We demonstrate that the supraphysiological levels of nutrients interfere with endodermal differentiation. Refinement of media formulations has a potential application in maturity modulation of stem cell-derived β-cells (SC-β) generation in vitro. To address these issues, we established a defined culture system to derive SC-β-cells using a blood amino acids-like medium (BALM). Human induced pluripotent stem cells (hiPSCs) can be efficiently differentiated into the definitive endoderm, pancreatic progenitors, endocrine progenitors, and SC-β in BALM-based med. The differentiated cells secreted C-peptide in vitro in response to high glucose levels and expressed several pancreatic β-cell markers. In conclusion, amino acids at the physiological levels are sufficient for deriving functional SC-β cells.Traditional cell culture media do not accurately represent the availability of the nutrients in plasma. They usually contain a supraphysiological concentration of nutrients such as glucose, amino acids, etc. These high nutrients can alter the metabolism of cultured cells and induce metabolic phenotypes that do not reflect  Summary: We used a blood amino acids-like medium and found that it is useful to efficiently differentiate human iPSCs into the definitive endoderm, and to generate functional pancreatic beta-cells. Previous reports have shown that deprivation of nutrients can eliminate residual undifferentiated cells and potentiate differentiation. This potential of metabolic approaches for modulating differentiation efficiency and the initial cell fate specification receives attention .in vivo conditions (in vitro generation of pancreatic β-cells from human induced pluripotent stem cells (hiPSCs).While metabolism offers a potential approach to improving the efficacy of cellular therapy, traditional cell culture media do not accurately represent the nutrients available in plasma. They usually contain supraphysiological concentrations of nutrients such as glucose, amino acids, etc. . These hnditions . Effortsnditions . RefinemSeveral recent protocol modifications to improve the maturation and homogeneity of hiPSCs-derived β (SC-β) cells were achieved by altering culture conditions for differentiation or using small-molecule screening . Metabolin vitro in response to various insulin secretagogues and high glucose levels and expressed several pancreatic β-cell markers.To address these issues, we used a culture system to derive SC-β cells using a blood amino acids-like medium (BALM). We demonstrated that supraphysiological nutrient levels interfere with endodermal differentiation. hiPSCs can be efficiently differentiated into DE, pancreatic progenitors, endocrine progenitors, and SC-β in the BALM-based medium. The differentiated cells secreted C-peptide in vivo conditions. We refer to a report to develop a chemically defined medium with amino acids and glucose at concentrations generally found in human plasma (Table S1. We compared using BALM and DMEM F12-based medium and assayed for their effects on differentiation of the human-induced pluripotent stem cells (hiPSCs) RPChiPS771 cells. To examine the impact of BALM on differentiation, we assessed SOX17 and OCT3/4 expression of day 3 clusters. Immunofluorescence revealed an increase in the ratio of SOX17-positive cells cultured in BALM compared to those cultured in DMEM F12-based medium. OCT3/4-positive cells significantly reduced when hiPSCs were differentiated in BALM compared to DMEM F12  showed significantly decreased levels in cells cultured in BALM compared to those cultured in DMEM F12-based medium. The definitive endoderm (DE) marker genes  revealed a significant increase in cells cultured in BALM compared to those in DMEM F12   was repoDMEM F12 . The resPDX1, SOX9, and endocrine progenitor marker genes, NGN3 and NKX6.1 . hiPSC-derived DE cells were differentiated into PP cells (day 9). Differentiation of hiPSC in BALM-based medium yielded (76.0%±7.6%) PDX1+ PP cells and (42.7%±7.4%) SOX9+ cells A,B. Furtd NKX6.1 E. The hid NKX6.1 F. The rePDX1, MAFB, CHGA, SST, PCSK1, GCK, and ISL1 levels than human islets. INS and MAFA expressions were lower than those of the human islets. GCG, NKX6.1, ABCC8, PCSK2, and SLC30A8 expressions were comparable with human islets. Gene expressions are presented as fold-changes compared to human islets (human islet=1)  INS+ cells, (39.2%±4.3%) NKX6.1+ cells, and (18.9%±5.0%) INS/NKX6.1 double-positive cells A,B. We eislet=1) C. The reislet=1) .We tested the function of stage 6 cells generated in a BALM-based medium using a static GSIS assay. The SC-β cells secreted insulin when challenged with high glucose D. The dain vitro differentiation is crucial for the clinical translation of SC-β cells toward curative diabetes treatment  expression was considerably lower in cells cultured in BALM than in those cultivated in DMEM F12-based medium. When comparing cells grown in BALM to those cultured in DMEM F12, the DE marker genes  exhibited a significant increase in cells cultured in a BALM-based medium. Our findings indicate that supraphysiological concentrations of nutrients suppressed DE differentiation. The use of physiological levels of nutrients can lead to modulating cell function.This study shows that through refinements of the nutrient composition of BALM-based medium, we successfully promoted hiPSCs differentiation into the DE. The physiological-like culture medium formulation significantly reduced the undifferentiated cell population. Moreover, pluripotency markers , and resuspended in StemFit AK02N medium supplemented with 10 µM ROCK inhibitor  and replated at 1-2×106 cells per dish.RPChiPS771 human induced pluripotent stem cells  were maintained in StemFit AK02N medium (Ajinomoto) on 100 mm CellBIND cell culture dishes  precoated with vitronectin . Cell passages were performed as follows. hiPSCs were washed, dissociated with TrypLE6 cells per well onto six-well suspension culture plates  in AK02N medium supplemented with 10 µM ROCK inhibitor, and cultured at 37°C for 24 h to form sphere on a rotating orbital shaker at 95 RPMs. Endodermal differentiation was initialized 24 h post-seeding by changing with differentiation medium : S1 medium + 100 ng/ml Activin A (cell guidance system GFH6-1000) + 3 μM Chir99021 (Wako 034-23103) + 10 µM Y27632 for 1 day. S1 medium + 100 ng/ml Activin A for 2 days. Stage 2 (3 days): S2 medium + 50 ng/ml KGF . Stage 3 (2 day): S3 medium + 50 ng/ml KGF + 200 nM LDN193189 (WAKO SML0559-5MG) + 500 nM PdBU  + 2 μM Retinoic Acid (Stemgent 04-0021) + 0.25 μM Sant1  + 10 µM Y27632. Stage 4 (5-6 days): S4 medium + 5 ng/ml Activin A + 50 ng/ml KGF + 0.1 µM Retinoic Acid + 0.25 µM SANT1 + 10 µM Y27632. Stage 5 (8 days): S5-1 medium (4 days) + 10 µM ALK5i II (TOCRIS 3742) + 20 ng/ml Betacellulin  + 0.1 µM Retinoic Acid + 1 µM T3  + 1 µM XXI (Sigma 595790), 10 µM Heparin + 0.25 µM SANT1 + 10 µM Y27632. S5-2 (4 days) medium + 10 µM ALK5i II (TOCRIS 3742) + 20 ng/ml Betacellulin  + 0.1 µM Retinoic Acid + 1 µM T3  + 1 µM XXI  10 µM Heparin + 10 µM Y27632. Stage 6 (14 days): on the first day of stage 6, reaggregation of cell spheres was performed. Cell spheres were washed in PBS and incubated in TrypLE Select Enzyme  for 10 min at 37C. Spheres were mechanically dissociated using a P1000 pipette. Cells were washed with S6 medium + Y27632 (10 µM), resuspended in S6 medium + Y27632, then passed through a 100-µm CellStrainer (pluriSelect) to remove any residual undissociated clusters. The dissociated single cells were counted and seeded into a six-well suspension culture plate . Differentiation media formulations used were the following. S1 medium: BALM supplemented with 1% Penicillin-Streptomycin solution , 1% (v/v) B27 insulin (-) , Recombinant IGF1 100 ng/ml . S2 medium: BALM supplemented with 1% PS, 1% (v/v) B27 , S3 medium: BALM supplemented with 1% PS, 1% (v/v) B27 , S4 medium: BALM supplemented with 1% PS, 1% (v/v) B27 minus vitamin A , S5 medium: BALM supplemented with 1% PS, 1% (v/v) . S6 medium: BALM supplemented with 1% PS, 2% (w/v) fatty acid-free (FAF)-BSA , 0.44 g/L glucose, 1 µM ZnSO4 , 10 µM Heparin, 0.1% Trace Elements A , and 0.1% Trace Elements B .We performed pancreatic differentiation following a previously reported protocol with modifications . RPChiPSRNA was extracted from human iPS cells using the RNeasy mini-kit, or All prep (DNA/RNA) Mini Kit  and then treated with DNaseI (Qiagen). 200 ng RNA was reverse-transcribed using PrimeScript™ RT Master Mix . For real-time PCR analysis, mRNA expressions were quantified with SyberGreen on a StepOne Plus .The PCR conditions were as follows: initial denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, for up to 40 cycles. Target mRNA levels were expressed as arbitrary units and were determined using the ΔΔ CT method.Primer sequences used for the detection of endoderm and pancreatic differentiation and internal controls are listed in For human islet cDNA three batches of first strand cDNA from non-diabetic human islets (Cosmo Bio): HIcDNA149, HIcDNA171T, and HIcDNA171) were used.hiPSC-derived cell clusters were fixed with 4% paraformaldehyde  in phosphate-buffered saline (PBS), permeabilized with 1% Triton X-100, and blocked with 20% Blocking One  and PBS-T (0.1% Tween-20 in PBS) for 1-2 h. After washing the cells in PBS-T three times, cells were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics). Images were captured using an ImageXpress Micro scanning system . Quantitative analysis of positive cells versus total cells (DAPI-positive cells) was performed using the MetaXpress cellular image analysis software (Molecular Devices). The primary and secondary antibodies used are summarized in C-peptide release and C-peptide content assay were performed as described previously with minor modifications . BrieflyN=3) and repeated multiple times. Differences between groups were analyzed by Student's t-tests; differences are shown as *P<0.05, **P<0.01, ***P<0.001 or ****P<0.0001.Individual data are shown or expressed as the mean± standard deviation (s.d.). Each experiment was conducted with three biological replicates (10.1242/biolopen.059581_sup1Supplementary informationClick here for additional data file.


