The time course of the events after application of exogenous H2S was also evaluated. RESULTS: Our findings revealed that in these pathologically important vessels (1) endogenously produced H2S is not a vasodilator, but a moderate vasoconstrictor; (2) H2S has a biphasic effect: low concentrations are moderate vasoconstrictors, while at higher concentrations the initial contraction is followed by dilatation; (3) that vasodilation is prevented by DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid disodium, an inhibitor of the Cl-/HCO3 - exchanger). CONCLUSION: These studies confirm that H2S should be taken into consideration as a modulator of cerebral arteriolar tone in mammals.

Band 3/anion exchanger 1/solute carrier family 4 member 1 expression as determinant of cellular sensitivity to selenite exposure.

Selenium is a chalcogen element that is essential in animals, but is highly toxic when ingested above the nutritional requirement. Selenite is used as a supplement in patients receiving total parenteral nutrition. However, the therapeutic and toxic doses of selenite are separated by a narrow range. This ambivalent character of selenite implies the presence of cellular mechanisms that precisely control selenite homeostasis. Here, we investigated mechanisms that determine cellular susceptibility to selenite exposure. The resistance to selenite exposure was significantly different among cell lines. We determined the expression levels of TPMT (thiopurine S-methyltransferase) and SLC4A1 (solute carrier family 4 member 1), which encode selenium methyltransferase and selenite transporter, respectively. We also examined the effect of inhibition of Band 3 protein activity, which is encoded by SLC4A1, on the cellular sensitivity to selenite. The data suggest that the expression level of SLC4A1 is the determinant of cellular sensitivity to selenite.

Toxicological analysis of stilbenes against the fall armyworm, Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is a global pest of multiple economically important row crops and the development of resistance to commercially available insecticidal classes has inhibited FAW control. Thus, there is a need to identify chemical scaffolds that can provide inspiration for the development of novel insecticides for FAW management. This study aimed to assess the sensitivity of central neurons and susceptibility of FAW to chloride channel modulators to establish a platform for repurposing existing insecticides or designing new chemicals capable of controlling FAW. Potency of select chloride channel modulators were initially studied against FAW central neuron firing rate and rank order of potency was determined to be fipronil > lindane > Z-stilbene > DIDS > GABA > E-stilbene. Toxicity bioassays identified fipronil and lindane as the two most toxic modulators studied with topical LD50's of 41 and 75 ng/mg of caterpillar, respectively. Interestingly, Z-stilbene was toxic at 300 ng/mg of caterpillar, but no toxicity was observed with DIDS or E-stilbene. The significant shift in potency between stilbene isomers indicates structure-activity relationships between stilbene chemistry and the binding site in FAW may exist. The data presented in this study defines the potency of select chloride channel modulators to FAW neural activity and survivorship to establish a platform for development of novel chemical agents to control FAW populations. Although stilbenes may hold promise for insecticide development, the low toxicity of the scaffolds tested in this study dampen enthusiasm for their development into FAW specific insecticides.

Understanding the Functional Expression of Na+-Coupled SLC4 Transporters in the Renal and Nervous Systems: A Review.

Acid-base homeostasis is crucial for numerous physiological processes. Na+/HCO3- cotransporters (NBCs) belong to the solute carrier 4 (SLC4) family, which regulates intracellular pH as well as HCO3- absorption and secretion. However, knowledge of the structural functions of these proteins remains limited. Electrogenic NBC (NBCe-1) is thought to be the primary factor promoting the precise acid-base equilibrium in distinct cell types for filtration and reabsorption, as well as the function of neurons and glia. NBC dysregulation is strongly linked to several diseases. As such, the need for special drugs that interfere with the transmission function of NBC is becoming increasingly urgent. In this review, we focus on the structural and functional characteristics of NBCe1, and discuss the roles of NBCe1 in the kidney, central nervous system (CNS), and related disorders, we also summarize the research on NBC inhibitors. NBCe1 and the related pathways should be further investigated, so that new medications may be developed to address the related conditions.

DOCK8-related Immunodeficiency Syndrome (DIDS): Report of Novel Mutations in Iranian Patients.

DOCK8 immunodeficiency syndrome (DIDS) is a rare autosomal recessive (AR) disorder characterized by elevated serum IgE levels, eosinophilia, recurrent cutaneous infections, severe eczema, and sinopulmonary and gastrointestinal infections. This syndrome is a multisystem disease that is associated with both immune deficiency and neurological complications. In this study, we describe the clinical characteristics of two Iranian patients with DOCK8 deficiency and propose possible mechanisms for this condition. By using whole exome sequencing (WES), we identified two novel mutations, namely c.3233_3234del AG (p.Q1078fs) in exon 6 and a large deletion with 94 kb (c.405-3231 deletion, p.K135fs), in these two patients. These variations are confirmed with Sanger sequencing and CGH array. Subsequent co-segregation analysis is performed to identify inheritance patterns. Both patients were homozygote and their parents were heterozygote for the variations. For further investigation, prediction tools were applied to identify the pathogenicity of the variations and also for modeling the truncated proteins. The patients did not show neurological abnormalities associated with a deficiency of the N terminal region of DOCK8. The absence of neurological complications in the first patient is justifiable due to the maintenance of the proline-rich region in DOCK8, but for the second patient with expanded deletion which is almost like null DOCK8 protein, it is not presumable, pointing to the fact that the C terminal region of the protein might have functions in the proliferation and migration neurons in the peripheral nervous system. Alternatively, it is possible that neurological abnormalities follow an age-dependent pattern, leading to the appearance of related symptoms later in life. Further multiple functional studies are needed to model different identified variants in animal models to confirm our results and suggest possible mechanisms associated with DOCK8 deficiency in this study.

Rapid volume pulsation of the extracellular space coincides with epileptiform activity in mice and depends on the NBCe1 transporter.

KEY POINTS: Extracellular space (ECS) rapid volume pulsation (RVP) accompanying epileptiform activity is described for the first time. Such RVP occurs robustly in several in vitro and in vivo mouse models of epileptiform activity. In the in vitro 4-aminopyridine model of epileptiform activity, RVP depends on the activity of the electrogenic Na+ /HCO3- cotransporter (NBCe1). NBCe1 pharmacological inhibition suppresses RVP and epileptiform activity. Inhibition of changes in ECS volume may be a useful target in epilepsy patients who are resistant to current treatments. ​ ABSTRACT: The extracellular space (ECS) of the brain shrinks persistently by approximately 35% during epileptic seizures. Here we report the discovery of rapid volume pulsation (RVP), further transient drops in ECS volume which accompany events of epileptiform activity. These transient ECS contractions were observed in multiple mouse models of epileptiform activity both in vivo (bicuculline methiodide model) and in vitro (hyaluronan synthase 3 knock-out, picrotoxin, bicuculline and 4-aminopyridine models). By using the probe transients quantification (PTQ) method we show that individual pulses of RVP shrank the ECS by almost 15% in vivo. In the 4-aminopyridine in vitro model, the individual pulses of RVP shrank the ECS by more than 4%, and these transient changes were superimposed on a persistent ECS shrinkage of 36% measured with the real-time iontophoretic method. In this in vitro model, we investigated several channels and transporters that may be required for the generation of RVP and epileptiform activity. Pharmacological blockages of Na+ /K+ /2Cl- cotransporter type 1 (NKCC1), K+ /Cl- cotransporter (KCC2), the water channel aquaporin-4 (AQP4) and inwardly rectifying potassium channel 4.1 (Kir4.1) were ineffective in halting the RVP and the epileptiform activity. In contrast, pharmacological blockade of the electrogenic Na+ /HCO3- cotransporter (NBCe1) by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminated both the RVP and the persistent ECS shrinkage. Importantly, this blocker also stopped the epileptiform activity. These results demonstrate that RVP is closely associated with epileptiform activity across several models of epileptiform activity and therefore the underlying mechanism could potentially represent a novel target for epilepsy management and treatment.

VDAC1 as a target in cisplatin anti-tumor activity through promoting mitochondria fusion.

Cisplatin is one of the most effective anti-cancer drugs, but its efficacy is limited by the development of resistance. Previous studies have shown that mitochondria play critical roles in cisplatin cytotoxicity, however, the exact mechanism of mitochondria involved in cisplatin sensitivity has not been clarified. In this study, cisplatin triggered mitochondrial oxidative stress and the decrease of mitochondria membrane potential in human cervical cancer cells. Then we screened a series of mitochondrial relevant inhibitors, including mitochondrial mPTP inhibitors DIDS and CsA, and mitochondrial respiratory complex inhibitors Rot and TTFA. Among these, only DIDS, as the inhibitor of mitochondrial outer membrane protein VDAC1, showed strong antagonism against cisplatin toxicity. DIDS mitigated cisplatin-induced MFN1-dependent mitochondrial fusion, mitochondrial dysfunction and oxidative damage. These findings demonstrated that VDAC1 may serve as a potential therapeutic target in the increase sensitivity of cisplatin, which provides an attractive pharmacological therapy to improve the effectiveness of chemotherapy.

DIDS inhibits Vibrio vulnificus cytotoxicity by interfering with TolC-mediated RtxA1 toxin secretion.