Raphignathus Dugès are hereby proposed: Raphignathus (Raphignathus), subgen. nov., Raphignathus (Monoraphignathus), subgen. nov., Raphignathus (Diraphignathus), subgen. nov., and Raphignathus (Triraphignathus), subgen. nov. These subgenera are diagnosed by the number of setae on the interscutal membrane of females. A new species, R. (D.) neohecmatanaensissp. nov., is described and illustrated based on females collected from Ziziphusspina-christi Mill. (Rhamnaceae). The taxonomic status of the monotypic genus Neoraphignathus Smiley & Moser and three species  are discussed. A key to world species of the family Raphignathidae is given.Four new subgenera in the genus  Raphignathidae Kramer  are active predators feeding on small arthropods (Raphignathus Dugès (76 species) and Neoraphignathus Smiley & Moser   . These gana, USA .Raphignathus (type species: R.ruberrimus Dugés) was diagnosed as having three, or sometimes four, dorsal shields . A new species, R. (D.) neohecmatanaensis sp. nov. is described and illustrated based on females. Some taxonomic notes on the monotypic genus, Neoraphignathus Smiley & Moser, and the identity of three species  are concisely discussed. A diagnostic key to the world species is also provided.In the present study, four new subgenera of Raphignathidae was critically reviewed to confirm the validity of the species, subgeneric divisions and to prepare a diagnostic key of world species. The new raphignathoid species was collected by shaking foliage of Ziziphusspina-christi Mill. (Rhamnaceae) over a white sheet of paper; mite specimens were preserved in small vials containing 70% ethanol. The specimens were permanently mounted on glass slides in Hoyer’s medium and identified under a phase-contrast microscope . All measurements of the holotype specimen are given in micrometers (μm), followed by those of paratypes in the parenthesis. The terminology and abbreviations used in the description of the new species follow those of King Saud Museum of Arthropods , Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia.All published taxonomic literature on the family Raphignathus are proposed: Raphignathus (Raphignathus), subgen. nov., Raphignathus (Monoraphignathus), subgen. nov., Raphignathus (Diraphignathus), subgen. nov., and Raphignathus (Triraphignathus), subgen. nov., on the basis of the number of setae on the interscutal membrane, a prominent and consistent morphological character. A new species, R. (D.) neohecmatanaensis sp. nov., is described and illustrated based on adult females. Furthermore, taxonomic notes on the status of the monotypic genus, Neoraphignathus Smiley & Moser, and the identity of three species  are discussed. A diagnostic key to the world species is also presented.Four new subgenera of the genus Taxon classificationAnimaliaProstigmataRaphignathidaeFamilyKramer, 18771464681B-3E4D-5CAF-92DB-EB3727AA4A1CRaphignathidae Kramer, 1877: 215Raphignathus Dugès, 1834: 53based on . PeritreRaphignathoidea while describing seven Raphignathus species. This author also provided comprehensive diagnosis of Raphignathus and stated that the number of setae on the shields and the interscutal membrane is a consistent and important diagnostic character. The number of setae on the interscutal membrane is considered to be a strong character and has been used in identification keys to distinguish species , subgen. nov. without setae (11 spp.), Raphignathus (Monoraphignathus), subgen. nov. with one seta (14 spp.), Raphignathus (Diraphignathus), subgen. nov. with two setae (33 spp.), and Raphignathus (Triraphignathus), subgen. nov. with three setae (10 spp.).In the current study, we categorize species of the genus Taxon classificationAnimaliaProstigmataRaphignathidae9647ECD3-9FB0-5F1B-8310-4C401721C64Dhttps://zoobank.org/4A81EA35-DC89-4971-BD24-0459A27A8621Raphignathusruberrimus Dugès, 1834: 53.Interscutal membrane without setae.The subgeneric epithet refers to the nominotypical subgenus.Taxon classificationAnimaliaProstigmataRaphignathidaeE0F96820-6E72-5ADB-A958-0652A1A3C16Ahttps://zoobank.org/D332A8E8-0390-4D39-95D4-A8C6E7E0B8D5Raphignathusbathursti Meyer & Ryke, 1960: 229.Interscutal membrane with one pair of setae.The subgeneric epithet refers to the one pair of setae on interscutal membrane.Taxon classificationAnimaliaProstigmataRaphignathidae9FADAE20-20A8-59F6-9FE7-7314FC6A9584https://zoobank.org/0D7C8B12-10E8-4019-A2FA-591934FD9B17Raphignathusgracilis : 281.Interscutal membrane with two pairs of setae.The sub-generic epithet refers to the two pairs of setae on interscutal membrane.The species included in this new subgenus are widely distributed over the world.Taxon classificationAnimaliaProstigmataRaphignathidae56BD0366-5B72-5D0D-8593-9BE41561D9EEhttps://zoobank.org/80C632B9-6521-47BB-A6E2-3EA44EE761DDRaphignathusdomesticus Shiba, 1969: 157.Interscutal membrane with three pairs of setae.The subgeneric epithet refers to the interscutal membrane with three pairs of setae.Raphignathidae has included two genera, Raphignathus and Neoraphignathus, which have been differentiated based on the presence or absence of shields on the dorsum. The monotypic genus, Neoraphignathus (type species: N.howei Smiley & Moser) was erected in 1968, based on a single female holotype specimen without detailed description and illustration. Based on observations and the collection of the immature specimens of the genus Raphignathus, prodorsal shields are weakly developed or absent in immatures. N.howei, has not been redescribed, nor have new Neoraphignathus species been described. We suggest that the type specimen of N.howei be re-examined and also that more specimens be collected from the type locality to confirm the absence of a dorsal shield to confirm the validity of Neoraphignathus.To date, the family Raphignathusevidus Fan, R.hsiufui Fan, and R.johnstoni Womersley are doubtful. These species were originally described based on single specimens, minor differential characteristics , and small opisthosomal shields. In contrast, all other Raphignathus species have three pairs of setae on the lateral prodorsal shields along with the pores (ia). The immature stages of Raphignathus gradually develop the prodorsal shields, striation patterns, and leg setae  and small lateral prodorsal shields with weakly developed striations. Moreover, we also observed the immatures from more than 10 populations of Raphignathus and found reduced size of weakly sclerotized lateral prodorsal shields and setae set on the edges of shields. Based on this evidence, R.evidus, R.hsiufui, and R.johnstoni should be revised and more specimens collected to confirm their validity.The taxonomic identity of eg setae . For insTaxon classificationAnimaliaProstigmataRaphignathidaesp. nov.4E0A4286-D871-5265-AAD6-99D360ED0DEFhttps://zoobank.org/12E13CEE-478F-4A3F-A47E-B3C7F34C73FAFemale: endopodal shield absent between coxae I–IV; two small shields present posterolateral to median prodorsal shield; palp femora with two setae; femora 6–5–3–2; genua 5(+κ)-5(+κ)–4–4; tibiae 5(+φρ)–5(+φρ)–4(+φρ)–4 (+φρ); tarsi 21(1ω) –20(1ω)–15–14.Female (n = 4). Idiosoma oval, length of body (including gnathosoma) 533 (525–545); width of body 345 (338–353).Dorsum  and sci, sce and seta c2 seta; opisthosomal setal pairs d1, e1 and the pair of cupuli (im); posterior opisthosomal shield large, rectangular, bearing four pairs of setae  and one pair of the cupule (ip); all dorsal shields finely punctate; dorsal body setae setiform, smooth and acute; pseudanal setae ps1 dorsally located. Lengths of dorsal setae: vi 25 (23–27), ve 28 (26–30), sci 27 (26–29), sce 28 (26–29), c1 23 (17–19), c2 25 (19–23), d1 21 (20–23), e1 22 (21–23), f1 21 (19–22), h1 24 (23–25), h2 25 (23–25), h3 22 (21–24), ps1 23 (21–25); distances between dorsal 195 setae: vi–vi 27 (29–31), sci–sci 123 (118–128), vi– sci 70 (66–72), sce–c2 68 (65–72), c1–c1 30 (28–32), d1–d1 99 (92–101), c1–d1 53 (49–55), d1–e1 22 (21–24), f1–f1 88 (83–90), e1–f1 65 (61–68), h1–h1 45 (42–48), h1–h2 43 (41–46), h2–h2 77 (72–80), h3–h3 101 (98–106).sum Fig. . PropodoVenter , 1b 38 (35–39), 1c 34 (33–36), 2b 36 (30–34), 2c 32 (30–34), 3a 24 (23–26), 3b 18 (17–20), 3c 36 (34–38), 4a 22 (23–27), 4c 38 (35–40); two pairs of aggenital setae (ag1–2) with one cupule (ih) on each side of the genital shield; anal opening and genital shields separate; genital shield prominent with a few punctations, bearing three pairs of genital setae (g1–3); anal opening terminal, with three pairs of setae (ps1–3), ps1 dorsal ps3 and ps2 ventral; ventral setal lengths: ag1 29 (27–32); ag2 27 (25–28); g1 31 (28–33); g2 25 (23–28); g3 20 (19–22); ps2 22 (21–23); ps3 21 (20–22). Distances between ventral setae: 1a–1a 55 (53–58), 3a–3a 115 (97–109), 4a–4a 70 (68–73), ag2–ag2 42 (41–43), g1–g1 38 (35–40), g2–g2 52 (48–55), g3–g3 75 (74–75), 2b–2c 30 (25–29), 1a–3a 50 (49–53), 3a–4a 72 (68–75), 4a–ag1 85 (82–88), ag1–ag2 65 (63–68), ag2–g1 63 (60–65), g1–g2 15 (14–17), g2–g3 22 (21–25), ag–g1 42 (40–45), g3–ps3 16 (15–18), ps2– ps3 16 (14–18).ter Fig. . Venter ter Fig. ; ventralGnathosoma , m 40 (39–42), n 52 (49–54) and two pairs of pilose adoral setae (or1–2), or1 23 (21–24), or2 20 (19–22) , m 40 9–42, n 5Legs  by Eid M. Khan, Jawwad H. Mirza & Hafiz S. Mushtaq.R. (D.) hecmatanaensis; neo = new.The specific epithet is in reference to the similarity of the new species to Raphignathus (D.) neohecmatanaensis sp. nov., belongs to the subgenus Diraphignathus subgen. nov. The new species resembles to R. (D.) hecmatanaensis Khanjani & Ueckermann in having two pairs of setae (d1 and e1) on the interscutal membrane, two setae on the palp femora, and two small plates present posterolateral to the median prodorsal shield. However, the new species differs from R. (D.) hecmatanaensis in the absence of an endopodal shield (vs present), femur IV with two setae (vs three), and leg tarsus I with one solenidion (vs two solenidia).Raphignathuslongimanus (Koch), R.impressus (Koch), R.hispidus (Dugès), and R.deserticula (Trägårdh) because their descriptions are incomplete, and R.lanuginosus Atyeo is excluded, as it was described on the male.Five species are not included in the key. Raphignathus are revised, and for the first time, the genus is divided into four subgenera by considering the morphologically valid, persistent, and prominent characters (Raphignathusevidus, R.hsiufui, and R.johnstoni are considered doubtfully valid. They were described based on size of lateral prodorsal shields and number of setae, but these in these characteristics they resemble immature stages (Neoraphignathus was erected based on only the single type specimen with a restricted geographical region and its description is brief. We suspect it might have been described based on the immature stage of a Raphignathus species, and we suggest that the type species be revised and more specimens collected to confirm the validity of the species and genus.The taxonomic classification of predatory mites of the genus aracters . The usee stages . The mon