Vibrio vulnificus (V. vulnificus) infection, frequently resulting in fatal septicemia, has become a growing health concern worldwide. The present study aimed to explore the potential agents that could protect against V. vulnificus cytotoxicity, and to analyze the possible underlying mechanisms. First, we observed that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) significantly suppressed V. vulnificus cytotoxicity to host cells by using a lactate dehydrogenase (LDH) assay. DIDS did not exhibit any effect on host cell viability, bacterial growth, microbial adhesion and swarming motility. DIDS effectively lowered V. vulnificus RtxA1 toxin-induced calcium influx into host mitochondria and RtxA1 binding to host cells. To further elucidate the underlying mechanism, the synthesis and secretion of RtxA1 toxin were investigated by Western blotting. Intriguingly, DIDS selectively inhibited the secretion of RtxA1 toxin, but did not influence its synthesis. Consequently, the outer membrane portal TolC, a key conduit for RtxA1 export coupled with tripartite efflux pumps, was examined by RT-PCR and Western blotting. We found that DIDS significantly reduced the expression of TolCV1 protein at the transcriptional level. Taken together, these results suggest that DIDS is a promising new paradigm as an antimicrobial drug that targets TolC-mediated toxin.

Voltage-gated chloride channel blocker DIDS as an acaricide for Varroa mites.

The Varroa mite is a primary driver behind periodical losses of honey bee colonies. These mites require honey bees for food and reproduction and, in turn, elicit physiological deficiencies and diseases that compromise colony health. Current acaricides for Varroa mite control, such as Apistan® (the pyrethroid tau-fluvalinate), CheckMite+® (the organophosphate coumaphos), and Apivar® (the formamidine amitraz) target the nervous system, can have adverse health effects on honey bees, and have limited effectiveness due to reported resistance issues. New target sites are needed to circumvent these obstacles in Varroa mite management, and voltage-gated chloride channels (VGCCs) are promising candidates due to their important role in the maintenance of nerve and muscle excitability in arthropod pests. Toxicological analysis of Varroa mites sensitive to tau-fluvalinate and coumaphos and Varroa mites with reduced sensitivity to these acaricides showed a significant increase in metabolic detoxification enzyme activities for the latter. Acetylcholinesterase activity in the Varroa mites exhibiting reduced mortality to coumaphos was significantly less sensitive to coumaphos-oxon compared to coumaphos-sensitive Varroa mites, which suggests target-site insensitivity to the acaricide. Voltage-gated chloride channel blocker DIDS had significantly greater field efficacy compared to Apistan® and CheckMite+® against Varroa mites from honey bee hives where tau-fluvalinate and coumaphos were observed to be ineffective, respectively. These data suggest that DIDS, and potentially other stilbene chemistries, might serve as candidates for continued field efficacy testing of alternative acaricides in apiaries where Apistan®- and CheckMite+® efficacy has been. reduced or lost for Varroa mites.

4,4'-Diisothiocyanato-2,2'-Stilbenedisulfonic Acid (DIDS) Modulates the Activity of KCNQ1/KCNE1 Channels by an Interaction with the Central Pore Region.

BACKGROUND/AIMS: The cardiac current IKs is carried by the KCNQ1/KCNE1-channel complex. Genetic aberrations that affect the activity of KCNQ1/KCNE1 can lead to the Long QT Syndrome 1 and 5 and, thereby, to a predisposition to sudden cardiac death. This might be prevented by pharmacological modulation of KCNQ1/KCNE1. The prototypic KCNQ1/KCNE1 activator 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) represents a candidate drug. Here, we study the mechanism of DIDS action on KCNQ1/KCNE1. METHODS: Channels were expressed in Xenopus oocytes and iPSC cardiomyocytes. The role of the central S6 region was investigated by alanin-screening of KCNQ1 residues 333-338. DIDS effects were measured by TEVC and MEA. RESULTS: DIDS-action is influenced by the presence of KCNE1 but not by KCNQ1/KCNE1 stochiometry. V334A produces a significant higher increase in current amplitude, whereas deactivation (slowdown) DIDS-sensitivity is affected by residues 334-338. CONCLUSION: We show that the central S6 region serves as a hub for allosteric channel activation by the drug and that DIDS shortens the pseudo QT interval in iPSC cardiomyocytes. The elucidation of the structural and mechanistic underpinnings of the DIDS action on KCNQ1/KCNE1 might allow for a targeted design of DIDS derivatives with improved potency and selectivity.

4,4'-Diisothiocyanatostilbene-2,2'-disulfonate modulates voltage-gated K+ current and influences cell cycle arrest in androgen sensitive and insensitive human prostate cancer cell lines.