Here, we aimed to investigate ERα and ERβ subtypes in the regulation of E2-modulated colonic epithelial HCO3− and Cl− secretion and epithelial restitution. Through physiological and biochemical studies, in combination of genetic knockdown, we showed that ERα attenuated female colonic Cl− secretion but promoted Ca2+-dependent HCO3− secretion via store-operated calcium entry (SOCE) mechanism in mice. However, ERβ attenuated HCO3− secretion by inhibiting Ca2+via the SOCE and inhibiting cAMP via protein kinases. Moreover, ERα but not ERβ promoted epithelial cell restitution via SOCE/Ca2+ signaling. ERα also enhanced cyclin D1, proliferating cell nuclear antigen, and β-catenin expression in normal human colonic epithelial cells. All ERα-mediated biological effects could be attenuated by its selective antagonist and genetic knockdown. Finally, both ERα and ERβ were expressed in human colonic epithelial cells and mouse colonic tissues. We therefore conclude that E2 modulates complex colonic epithelial HCO3− and Cl− secretion via ER subtype-dependent mechanisms and that ERα is specifically responsible for colonic epithelial regeneration. This study provides novel insights into the molecular mechanisms of how ERα and ERβ subtypes orchestrate functional homeostasis of normal colonic epithelial cells.Although it was described previously for estrogen (E Intestinal epithelium either absorbs electrolytes or secretes anions  , etc.3− and Cl− secretion), which in turn is important for nutrient absorption and mucosal protection  in murine duodenal epithelium (2) may protect human duodenum against the acid-induced injury by mediating duodenal HCO3− secretion likely via ER activation, which explains the lower incidence of duodenal ulcer in women than age-matched men  may cause not only imbalance of pH values and electrolytes in whole body but also local disruption of colonic environments, such as epithelial barrier and microbiome  and ERα selective activator propyl pyrazole triol  stimulated rapid duodenal HCO3− secretion; however, ERβ selective activator diarylpropionitrile  did not (A). Second, we tested if there is any regional heterogeneity for the ER subtype-mediated HCO3− secretion in between the duodenum and distal colon. Like in the duodenum, both E2 and PPT stimulated colonic HCO3− secretion but DPN alone did not (B). Moreover, compared to PPT alone, PPT plus DPN did not further stimulate additional colonic HCO3− secretion (B). Thus, ERα activation stimulated HCO3− secretion from the duodenum and distal colon, which has no regional heterogeneity in GI tract. In the subsequent experiments, distal colonic HCO3− secretion was focused because duodenal HCO3− secretion has been extensively studied - or forskolin -stimulated HCO3− secretion. As shown in A and B, the selective ERα activator PPT (10 nM) pretreatment did not affect CCh (100 μM)- or forskolin (20 μM)-induced colonic HCO3− secretion, indicating that ERα-stimulated HCO3− secretion did not utilize the same signaling pathways as CCh or forskolin. In contrast, although the selective ERβ activator DPN (10 nM) did not alter basal colonic HCO3− secretion, it almost abolished both CCh (100 μM)- and forskolin (20 μM)-induced colonic HCO3− secretion (C and D), suggesting that ERβ activation may alter Ca2+- and cAMP-mediated colonic HCO3- secretion.It is well-known that Caecretion , 20, 21.ectively , we examecretion , C and D3− secretion, we further elucidated the underlying mechanisms. Since protein kinase, such as tyrosine kinase (TK), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) pathways play critical roles in intestinal HCO3− secretion , a commonly used TK inhibitor, inhibited basal HCO3− secretion, suggesting its role in basal HCO3− secretion, consistently with the report by Osamu Furukawa et al (3- secretion (E and F), suggesting that ERβ activation inhibits Ca2+- and cAMP-mediated colonic HCO3− secretion likely via TK pathway. Although genistein may also activate ER , suggesting its role in regulating basal HCO3− secretion, which is consistent with the report by Odes HS et al (3− secretion (B and D). Neither rottlerin nor wortmannin affected DPN inhibition of CCh-induced male colonic HCO3− secretion (A and B). In contrast to CCh-induced colonic HCO3− secretion, both rottlerin and wortmannin reversed DPN inhibition of forskolin-induced colonic HCO3− secretion (C and D). These data suggest that ERβ activation inhibits cAMP-mediated colonic HCO3− secretion via PKC and PI3K pathways, which are not involved in ERβ inhibition of Ca2+-mediated HCO3− secretion.Second, rottlerin (10 μM), a selective PKC inhibitor , attenuaecretion , A and CHS et al . HoweverHS et al  did not ecretion , B and Decretion , A and Becretion , C and D3− secretion, we examined their roles in colonic Cl− secretion that is poorly understood although it is known about E2 inhibition of Cl− secretion in rat distal colonic epithelium with a gender-specific mechanism (2 (1 μM), PPT (500 nM), and DPN (500 nM) at high concentrations affected basal colonic short-circuit current (Isc) in both sexes (A and B). Moreover, none of them affected CCh- and forskolin-induced male colonic Isc (C and D), consistently with the previous reports that E2 did not alter colonic epithelial Cl− secretion in male rats  significantly inhibited CCh- and forskolin-induced Isc of female mouse colon, which is consistent with the previous reports that E2 inhibited colonic epithelial Cl− secretion through KCNQ1 channels in female rats . Taken together, ERα subtype is specifically responsible for the inhibition of female colonic Isc.We further examined the role of ER subtypes in female colonic Clale rats , 15, 28.se colon , G and H2+ signaling triggers intestinal HCO3− secretion  plays a pivotal role in controlling Ca2+ signaling. Initially, we applied single cell Ca2+ imaging to characterize the SOCE in human colonic epithelial cell line (HCoEpiC). Cyclopiazonic acid , an inhibitor of the sarcoendoplasmic reticulum Ca2+ ATPase and a commonly used SOCE activator, significantly induced free cytoplasmic Ca2+ [Ca2+]cyt in Ca2+-free solutions and then a sustained [Ca2+]cyt increase in Ca2+-containing solutions, suggesting that intracellular Ca2+ release causes extracellular Ca2+ influx in HCoEpiC  (A). Pretreatment with three different SOCE blockers, GSK7975A (10 μM) (B–J), verifying SOCE function in HCoEpiC.It is well documented that Caecretion , 20, 29,e, SOCE) A. Pretre (10 μM) , ML-9 (1 (10 μM) , and SKF (10 μM) , markedl (10 μM) , B–J, ve2+ transient produced by CPA in Ca2+-free solutions is presumably Ca2+ release from stores, it was reduced by pretreatment with SOCE blockers . We assume it is because the SOCE is important to refill the intracellular Ca2+ store, and Ca2+ release from stores would be reduced by pretreatment with SOCE blockers. To test this possibility, we added SOCE blockers after the store depletion but before extracellular Ca2+ influx. Indeed, SKF96365 (50 μM) markedly reduced CPA-induced Ca2+ influx (K–M). These data verify not only SOCE function per se but also its important role in refilling intracellular Ca2+ store in HCoEpiC.Although the initial Ca+ influx , K–M. Th2+ signaling. ERα selective activator PPT (10 nM) significantly stimulated [Ca2+]cyt in Ca2+-containing solutions (A and C), but ERβ selective activator DPN (10–500 nM) did not alter basal [Ca2+]cyt (B and C). However, DPN (10 nM) significantly inhibited CPA (5 μM)-induced SOCE (D–G). Together, these data suggest that ERα triggers Ca2+ signaling, but ERβ inhibits Ca2+ signaling likely via the SOCE.After characterizing the SOCE in HCoEpiC, we next examined whether ER stimulates colonic epithelial Caolutions , A and CCa2+]cyt , B and Cced SOCE , D–G. To2+ signaling in HCoEpiC. Like SOCE inducer CPA, PPT (10 nM) significantly stimulated [Ca2+]cyt in Ca2+-free solutions and then a sustained [Ca2+]cyt increase in Ca2+-containing solutions (A). Further studies showed that either MPP dihydrochloride (MPP) at 5 to 10 μM (B–E), a ERα selective inhibitor, or ML-9 (100 μM) (F–H) and SKF96365 (50 μM) (I–K), two different inhibitors of the SOCE, markedly eliminated PPT-induced Ca2+ release and Ca2+ influx, suggesting that ERα activation triggers [Ca2+]cytvia the SOCE. Moreover, SKF96365 (50 μM) markedly reduced PPT-induced Ca2+ entry when it was added after the store depletion but before extracellular Ca2+ entry (L–N). Finally, after shERα was applied to successfully knock down the protein expression of ERα in HCoEpiC (O), PPT-induced [Ca2+]cyt was significantly attenuated by shERα-3 as well (P–S), verifying ERα activation of the SOCE in colonic epithelial cells.We further elucidated the underlying mechanisms of ERα-induced Caolutions A. Furtheto 10 μM , B–E, a (100 μM) , F–H and (50 μM) , I–K, tw2+ entry , L–N. Fi HCoEpiC O, PPT-in as well , P–S, veA), which was attenuated by ERα selective inhibitor MPP  and shERα , but not by ERβ selective inhibitor PHTPP (5 μM) . However, DPN at 1 to 50 nM did not affect proliferation of HCoEpiC . Finally, [Ca2+]cyt chelator BAPTA-AM (1 μM) also inhibited PPT-induced cell proliferation . MPP (1 μM), PHTPP (5 μM), and BAPTA-AM (1 μM) were chosen at these concentrations because they per se did not directly alter HCoEpiC proliferation .Since proliferation and migration of epithelial cells are critical for the restitution of injured intestinal epithelium , 34, we Fig. 8I, the pretreatment with PPT (10 nM) for 48 h enhanced the expression of cyclin D1, PCNA, and β-catenin. However, DPN (10 nM) did not affect their expression . Therefore, ERα promotes Ca2+-dependent proliferation of HCoEpiC.Since cyclin D1, proliferating cell nuclear antigen (PCNA), and β-catenin play crucial roles in enterocyte proliferation , 36, we Fig. 8A and B, PPT at 10 to 50 nM promoted cell migration, but DPN (10–50 nM) did not affect it. Moreover, ERα selective inhibitor MPP (1 μM) and BAPTA-AM (1 μM) and shERα abolished PPT-induced cell migration (C–E). Therefore, ERα also promotes Ca2+-dependent migration of HCoEpiC.Next, we performed cell scratch assays to examine the roles of ER subtypes in HCoEpiC migration. As shown in igration , C–E. ThA and B, both ERα and ERβ mRNA expression were detected in HCoEpiC. Western blots analysis further confirmed ERα and ERβ protein expression in HCoEpiC like in HUVEC (C and D). Moreover, we also performed immunofluorescence to examine the expression and localization of ER subtypes in HCoEpiC. As shown in Figure 10E, ERα proteins were predominately expressed in the cytoplasm and nucleus, but ERβ were mostly expressed in the cytoplasm compared to the nucleus. However, the immunofluorescence staining was not observed without the primary antibodies against ERα and ERβ in the negative control, indicating specific staining on these proteins in HCoEpiC.Since there is no information available in literature on ER subtype expression in HCoEpiC, we examined ER subtype expression in HCoEpiC and human umbilical vein endothelial cells (HUVECs) as a positive control , 38. As in HUVEC , C and DF and G, both proteins were expressed in native colonic epithelia in male and female mice, further supporting both ERα and ERβ expression in mice  ERβ attenuates E2-mediated HCO3− secretion by inhibiting SOCE/Ca2+ signaling and by inhibiting cAMP signaling via activation of TK, PKC, and PI3K, (4) Erα but not Erβ promotes E2-mediated epithelial cell restitution via the SOCE, and (5) ERα enhances the expression of cyclin D1, PCNA, and β-catenin in HCoEpiC.Although sex-based differences in Eescribed , 15, 16,2 stimulated murine duodenal HCO3− secretion  to exert various biological actions through genomic and nongenomic pathways  without triggering excessive Cl− secretion that might induce the unwanted diarrheal side effects.We previously showed that activation of the Cadecrease ; the endignaling , 19. Det2+-dependent cell proliferation and migration, leading to colonic epithelial restitution; a process that may be beneficial to colonic disease like UC. Moreover, we found that activation of ERα but not ERβ increases protein levels of cyclin D1, PCNA, and β-catenin, indicating increased proliferation and migration.While epithelial restitution plays a critical role in the healing process of intestinal mucosa , little 2 modulates colonic epithelial ion transports and epithelial restitution via ER subtype-dependent mechanisms. Since ERα and ERβ subtypes usually play opposite roles in regulating colonic epithelial function, E2 may coordinate different ER subtypes to orchestrate functional homeostasis of colonic epithelial cells. Disruption of ERα- and ERβ-coordinated gut homeostasis may have underpinnings behind sex-based differences in GI disease, especially those involving epithelial injury and repair. While this requires further study, we have provided new insights into the cellular mechanisms of E2-mediated colonic epithelial ion transports and epithelial repair via different ER subtypes.In conclusion, we demonstrate for the first time that E2.Human colonic epithelial cells (HCoEpiC) (Cat #2950) and HUVEC (Cat #8000) were purchased from ScienCell Research Laboratory. Cells were cultured in DMEM-HIGH GLUCOSE or RPMI-1640 (HyClone) supplemented with 10% fetal bovine serum (HyClone) in a 37 °C humidified atmosphere containing 5% COAll animal studies were approved by the Ethics Committee of the Qingdao University Medical College. All animal care and experimental procedures complied with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health. Animal studies are reported in compliance with the ARRIVE guidelines . The C572). The experiments were performed under continuous short-circuited conditions , and luminal pH was maintained at 7.0 by the continuous infusion of 5 mm HCl under the automatic control of a pH-stat system . The volume of the titrant infused per unit time was used to quantitate HCO3− secretion. Measurements were recorded at 5-min intervals, and mean values for consecutive 5- or 10-min periods were averaged. The rate of luminal HCO3− secretion is expressed as micromoles per square centimeter per hour. The transepithelial Iscs were measured via an automatic voltage clamp, in which μA was used for the original recordings, but μA·cm-2 was used for summary data. After a 30 min basal period, inhibitor or control vehicle was added for another 30 min, followed by addition of stimulus to both sides of the tissue. Electrophysiological parameters and HCO3− secretion were then recorded for 60 min. The mucosal solution contained the following (mM): 115 NaCl, 25 sodium-D-gluconate, 5.2 potassium-D-gluconate, 1.2 CaCl2, 1.2 MgCl2, and 10 mannitol. The serosal solution contained the following (mM): 115 NaCl, 25 NaHCO3, 2.2 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, 0.8 KH2PO4, 10 glucose, and 0.01 indomethacin. The osmolalities for both solutions were ∼300 mosmol·kg-1 of H2O.Ussing chamber experiments were performed as previously described , 53. C5750 and the data from others’ reports.The maximal volumes of concentrated stock solutions of all compounds added to 3 ml Ussing chamber solutions were less than 30 μl. All added compounds did not alter pH values in chamber solutions, which is consistent with other report . The con2+]cyt imaging experiments were performed as previously described . Fluorescence signals were imaged using an intensified CCD camera (ICCD200) attached to an inverted fluorescence microscope (Leica) and recorded with MetaFluor software . Images were acquired every 3 s. The dual wavelength excitation method for the measurement of fura-2 fluorescence was used. The excitation wavelengths were 340 and 380 nm, and the emitted fluorescence was collected at 510 nm. [Ca2+]cyt was presented as fluorescence ratios (F340/F380) after background subtraction. The PSS contained the following: 140 mM Na+, 5 mM K+, 2 mM Ca2+, 147 mM Cl−, 10 mM Hepes and 10 mM glucose (pH 7.4). The 0 Ca2+ solution (0 Ca2+) contained the following: 140 mM Na+, 5 mM K+, 145 mM Cl−, 0.5 mM EGTA, 10 mM Hepes, and 10 mM glucose (pH 7.4). The osmolality for the solution was ∼300 mosmol.kg−1 of H2O.[Caescribed . BrieflyWestern blotting was performed as previously described , 54. BriQuantitative real-time PCR was performed as previously described , 55. BriERα: 5′- GGGAAGTATGGCTATGGAATCTG-3′ (Forward),ERα: 5′- TGGCTGGACACATATAGTCGTT-3′ (Reverse).ERβ: 5′-AGCACGGCTCCATATACATACC-3′ (Forward),ERβ: 5′- TGGACCACTAAAGGAGAAAGGT-3′ (Reverse).GAPDH: 5′- ACAACTTTGGTATCGTGGAAGG-3′ (Forward),GAPDH: 5′- GCCATCACGCCACAGTTTC-3′ (Reverse).After fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and blocked with bovine serum albumin, the HCoEpiC were incubated with anti-ERα (1:100)  or anti-ERβ (1:200)  antibody overnight at 4 °C. Then, the cells were incubated with Cy3 labeled anti-rabbit secondary antibody . Finally, nuclei were stained with DAPI for 5 min, and images were captured using confocal microscope.Cell proliferation assay was performed as previously described . BrieflyLentiviruses were purchased from Sangon Biotech Co, Ltd. The sequences for ERα shRNA and NC were as follows: shRNA-1 (5′-TTGTGTGCCTCAAATCTATTA-3′), shRNA-2 (5′-AGGCCAAATTCAGATAATCGA-3′), shRNA-3 (5′- CAGGTCCACCTTCTAGAATGT-3′), shNC (5′- TTCTCCGAACGTGTCACGT -3′). HCoEpiC were infected with lentiviruses according to the protocol of the manufacturer.2, forskolin, genistein, rottlerin, wortmannin, GSK-7975A, ML-9, SKF-96365, CPA, and BAPTA-AM were purchased from MedChemExpress and dissolved in DMSO. CCh and indomethacin were purchased from Sigma-Aldrich. CCh was dissolved in ultrapure water. Indomethacin was dissolved in anhydrous alcohol. All salts were supplied by Sangon Biotech and dissolved in ultrapure water.PPT, DPN, MPP, PHTPP, ESCR_002798, USA) software was used for analysis and graph generation. All results shown are means ± SD. All experiments were repeated at least three times. The number of biological repeats (n) in the figures is the number of individual tissues or cells obtained from at least three mice or three independent experiments. The statistical significance of differences in the means of experimental groups was determined using Student's t test or one-way ANOVA followed by post hoc test for multiple pairwise comparisons. Significant differences (∗p < 0.05) are expressed in the figures and figure legends.GraphPad Prism 7.0 (RRID: The data supporting the findings of this study are available within the article.The authors declare no conflicts of interest with the content of this article.


Xenopus oocytes in 96-well microtiter plates. We employed guinea pig, human and Xenopus laevis ENaC orthologs that display specific magnitudes of SSI. While demonstrating some limitations over traditional TEVC systems with customized perfusion chambers, the automated TEVC system was able to detect the established SSI characteristics of the employed ENaC orthologs. We were able to confirm a reduced SSI in a gene variant, leading to C479R substitution in the human α-ENaC subunit that has been reported in Liddle syndrome. In conclusion, automated TEVC in Xenopus oocytes can detect SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended.The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene variants associated with hypertension, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC activity and SSI. We evaluated a commercially available automated two-electrode voltage-clamp (TEVC) system that records transmembrane currents of ENaC-expressing  The epithelial sodium channel (ENaC) is a sodium-selective ion channel in the apical membrane of the epithelial cells of the aldosterone-sensitive distal nephron, lung and colon, where it mediates the rate-limiting step for transepithelial sodium absorption. ENaC is a key component of the renin–angiotensin–aldosterone system, and hormonally controlled ENaC-activity matches dietary sodium intake to its excretion rate. ENaC is therefore a key protein in the control of sodium homeostasis. Since plasma sodium concentrations correlate with the extracellular fluid volume and systolic blood pressure , ENaC coA reduction in dietary sodium intake has been suggested to lower blood pressure; however, individual blood pressure correlations with dietary sodium intake vary. Some individuals respond strongly to an increase in dietary sodium intake with increased blood pressure, a condition described as salt-sensitive hypertension . Recent O) [SCNN1A gene), β-ENaC (SCNN1B gene), γ-ENaC (SCNN1G gene) and δ-ENaC (SCNN1D gene). Recent cryo-electron microscopy-derived structures revealed that each subunit resembles a clenched hand holding a central ball of beta-sheets [Xenopus oocytes have shown that αβγ- and δβγ-subunit assemblies form functional ENaCs [O [O [ENaC activity is affected by the channel subunit assembly as well as intra- and extracellular stimuli controlling its membrane abundance and open probability (PO) . There aa-sheets ,10. The al ENaCs ,12, withal ENaCs ,14,15. Aal ENaCs  and trigENaCs [O . This meENaCs [O . Many adCs [O [O . SSI is Xenopus oocytes using the two-electrode voltage-clamp (TEVC) technique . Here, Xenopus oocytes in 96-well microtiter plates [With an increasing number of identified ENaC gene variants, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC SSI. We therefore aimed to establish a simple assay allowing us quantify SSI using a commercially available automated TEVC system recording transmembrane currents of ENaC expressing r plates . While tXenopus laevis ENaC subunits  were present in the pTNT vector . Plasmids were cleaned using the Monarch PCR and DNA Cleanup Kit  according to the manufacturer’s instructions, eluted in nuclease-free water and stored at −20 °C. Cleaned plasmids served as templates for cRNA synthesis using the mMESSAGE mMACHINE T7 Kit  in accordance with the manufacturer’s instructions. The cRNA was treated with TURBO DNase (New England Biolabs) to remove the template DNA, cleaned using the Monarch® RNA Cleanup Kit (New England Biolabs) or MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific) according to manufacturer’s instructions and eluted in nuclease-free water. The subunit cRNAs