The stilbene derivative, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), an anion channel blocker is used in the present study to evaluate its modulatory effect on voltage-gated K+ current (IK) in human prostate cancer cell lines (LNCaP and PC-3). Voltage-gated K+ (KV) channels in the plasma membrane are critically involved in the proliferation of tumor cells. Therefore, KV channels are considered as a novel potential target for cancer treatment. The results of the present study show that the external perfusion of DIDS activates IK in a concentration-dependent manner, although the known K+ channel blocker TEA failed to block the DIDS activated IK in PC-3 cells. Whereas, in LNCaP cells, the higher concentration of DIDS blocked IK, though this effect was not completely recovered after washout. The difference in function of DIDS might be due to the expression of different Kv channel isoforms in LNCaP and PC-3 cells. Further, the anticancer studies show that treatment of DIDS significantly induced G2/M phase cell cycle arrest and induced moderate and low level of cell death in LNCaP and PC-3 cells respectively. This finding reveals that DIDS modulates IK and exerts cell cycle arrest and cell death in LNCaP and PC-3 cells.

The teleost fish intestine is a major oxalate-secreting epithelium.

Oxalate is a common constituent of kidney stones, but the mechanism of its transport across epithelia is not well understood. With prior research on the role of the intestine focused on mammals, the present study considered oxalate handling by teleost fish. Given the osmotic challenge of seawater (SW), marine teleosts have limited scope for urinary oxalate excretion relative to freshwater (FW) taxa. The marine teleost intestine was hypothesized as the principal route for oxalate elimination, thus demanding epithelial secretion. To test this, intestinal 14C-oxalate flux was compared between FW- and SW-acclimated sailfin molly (Poecilia latipinna). In SW, oxalate was secreted at remarkable rates (367.90±22.95 pmol cm-2 h-1), which were similar following FW transfer (387.59±27.82 pmol cm-2 h-1), implying no regulation by salinity. Nevertheless, this ability to secrete oxalate at rates 15-19 times higher than the mammalian small intestine supports this proposal of the teleost gut as a major, previously unrecognized excretory pathway.

Isothiocyanate groups of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) inhibit cell penetration of octa-arginine (R8)-fused peptides.

Delivering biomolecules, such as antibodies, proteins, and peptides, to the cytosol is an important and challenging aspect of drug development and chemical biology. Polyarginine-a well-known cell-penetrating peptide (CPP)-is capable of exploiting its positive charge and guanidium groups to carry a fused cargo into the cytosol. However, the precise mechanism by which this occurs remains ambiguous. In the present study, we established a new method of quantitatively assessing cell penetration. The method involves inducing cell death by using a polyarginine (R8) to deliver a peptide-ie, mitochondrial targeting domain (MTD)-to the cytosol. We found that 4,4'-diisothiocyanatostilbene-2,2'-di-sulfonate (DIDS)-an anion channel blocker-inhibited the ability of octa-arginine (R8)-fused MTD to penetrate cells. Other anion channel blockers did not inhibit the penetration of peptides fused with R8. Comparison of DIDS with other structurally similar chemicals revealed that the isothiocyanate group of DIDS may be primarily responsible for the inhibitory effect than its stilbene di-sulfonate backbone. These results imply that the inhibitory effect of DIDS may not be derived from the interaction between stilbene di-sulfonate and the anion channels, but from the interaction between the isothiocyanate groups and the cell membrane. Our new MTD method enables the quantitative assessment of cell penetration. Moreover, further studies on the inhibition of CPPs by DIDS may help clarify the mechanism by which penetration occurs and facilitate the design of new penetrative biomolecules.

Cryo-EM structures of DrNKCC1 and hKCC1: a new milestone in the physiology of cation-chloride cotransporters.

New milestones have been reached in the field of cation-Cl- cotransporters with the recently released cryo-electron microscopy (EM) structures of the Danio rerio (zebrafish) Na+-K+-2Cl- cotransporter (DrNKCC1) and the human K+-Cl- cotransporter (hKCC1). In this review we provide a brief timeline that identifies the multiple breakthroughs in the field of solute carrier 12 transporters that led to the structure resolution of two of its key members. While cation-Cl- cotransporters share the overall architecture of carriers belonging to the amino acid-polyamine-organocation (APC) superfamily and some of their substrate binding sites, several new insights are gained from the two individual structures. A first major feature relates to the largest extracellular domain between transmembrane domain (TMD) 5 and TMD6 of KCC1, which stabilizes the dimer and forms a cap that likely participates in extracellular gating. A second feature is the conservation of the K+ and Cl- binding sites in both structures and evidence of an unexpected second Cl- coordination site in the KCC1 structure. Structural data are discussed in the context of previously published studies that examined the basic and kinetics properties of these cotransport mechanisms. A third characteristic is the evidence of an extracellular gate formed by conserved salt bridges between charged residues located toward the end of TMD3 and TMD4 in both transporters and the existence of an additional neighboring bridge in the hKCC1 structure. A fourth feature of these newly solved structures relates to the multiple points of contacts between the monomer forming the cotransporter homodimer units. These involve the TMDs, the COOH-terminal domains, and the large extracellular loop for hKCC1.