were combined as αβγ- or δβγ-ENaC combinations at a concentration of 5 ng/µL per ENaC subunit and stored at −80 °C.Coding DNA sequences of human, guinea pig and E. coli (New England Biolabs) and prepared using the Monarch® Plasmid DNA Miniprep Kit (New England Biolabs) according to manufacturer’s instructions. Successful C479R substitution was confirmed using the sequencing of plasmid DNA, and the corresponding plasmids were employed as templates for cRNA synthesis as described in Cysteine 479 in the human α-ENaC subunit was substituted by arginine using the Q5 Site-directed Mutagenesis Kit (New England Biolabs) according to manufacturer’s instructions. The forward primer sequence was 5’-CCGGAAGCCAcggAGCGTGACCA-3’, and the reverse primer sequence was 5’-CACTTGGTGAAACAGCCCAGG-3’. After DpnI digest of the template plasmid DNA, the mutated plasmids were heat-transformed into NEB 5-alpha Competent Xenopus laevis oocytes were purchased from Ecocyte Bioscience  and stored in Modified Barth’s Solution 2, 0.41 CaCl2 and 10 HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH = 7.5, and supplemented with 20 µg/mL gentamycin) at 4 °C until use. The MBS was exchanged after every 48 h. For injection of cRNA, the oocytes were placed into a N-Methyl-D-Glutamine Oocyte Ringer Solution  10 NaCl, 1 KCl, 2 CaCl2, 2.5 Na+-pyruvate, 80 NMDG, 5 HEPES, pH = 7.4, and supplemented with 20 µg/mL gentamycin). The oocytes were manually injected with 18.4 nL of cRNA using a Nanoject II . Oocytes were stored in NMDG-ORS at 16 °C for 24 h.Stage V/VI 2, 89 NMDG, 5 HEPES, pH = 7.4). Oocytes were clamped at a holding potential of −60 mV, and whole-cell transmembrane current signals (IM) were recorded at a sampling rate of 20 Hz. ENaC SSI was in the range of several seconds [2, 5 HEPES, pH = 7.4) using the integrated Roboocyte 2 perfusion system. The perfusion rate yielding robust detection of SSI was tested in the initial experiments and eventually set to 6 mL/min  unless otherwise stated. Recordings were performed at buffer temperatures of 19 °C (room temperature) or 24 °C. To achieve 24 °C at the oocytes, the buffer reservoirs were placed in a water bath, and the outflow temperature at the measuring head was controlled. All recordings were performed using the Roboocyte 2 automated recording mode according to manufacturer’s instructions.Automated TEVC recordings were performed using a Roboocyte 2 and corresponding measuring heads carrying the recording and bath electrodes . The capillaries of the recording electrodes were replaced by microelectrodes that were pulled from borosilicate glass capillaries  using a PP-83  or DMZ  puller. Recording electrodes were filled with 3 M KCl. Resistance of the current and voltage electrodes ranged between 100 and 2000 Ohm. Oocytes were placed in a 96-well microtiter plate containing 200 µL of low-sodium ORS (containing (in mM) 1 NaCl, 1 KCl, 2 CaCl seconds ,22 and tAll buffer ingredients were purchased from Carl Roth , except for NMDG, which was obtained from SIGMA, and sodium pyruvate and gentamycin which were obtained from Thermo Fisher Scientific . The ENaC inhibitor amiloride  was prepared as a 100 mM stock solution in water and employed in a final concentration of 100 μM to assure maximum block of both αβγ-ENaC and δβγ-ENaC isoforms.−4 kJ·mol−1·Å−1.Mutation C479R was introduced in silico into the cryo-EM structure of the α subunit of the extracellular domain of αβγ-ENaC (PDB: 6WTH)  using thXenopus oocyte deliveries and the number of experimental repeats is given as n. Normal distribution of data was analyzed using the D’Agostino and Person test and corresponding parametric or non-parametric statistical analyses were performed as specified in the figure legends. All statistical analyses were performed using the GraphPad Prism version 9.5.1 , and a p-value ≤ 0.05 was considered statistically significant.Data are presented as means ± standard deviation (SD). Experiments were performed using at least two independent Xenopus laevis αβγ- or δβγ-ENaCs, since these ENaC orthologs display characteristic magnitudes of SSI. We performed experiments at 19 °C and 24 °C since SSI is a temperature-dependent mechanism. To record SSI, ENaC-expressing oocytes were first perfused in a low-sodium ORS containing 1 mM NaCl for 30 s. The perfusion was subsequently switched to high-sodium ORS containing 90 mM NaCl for 3 min. Afterwards, the ENaC inhibitor amiloride (100 μM) was added to the high-sodium ORS to determine the baseline for ENaC-mediated inward currents. SSI was detectable in recordings from oocytes expressing guinea pig αβγ-ENaC (n = 12), and it was 65.66 ± 10.95 % (n = 17) at 24 °C. Based on manual TEVC recordings, we have previously reported that guinea pig δβγ-ENaC has a strongly reduced SSI of approx. 16 % [n = 9) at 19 °C, and −2.40 ± 8.36 % (n = 11) at 24 °C  at 19 °C and 67.04 ± 11.96 % (n = 9) at 24 °C, whereas SSI of Xenopus laevis αβγ-ENaC was significantly lower with 10.57 ± 11.33 % (n = 11) at 19 °C, and 26.19 ± 2.8.09 % (n = 8) at 24 °C  at 19 °C and of 55.16 ± 9.36 % (n = 14) at 24 °C. SSI of human δβγ-ENaC was significantly lower (24.37 ± 9.21 % (n = 17) at 19 °C, and 5.37 ± 14.33 % (n = 11) at 24 °C)  at 19 °C and 25.74 ± 17.02 % (n = 13) at 24 °C. In summary, these data demonstrate that the automated TEVC on ENaC-expressing Xenopus oocytes in 96 well microtiter plates can be employed to investigate the effects of ENaC subunit gene variants on SSI.Using automated TEVC, we recorded SSI of human αβγ-ENaC of 39.24 ± 11.97 % (t 24 °C) . Human αXenopus oocytes using automated TEVC electrophysiology. SSI is a regulatory mechanism adjusting ENaC PO to the extracellular sodium concentrations. A putative sodium binding site was identified in an ‘acidic cleft’ located between the ‘knuckle’ and ‘finger’ domains of the extracellular region in the mammalian α-ENaC [Xenopus δ-ENaC subunits [O state [In this study, we aimed to record ENaC SSI in n α-ENaC ,16 and Xsubunits . It is s[O state ,16,30. AXenopus laevis ENaC orthologs and confirmed, consistent with previous publications [Xenopus laevis) SSI in δβγ-ENaCs compared with the corresponding αβγ-ENaCs. However, the herein-employed system shows limitations over traditional TEVC systems with optimized perfusion chambers, since SSI in guinea pig δβγ-ENaCs was almost absent in the automated TEVC recordings, whereas 16% SSI could previously be recorded using traditional TEVC systems [Recording of SSI requires the separation of sodium-mediated ion currents from the inhibition of ion currents due to sodium binding to ENaC. We have previously recorded SSI in ENaC-expressing oocytes using traditional TEVC systems ,30. In tications ,20, the  systems . To exclC479Rβγ-ENaC displayed larger currents than wild type αβγ-ENaC when expressed in Xenopus oocytes [O of αC479Rβγ-ENaC. Furthermore, a reduction in SSI was described for this ENaC variant [C479Rβγ-ENaC [C479Rβγ-ENaC is consistent with a reduced sensitivity to trypsin [C479Rβγ-ENaC yielded low transmembrane currents in our experiments; however, our experiments were not designed to compare overall transmembrane current signals, and SSI is independent of ENaC expression levels. Reduced SSI hampers ENaC from decreasing its PO under conditions of high extracellular sodium concentrations. Consequently, enhanced sodium retention in the distal nephron provides a mechanistic explanation for hypertension in patients carrying this ENaC variant.We therefore aimed to record changes in SSI in a human α-ENaC variant C479R) that was identified in a family with Liddle syndrome . Salih e oocytes . The aut variant . Molecul variant ,31,32,33R that wa variant . In agre variant ,21, we dRβγ-ENaC , suggest trypsin  since pr trypsin ,35. NotaXenopus oocytes can reveal alterations in SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended.In conclusion, automated TEVC in


H-deprotonation of a 10-aza-9-borabenzo[h]quinoline yields a bidentate ligand that was used for the synthesis of a 2:1 coordination complex with zinc. Its crystal packing is dominated by intense intra- and intermolecular π–π stacking interactions.The N 20H18BN2)2] (ZnL2), is an overall uncharged chelate that consists of two units of an NH-deprotonated 10-aza-9-borabenzo[h]quinoline ligand (L) per ZnII center. It was synthesized in two steps by treating the protonated ligand HL with lithium bis(trimethylsilyl)amide and further conversion with diethylzinc. Its asymmetric unit comprises one ZnL fragment; the molecule is completed by application of inversion symmetry at Zn. Due to the fourfold coordination with nitrogen atoms, the zinc(II) ion is located in a distorted tetrahedral environment. Besides the relatively short N—Zn bonds, ZnL2 is characterized by the significant protrusion of the central ion from the plane of the ligand backbone. The crystal structure is consolidated by intra- and intermolecular π–π stacking interactions, while the polarized B—N bond is barely involved in any close atom contacts.The title compound, [Zn(C Several studies highlighted the BN-induced tailored adjustment of chemical, physical and optical properties, enabling the application of such heteroaromatics for instance as white-emitting layers in organic light-emitting diodes , containing one 1,2-azaborininyl- and one pyridyl subunit with both nitrogen atoms beneficially preorganized for chelation was presented. In the context of this communication, we report on the synthesis and crystal structure of the 2:1 coordination complex of ligand L with zinc(II).In a previous study  is illustrated in Fig. 1C2/c centrosymmetric space group with one zinc(II) cation and one ligand molecule in the asymmetric unit, being completed by the application of inversion symmetry at ZnII. The latter is fourfold coordinated by two types of N donors, namely the azaborinine and pyridine subunits comprised in the BN-benzo[h]quinoline. This results in a significantly distorted tetrahedral configuration  are oriented almost perpendicularly to each other, with an angle between their mean planes of 79.41 (4)°.The molecular structure of the title compound  and their pseudo-symmetrical counterparts at the bottom right of the two-dimensional fingerprint plot correspond to C—H⋯π interactions. These are also mapped by several red spots on the Hirshfeld surface  revealed that 654 crystal structures of six-membered carbocycles with 1-aza-2-bora substitution patterns have been reported. Among these, 101 structures comprising B-mesityl substituents have been deposited, which involves aromatic 1,2-azaborinine subunits for the most part. The crystal structures of 13 compounds with 1,2-azaborinine substructures and nitrogen–metal bonds have been published, of which different lithium solvates as well as potassium, beryllium, aluminum, gallium and tin complexes are included in one publication  was added, before a solution of diethylzinc  was added via a syringe. The mixture was heated to 428 K for 17 h while stirring. In a glove box, the volatiles were removed under reduced pressure. The residue was extracted with n-hexane (3 × 2 mL) and the solvent was removed. The crude product was dissolved in THF (500 µL) and n-hexane was allowed to diffuse into this solution over the course of 3 d. The light-yellow product  was obtained as air-sensitive crystals suitable for X-ray diffraction analysis by repeating this process twice. 1H NMR : δ = 8.37–8.32 , 8.07 , 7.79 , 7.38–7.35 , 7.33 , 6.90 , 6.04 , 5.61 , 1.83 , 1.79 , 1.40  ppm. 13C{1H} NMR : δ = 147.3 (C3), 145.8 (C1), 143.3 (C2), 143.0 (C10), 139.2 (C17), 139.2 (C5), 139.1 (C13), 134.8 (C15), 134.5 (C11), 131.2 (C8), 129.0 (C6), 127.1 (C14), 126.4 (C16), 126.1 (C9), 122.3 (C4), 116.1 (C7), 24.0 (C18), 23.8 (C20), 21.3 (C19) ppm. 11B{1H} NMR : δ = 38.6 ppm. MS (EI): m/z 658.3 (3%) [2ZnL]+, 298.2 (100%) [HL]+. HR-MS (EI): m/z calculated for C40H36B2N4Zn+ 658.24259, found 658.24253 . UV/Vis: λabs = 296, 343, 358 nm. Fluorescence: λfl = 488 nm (λexc = 350 nm). Further experimental details can be found in the Supporting Information.The synthesis of 6.3). Isotropic displacement parameters (Uiso) of these H atoms were fixed to 1.2 (C—H) or 1.5 (C—H3) of the values of the parent carbon atoms. Idealized methyl groups  were allowed to rotate.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023009192/dj2071sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023009192/dj2071Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989023009192/dj2071sup4.pdfExperimental details, unit cell packing views, NMR spectra, UV vis and fluorescence spectra. DOI: 2302109CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


RXR heterodimerize with type II nuclear receptors, including RAR, to regulate a vast gene array. Despite much effort, 9cRA has not been identified as an endogenous retinoid, other than in pancreas. By revising tissue analysis methods, 9cRA quantification by liquid chromatography-tandem mass spectrometry becomes possible in all mouse tissues analyzed. 9cRA occurs in concentrations similar to or greater than atRA. Fasting increases 9cRA in white and brown adipose, brain and pancreas, while increasing atRA in white adipose, liver and pancreas. 9cRA supports FoxO1 actions in pancreas β-cells and counteracts glucose actions that lead to glucotoxicity; in part by inducing Atg7 mRNA, which encodes the key enzyme essential for autophagy. Glucose suppresses 9cRA biosynthesis in the β-cell lines 832/13 and MIN6. Glucose reduces 9cRA biosynthesis in 832/13 cells by inhibiting Rdh5 transcription, unconnected to insulin, through cAMP and Akt, and inhibiting FoxO1. Through adapting tissue specifically to fasting, 9cRA would act independent of atRA. Widespread occurrence of 9cRA in vivo, and its self-sufficient adaptation to energy status, provides new perspectives into regulation of energy balance, attenuation of insulin and glucose actions, regulation of type II nuclear receptors, and retinoid biology.9- In contrast, 9cRA rises to abnormally high levels in glucose-intolerant mice with β-cell hypertrophy, including mice with diet-induced obesity, as well as ob/ob and db/db mice. These data established 9cRA as a pancreatic autacoid that modifies GSIS, and beg the question whether 9cRA occurs endogenously in other tissues.The question of 9cRA as an endogenous retinoid was partially resolved by its quantitation in pancreas in concentrations similar to or higher than atRA , 18. Pantabolism , 20, 21.in vivo would inform its therapeutic applications. As the drug alitretinoin, 9cRA exerts ameliorating effects on skin cancers, such as cutaneous T-cell lymphoma and Kaposi’s sarcoma, therapy-resistant lichen planus, and chronic hand eczema . The original saline/ethanol homogenization resulted in atRA isomerizing partially into 13cRA (D) and 9cRA isomerizing partially into atRA  and 13-cis-RA (13cRA) (F). In contrast, methanol-based homogenization maintained the integrity of extracted atRA and 9cRA (E and G). To verify reliability of the methanol-based procedure, we added 9cRA to a pancreas homogenate, extracted the sample, and compared it to a sample without added 9cRA  protocols for quantifying RA isomers in serum and tissues by returning to tissue homogenization with methanol, rather than with saline/ethanol, and modifying LC elution to improve resolution . The orito 13cRA D and 9cR (13cRA) F. In conand 9cRA , E and Gded 9cRA . The metm/z 301. Characteristic product ions were present at m/z 283, 255, 205, and 123. These ions identified the molecular weight as an RA, with loss of water, loss of the carbonyl group, presence of the side-chain plus a portion of the β-ionone ring and the β-ionone ring, respectively.To further confirm 9cRA authenticity, 46 pooled mouse pancreata were analyzed after methanol homogenization, followed by extraction with hexane. Endogenous 9cRA was resolved from other RA isomers by high-performance LC. Reinjection of the collected 9cRA fraction for a Q3 mass scan generated a mass spectrum consistent with that of a 9cRA standard  38). Th. Th38). A). First, we tested derivatizing atRA with 3-NPH using N-(3-dimethylaminopropryl)-N′-carbodiimide as catalyst. Mass transitions of atRA-3-NPH were determined by Q1 and Q3 scans (B and C).Derivatization with 3-nitropenylhydrazine (3-NPH) provided another independent confirmation of 9cRA authenticity A. First,Q3 scans . Having Q3 scans , B and Cm/z 301 by Q1, and quantification of the characteristic product ion at m/z 205 in Q3. The ethanol protocol provided results equivalent to the original reports authenticating 9cRA in pancreas and presence of 9,13dcRA, but not 13cRA (A), and failing to observe 9cRA above the limits of quantification outside pancreas (B). The methanol-based protocol showed 9,13dcRA only in serum, confirming a previous report . The medium glucose concentration did not affect the amount of intracellular all-trans-retinol. Cells treated with 9-cis-retinol had both all-trans-retinol and 9-cis-retinol present. Fifteen millimolar glucose decreased the amount of all-trans-retinol by 49%, whereas it increased 9-cis-retinol by 1.8-fold compared to the 3 mM glucose-treated group. All-trans-retinol generated mostly all-trans-retinal and about half as much 9-cis-retinal (B). In contrast, 9-cis-retinol generated predominantly 9-cis-retinal, with minor amounts of all-trans-retinal. Retinal concentrations with 3 mM glucose exceeded those of 15 mM glucose, with exception of 9-cis-retinal during all-trans-retinol treatment. Both atRA and 9cRA were detected with all-trans-retinol substrate, with much higher atRA than 9cRA (C). 9-cis-retinol generated similar levels of atRA and 9cRA. Fifteen millimolar glucose decreased 9cRA biosynthesis from 9-cis-retinol ∼40%.Mouse models with depleted β-cell populations indicate that β-cells account for ∼70% of pancreas 9cRA. Moreover the β-cell model 832/13 produces 9cRA from 9--retinol . GSIS in-retinol , 44, 45.-retinol . Exposur-retinol A. The me-retinal B. In conhan 9cRA C. 9-cis-Rdh5 widely and recombinant Rdh5 catalyzes conversion of 9-cis-retinol into 9cRA efficiently . Reductions after refeeding were not limited to pancreas (B). Six hours after refeeding Rdh5 mRNA decreased in the liver, kidney, and brown adipose tissue by 68, 93, and 92%, respectively, but increased 15% in eWAT and 80% in retinal pigment epithelium. Regulation of Rdh5 mRNA by glucose was tested in 832/13 cells (C). Glucose (15 mM) reduced Rdh5 mRNA ∼60% relative to 3 mM glucose after 6 h, without affecting Rdh10. We also evaluated the mouse pancreas cell line MIN6, another model of β-cell function (D) (Rdh5 mRNA in the presence of 3 mM glucose exceeded that of 15 mM glucose by 1.8-fold. Actinomycin D (ActD) prevented the increase in Rdh5 mRNA prompted by 3 mM glucose, indicating that low glucose permits activation of Rdh5 transcription (E). ActD did not decrease Rdh5 mRNA further than 15 mM glucose, indicating 15 mM glucose prevents Rdh5 transcription. To confirm that Rdh5 catalyzes 9cRA biosynthesis, 832/13 cells were transfected with Rdh5 (F). Rdh5 overexpressing cells increased Rdh5 mRNA and net conversion of 9-cis-retinol into 9cRA.Tissues express iciently . Decreashe liver , 41, 42.ectively A. Reductpancreas B. Six ho13 cells C. Glucosfunction D 47). R. RRdh5 wcription E. ActD dith Rdh5 F. Rdh5 oA). Insulin had no effect on Rdh5 mRNA, regardless of the medium glucose concentration. We then applied pharmacological approaches to major aspects of glucose signaling to identify pathways linking glucose to Rdh5 expression. Glucose increases cAMP in β-cells and 3-isobutyl-l-methylxanthine (IBMX) increases cAMP and cGMP . Glucagon-like peptide-1 (GLP-1) targets β-cells, among others, and increases β-cell cAMP , which encodes transcription factors that regulate gene expression in β-cells . Riociguat suppressed Rdh5 transcription only in the presence of low glucose conditions, indicating that cGMP suppresses Rdh5 transcription only during low glucose and confirming cAMP suppresses Rdh5 transcription in the presence of high glucose. Next, we investigated signaling downstream of glucose-ATP-Ca2+. During low glucose, the FoxO1 inhibitor AS1842856 reduced Rdh5 expression by ∼50%, that is, to the same extent as high glucose (E). With high glucose, the FoxO1 inhibitor suppressed Rdh5 expression another 50%. These data indicate that FoxO1 serves as the proximate inducer of Rdh5. During low glucose, neither the CaMK inhibitor nor the Akt inhibitor affected Rdh5 expression. In 15 mM glucose, CaMK and Akt inhibitors prevented Rdh5 repression, indicating involvement of both in Rdh5 transcription. This again indicates a difference in mechanism between low and high glucose. Inhibitors of PKA, GSK3β, or rapamycin had no effect on high glucose-mediated Rdh5 repression (G). These data suggest that CaMK transmits glucose signaling through Akt and Foxo1 to Rdh5.We tested insulin to determine the possibility of negative feedback on 9cRA biosynthesis A. Insuliand cGMP . IBMX deectively B. Glucagell cAMP . The GLP β-cells . IBMX inctivator D. Riocig glucose E. With hpression G. These Atg7 promoter (Atg7 mRNA relative to lower glucose (F). 9cRA restored Atg7 mRNA during high glucose to that of low glucose levels.Autophagy maintains β-cell mass and function . RAR indpromoter , 53. Hig glucose F. 9cRA rm/z 301 [M + H+], followed by verification of a characteristic product ion in Q3 at m/z 205; (2) a full-scan mass spectrum of endogenous 9cRA revealing the typical molecular ion and product ions after isolation by high-resolution LC; (3) derivatization and comigration of the endogenous 9cRA derivative with a synthetic 9cRA derivative during high-resolution LC. Each of these procedures alone would have authenticated the analyte as 9cRA. The three together should resolve unmistakably the presence of 9cRA in tissues. This work also resolved why 9cRA has not been observed previously in tissues other than pancreas. Apparently, a combination of ethanol/saline-based homogenization and tissue-specific matrix effects causes 9cRA to isomerize and/or to extract poorly, reducing it below limits of quantification. Methanol precipitates proteins more efficiently than the saline/ethanol mixture. Homogenization in methanol, with removal of precipitated proteins before hexane extraction, diminishes matrix effects and prevents isomerization.This report establishes existence of 9cRA in multiple tissues at concentrations similar to or greater than atRA. Identification rested on multiple criteria including the following: (1) selective ion monitoring of multiple tissues relying on comigration of endogenous 9cRA with synthetic 9cRA during high-resolution LC, accompanied by selection of the molecular ion in Q1 at atRA has been accepted as an autacoid with diverse actions, that is, an endogenous retinoid effector of diverse biological processes . Debate cis-retinol, and its relatively high mRNA expression in pancreas, this work examined the possibility that Rdh5 participates in pancreas 9cRA biosynthesis. Rdh5 is better known as one of the 11-cis-retinol dehydrogenases important to the visual cycle  with 9-cis-retinol and has widespread mRNA expression , a protein that reduces the insulin content of islets and contributes to impairing GSIS  and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich. Standard 9,13-di-g and 4 °C. Sera and tissues were snap-frozen in liquid nitrogen and stored at −80 °C until assayed. Animal experimental protocols were approved by the University of California Berkeley Animal Care and Use Committee.C57BL/6J (strain# 000664) mice were purchased from The Jackson Laboratory. Upon arrival, mice were fed a purified AIN93G diet  containing 4 IU retinyl acetate/g and 7% fat and bred in-house through >3 generations. Blood, liver, pancreas, kidney, testis, epididymal and inguinal white adipose tissues (eWAT and iWAT), brown adipose tissue (BAT), and brain samples were collected from male 8∼9 weeks-old-mice, fasted 16 h and compared to those refed 6 h after a 16 h fast. Mice were euthanized in an isoflurane chamber, followed by cervical dislocation. Blood samples, drawn from the vena cava, were allowed to clot on ice and centrifuged 30 min at 12,000g. The upper hexane phase was discarded. One hundred eighty microliters of 4 M HCl were added to the remaining aqueous phase. RAs were extracted with 10 ml hexane by vortexing 20 s. The upper hexane phase was transferred to a new round bottom glass tube and evaporated under a gentle stream of nitrogen in a water bath  at 25 to 30 °C. The residue was reconstituted in 40 μl acetonitrile with 20 s vortexing, spun for 10 s at 1200g, and transferred to a glass vial insert. One microliter was injected for LC-APCI-MS/MS analysis.Tissues (50∼150 mg) or sera (100 μl) were homogenized in ice-cold 0.9% saline using a glass pestle tissue grinder  with a motorized homogenizer  at 280 rpm to produce a 10% homogenate. The homogenate (0.5 ml) was transferred to a 23 ml round-bottom glass tube and mixed with 10 μl internal standard  and 3 ml of 25 mM KOH, prepared in ethanol, by vortexing for 10 s. Ten milliliters hexane was added. The mixture was vortexed 20 s and centrifuged 2 min at 2000g at room temperature. The supernatant was transferred to a clean glass round-bottom tube and mixed with 100 μl of 750 mM KOH, prepared in methanol, and vortexed 10 s. To remove hydrophobic impurities, 10 ml of hexane was added, vortexed 10 s, and spun 10 s at 1200g. The upper hexane layer was discarded. The bottom methanol phase was mixed with 180 μl 4 M HCl by vortexing 10 s and extracted with 10 ml hexane. The mixture was centrifuged 2 min at 1200g. The upper hexane layer was transferred to a new round-bottom glass tube and evaporated under a gentle stream of nitrogen with heating at 25 to 30 °C in a water bath. The residue was reconstituted in 40 μl of acetonitrile with 20 s vortexing, spun 10 s at 1200g, and transferred to a glass vial insert. One microliter was injected for LC-APCI-MS/MS analysis.Tissue samples (50∼150 mg) or sera (100 μl) were placed into a glass pestle tissue grinder with 1.5 ml of ice-cold methanol and 10 μl of the 100 nM internal standard, prepared in acetonitrile. Samples were homogenized using the motorized homogenizer at 280 rpm. The homogenate was transferred to a 15 ml glass conical tube  and centrifuged 2 min at 1200Chromatography was done with an Agilent 1290 series liquid chromatograph consisting of a binary pump, temperature-controlled column compartment, and temperature-controlled autosampler. The column compartment and autosampler were maintained at 40 °C and 10 °C, respectively. The mobile phase included two components: component A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). The following gradient was used: 0 to 3 min, hold at 70% B; 3 to 15 min, 70% B to 95% B; 15 to 20 min, hold at 95% B; 20 to 21 min, 95% B to 70% B; 21 to 25 min, re-equilibrate at 70% B. An Ascentis RP-Amide column resolved analytes  fitted with a preceding guard column , at a flow rate of 400 μl/min.Resolution of RA isomers was achieved with an analytical Ascentis Express RP-Amide column  fitted with a preceding guard column at a flow rate of 500 μl/min. The following were the mobile phases used: (A) 0.1% formic acid in water/methanol (6:4); (B) 0.1% formic acid in acetonitrile/methanol (6:4). A gradient was applied over 25 min: 0 to 2 min, 40% B; 2 to 10 min, 40 to 55% B; 10 to 18 min, 55 to 95%; 18 to 21 min, holding at 95% B; 21 to 23 min, 95 to 40% B; 23 to 25 min, holding at 40% B.https://sciex.com/products/software/analyst-software) and operated in multiple reaction–monitoring mode. RAs were monitored using a m/z 301.1 [M + H]+ to m/z 205.0 transition. The internal standard 3,3-dimethyl-RA was monitored using a m/z 329.4 [M + H]+ to m/z 151.3 transition. The dwell time for both RA and 3,3-dimethyl-RA was 150 ms. Optimum positive APCI conditions included the following: collision gas, 7 psig; curtain gas, 10 psig; gas1, 70 psig; nebulizer current, 3 μA; source temperature, 350 °C; declustering potential, 55 V; entrance potential, 10 V; collision energy, 17 eV; and collision exit potential, 5 V.Quantitative data were obtained using a Sciex API-4000 triple-quadrupole mass spectrometer, equipped with an APCI source, operated in positive ion mode. The instrument was controlled by Analyst v1.4 software (Standards of RA isomers (30 pmol each) were prepared in 50 μl acetonitrile. Remaining samples after the injection for RA extraction/quantification were pooled, evaporated under a gentle stream of nitrogen, and reconstituted in 50 μl acetonitrile. Twenty millimolar of 3-NPH was prepared in 40 mM HCl and ethanol (v/v 3:1). One hundred millimolar of EDC was prepared in pyridine and acetonitrile (v/v 1:1). In a 1.8 ml amber glass vial, 50 μl of sample was mixed with 100 μl 3-NPH and 75 μl EDC by vortexing 10 s, then incubated 1 h at room temperature. The reaction mixture was transferred to a 23 ml round-bottom glass tube and extracted with 10 ml hexane as described in the revised extraction section.Resolution of RA-3-NPH isomers was achieved with the analytical Ascentis Express RP-Amide column with the preceding guard column at a flow rate of 400 μl/min. The following were the mobile phases used: (A) 0.1% formic acid in water; (B) 0.1% formic acid in methanol. The following gradient was applied over 25 min: 0 to 21 min, 30 to 85% B; 21 to 22.5 min, holding at 85% B; 22.5 to 23.5 min, 85 to 40% B; 23.5 to 25 min, holding at 30% B.RA-3-NPH was monitored using an m/z 436.7 [M + H]+ to m/z 283.3 transition. Positive APCI conditions included the following: collision gas, 7 psig; curtain gas, 10 psig; gas1, 70 psig; nebulizer current, 1 μA; source temperature, 350 °C; declustering potential, 55 V; entrance potential, 10 V; collision energy, 20 eV; collision exit potential, 5 V.O-ethyloxime isomers were resolved via an Acclaim C30 HPLC Column  with a flow rate of 400 μl/min  at a gradient: 0 to 2 min, hold at 80% B; 2 to 2.5 min, 80% B to 85% B; 2.5 to 17.5 min, 85% B to 95% B; 17.5 to 20 min, hold at 95% B; 20 to 20.5 min, 95% B to 80% B; 20.5 to 25 min, re-equilibrate at 80% B at 40 °C.Retinol isomers were measured by HPLC/ultraviolet absorbance detection . Retinal2 in T75 flasks or 6-well plates. To minimize effects of unknown factors, cells were cultured in FBS-free growth medium containing 11.1 mM glucose 18 h prior to experiments. MIN6 cells were maintained in growth medium (Dulbecco's modified Eagle's medium containing 4.5 g/l glucose) under 5% CO2 in T75 flasks or 6-well plates.The rat insulinoma cell line 832/13 was a gift from the Newgard lab . Cells wActb, Mm00607939_s1; mouse Rdh5, Mm00506111_m1; rat Rdh5, Rn.PT.56a.11212766.g; rat Gusb, Rn.PT.39a.22214822.g, rat Rdh10, Rn00710727_m1; rat Crem, Rn01538528_m1) and run on CFX Connect Real Time System (Bio-Rad). Gene expression was analyzed by the ΔΔ-Ct method, normalized to Actb (mouse) or Gusb (rat), and expressed as fold-change relative to expression in reference condition.RNA was isolated from tissues and cells with TRI reagent  following manufacturer’s instructions. Complementary DNA (cDNA) was reverse-transcribed using iScript cDNA Synthesis Kit . Real-time quantitative PCR (qPCR) was prepared with PrimeTime Gene Expression Master Mix  and qPCR probes ; GSk3β, XXV ; PKA, 14 to 22 Amide ; Akt, triciribine hydrate ; mTOR, rapamycin ; CaMK, CK59 . 832/13 cells were seeded on 6-well plates and maintained in growth medium. FBS-starved cells were treated with serum-free growth medium containing 3 mM or 15 mM glucose ± inhibitors for 6 h, followed by RNA isolation, cDNA synthesis, and qPCR.Data are expressed as mean ± SEM. Statistical analyses were made by two-tailed unpaired t tests using GraphPad Prism ver. 9.5.1.All data are present in this manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The porous structure and interconnected conductive pathways accommodate a large amount of iodine, entrap polyiodides and guarantee its efficient utilization. While the Fe single atom catalyst efficiently catalyzes the iodine/polyiodide conversion.2 battery delivers a high capacity of 188.2 mAh g−1 at 0.3 A g−1, excellent rate capability with a capacity of 139.6 mAh g−1 at 15 A g−1 and ultra-long cyclic stability over 50,000 cycles with 80.5% initial capacity retained under high iodine loading of 76.72 wt%.With “confinement-catalysis” host, the ZnǀǀIThe online version contains supplementary material available at 10.1007/s40820-023-01093-7. 2) batteries have been promising energy storage technologies due to low-cost position and constitutional safety of zinc anode, iodine cathode and aqueous electrolytes. Whereas, on one hand, the low-fraction utilization of electrochemically inert host causes severe shuttle of soluble polyiodides, deficient iodine utilization and sluggish reaction kinetics. On the other hand, the usage of high mass polar electrocatalysts occupies mass and volume of electrode materials and sacrifices device-level energy density. Here, we propose a “confinement-catalysis” host composed of Fe single atom catalyst embedding inside ordered mesoporous carbon host, which can effectively confine and catalytically convert I2/I− couple and polyiodide intermediates. Consequently, the cathode enables the high capacity of 188.2 mAh g−1 at 0.3 A g−1, excellent rate capability with a capacity of 139.6 mAh g−1 delivered at high current density of 15 A g−1 and ultra-long cyclic stability over 50,000 cycles with 80.5% initial capacity retained under high iodine loading of 76.72 wt%. Furthermore, the electrocatalytic host can also accelerate the −/I2 and I2/I+ couples, and polyiodide intermediates conversions.Rechargeable aqueous zinc iodine (ZnǀǀIThe online version contains supplementary material available at 10.1007/s40820-023-01093-7. To date, various polar materials such as metal oxides, metal nitrides, metal phosphide and metal organic frameworks are reported as polarized hosts for ZnǀǀIatteries –22. Despl device , 24. It 2 battery with fast reaction kinetics and ultra-long cycling stability by embedding iron single atom catalyst (SAC) in ordered mesoporous conductive framework as a catalytic iodine host. With this design, the porous structure and interconnected conductive pathways accommodate a large amount of iodine, entrap polyiodides and guarantee its efficient utilization. While the Fe SAC efficiently catalyzes the iodine/polyiodide conversion. Considering the synergistic contribution of high catalytic and iodine/polyiodide adsorption ability from host framework, the ZnǀǀI2 battery achieves ultra-high-rate capability at 15 A g−1 with a capacity of 139.6 mAh g−1 delivered and ultra-long cycling stability over 50,000 cycles with 80.5% initial capacity retained at 5 A g−1 under 76.72 wt% iodine loading condition. This work opens a way to shortens the gap between research and application for ZnǀǀI2 batteries.In this work, we propose a “confinement-catalysis” strategy to enable a high iodine loading ZnǀǀI2) host, although the electrode achieves good electrical conductivity, the sluggish reaction kinetics, low active I2 content, low active I2 utilization and severe shuttle effects become the bottleneck to realize stably high energy ZnǀǀI2 batteries  with activated and isolated catalytic metal atoms presents the best catalytic sites utilizations for catalytic reactions. In this work, the Fe SAC on mesoporous nitrogen doped carbon (Fe SAC-MNC) is designed as active I2 host for aqueous ZnǀǀI2 batteries. The ordered porous structure can effective restrain I2/I3−/ZnI2, while the Fe SAC significantly reduce activation energy barrier for outstanding I2 reduction reaction (IRR) and boost electrochemical I2/I3−/ZnI2 redox kinetics. Owing to this peculiarity, ZnǀǀI2 batteries display high active I2 utilization, stable cycle, high coulombic efficiency and high energy density.With the traditional disordered activated carbon (AC) as insulating active iodine  and (101) reflections . In the Raman spectra, the ratio of D peak to G peak reveals the change of material structure in an extent. ID/IG ratio of Fe SAC-MNC (0.94) is close to MNC (0.95), declaring that the graphitization extent is not influenced by the introduction of Fe SAC . The uniform nanospheres with diameter of ~ 80 nm and bestrewing abundant ordered mesoporous are depicted by the transmission electron microscopy , Pyrrolic-N (400.7 eV), Graphitic-N (401.7 eV) and Oxidized-N (403.3 eV). The characteristic peaks of Fe 2p appeared at 712.25 eV (Fe 2p3/2) and 723.75 eV (Fe 2p1/2), confirming that the oxide state of Fe SAC is + 3  [2 cathode (MNC/I2 and KB/I2 as control groups), the I2 molecules are successfully confined in the hosts via thermal melt-diffusion routes under high temperature of 120 °C. The successful confinement of I2 in Fe-SAC-MNC is confirmed by XRD pattern, N2 adsorption/desorption isotherms and pore size distribution, in which the specific surface area is sharply decreased from 257.9 to 1.84 m2 g−1 and the content of pores is remarkably reduced with introduction of I2 molecules  [Fe SAC-MNC host is prepared by thermal pyrolysis of nanoemulsion-directed UiO-66-NH002) and 01 reflecFig. S3) . To prep and S4) . And TG −/I2/I3− species, supporting exact insight into restraining the shuttle effect. Compared with MNC and KB, the Fe SAC-MNC host exhibits the stronger physical and chemical absorption for all of I−/I2/I3− species  [Eab are listed specifically in Table S1. The optimized charge-density-difference of I−, I2, and I3− on Fe SAC-MNC, MNC and KB is displayed in inset of Figs. −, I2, and I3−. In light of abundant pore structure for physical confinement and strong chemical affinity, I2 molecules loading content in the Fe SAC-MNC/I2 and MNC/I2 can reach to high mass ratio of 76.72 and 77.82 wt%, respectively, higher than KB/I2 (63.35 wt%), which are evaluated by thermogravimetric analysis . Notably, comparing with the I2 weight content in various hosts as cathode of metal-iodine batteries reported previously, Fe SAC-MNC shows an outstanding competitive superiority in the iodine content for 76.72 wt%  calculation is performed to describe the interaction of the hosts of Fe SAC-MNC, MNC and KB with Iies Fig. e. The beFig. S5) . The the2, MNC/I2 and KB/I2 cathodes in the 2 M aqueous ZnSO4 electrolyte , demonstrating robust physio/chemisorption of Fe SAC-MNC host to I2.The ultraviolet–visible (UV–vis) is employed to explore the dissolution of iodine from the Fe SAC-MNC/Iyte Fig. g. The pe2/I−, cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) tests are measured in two-electrode configuration with Zn metal as counter electrode and reference electrode, catalyst deposited carbon fiber cloth (CFC) as working electrode and 2 M ZnSO4 + 0.02 M I2 aqueous solution as electrolyte [−2) and KB , the Fe SAC-MNC electrocatalyst exhibit high optimal reduction potential of 1.36 V (vs. Zn/Zn2+) and reduction current density of 0.44 mA cm−2  curves of Fe SAC-MNC, MNC or KB as working electrode. Meanwhile, the Fe SAC-MNC electrocatalyst presents smaller Tafel slope (η) of 59.07 mV dec−1, than that of MNC (86.39 mV dec−1) and KB (108.52 mV dec−1) . The Fe SAC-MNC electrocatalyst shows smaller Rct (54.47 Ω)  and lower slope of Arrhenius curves , compared with MNC (145.9 Ω) and KB (90.73 Ω). According to oxygen reduction reaction (ORR), the lower slope predicates the smaller activation energy (Ea). The specific Ea is calculated using the following Arrhenius equation:Rct is the charge transfer resistance, Ea is the activation energy, T is the temperature and R is the gas constant. The Ea of IRR by Fe SAC-MNC is calculated to be 27.878 kJ mol−1, smaller than that of MNC (31.386 kJ mol−1) and (39.654 kJ mol−1), representing the super-fast kinetic of electrocatalytic IRR under existing of Fe SAC  during I−/I2 reaction with Fe SAC-MNC, MNC and KB electrocatalysts employed, respectively. As shown in Fig. −, while the two peaks at 288 and 355 nm are ascribed to the formation of I3− in MNC and KB. In comparison, no I3− formed in Fe SAC-MNC catalyzed IRR, further certifying the forceful physio/chemical affinity of Fe SAC-MNC with iodine species and totality of reaction from I2 to I−. The rate of I2 precipitation on the electrode matrix is another key indicator for Zn-I2 batteries to evaluate the conversion kinetic of I3−/I2 [3− to solid I2 at 1.34 V , in comparison to the MNC (0.29 mA after 764.20 s) and KB  electrode. The capacities of the I2 precipitation on Fe SAC-MNC, MNC and KB are determined to be 57.6, 40.2 and 18.2 mAh g−1, affirming the remarkable nucleation of I2 at Fe SAC-MNC electrode during same period (9000 s).For investigating the effect of Fe SAC for electrocatalytic redox conversion of Ictrolyte , 42, 43.2 species conversion on the active centers of Fe SAC-MNC. The lower the Gibbs free energy (∆G) represents the deeper spontaneity trends and the faster reaction kinetics [2/I− redox reactions often proceed in two steps of I2 + 2/3 e− ↔ 2/3 I3− and I3− + 2e− ↔ 3I−, where the latter one is the rate-determining step for whole IRR. The ∆G value for I3−/I− is -3.4658 eV for Fe SAC-MNC electrocatalyst, which is lower than -2.973 eV for MNC and 1.7816 eV for KB electrocatalysts , indicating the easy break of Zn-I bond on Fe SAC-MNC. In contrast to MNC and KB, the Fe SAC-MNC is quite more metallic for a higher DOS at the Fermi level, giving evidence of satisfactory electrical conductivity of Fe SAC-MNC to markedly enhance the fast electrochemical reaction between I− and I2  of cathodic and anodic reaction against the square roots of scan rates (v1/2) are derived from the CV curves at different scan rate . The linear relationship declares that the redox reactions are controlled by mass diffusion transport. The resulting proton diffusivity (DH) values are calculated by the Randle-Sevcik equation for Fe SAC-MNC/I2, MNC/I2 and KB/I2. The DH values of Fe SAC-MNC/I2 is 1.83 × 10–10 cm2 s−1, larger than 9.05 × 10–11 cm2 s−1 for MNC/I2 and 8.36 × 10–11 cm2 s−1 for KB/I2 which declares that Fe SAC is a key point for I−/I2 redox reaction to boost iodine utilization and reaction kinetics . The Fe SAC-MNC/I2 cathode exhibits persistent high proton DH, even if at 75% depth of discharge (DOD) [Rct, 48.12 Ω) of Fe SAC-MNC/I2, compared with that of the MNC/I2 (68.65 Ω) and KB/I2 (77.06 Ω), which can be attributed to the introduction of Fe SAC  of Fe SAC-MNC/I2 cathode is 1,181.6 Hz (0.8467 ms) suggesting a faster charge response than that of MNC/I2 cathode  and KB/I2 cathode . Both enhanced proton diffusivity and boosted charge response accounts for the role of Fe SAC for I2/I− redox conversion.The current density (ge (DOD) . The cha2, MNC/I2 and KB/I2 electrode are used to evaluate the electrocatalytic effects at 5 mV s−1 from 0.45–1.65 V. In comparison of the cathodic and anodic peak for MNC/I2 (1.291 and 1.148 V) and KB/I2 (1.513 and 1.179 V), the Fe SAC-MNC/I2 displayed a pair of strongest current peaks for I2/I− redox reaction at 1.414 and 1.191 V, respectively . As shown in Fig. 2 cathode exhibits a smallest Tafel slope of 75.25 mV dec−1, compared with MNC/I2 (82.28 mV dec−1) and KB/I2 (89.72 mV dec−1) cathodes, which reveals the prominent improvement of I2/I− redox reaction kinetic by Fe SAC active sites.The cyclic voltammetry (CV) curves of Fe SAC-MNC/I2 battery shows preponderant capacity of 188.21, 180.92, 175.96, 168.53, 163.32, 151.33 and 139.60 mAh g−1 at 0.3, 0.5, 1, 3, 5, 10, and 15 A g−1, which are almost twice and three times as much as ZnǀǀMNC/I2 and ZnǀǀKB/I2 battery  and KB/I2 (35.52 mAh g−1) cathodes. It’s worth noting that the capacity of ZnǀǀFe SAC-MNC/I2 battery at such high rate (15 A g−1) outperforms most previous works [−1, the CE curves of ZnǀǀKB/I2 battery become greatly unstable, suggesting deficient I2/I− conversion reactions. The galvanostatic charge/discharge (GCD) curves of ZnǀǀFe SAC-MNC/I2 battery are displayed in Fig. −1), being company with ZnǀǀMNC/I2 and ZnǀǀKB/I2 battery . Along with the increasing of rate, the capacities of Fe SAC-MNC/I2 cathode show a small decay with uniformly clear charge/discharge plateaus, which is consistent with the CV curves . Meanwhile, the ZnǀǀFe SAC-MNC/I2 battery displays the smallest electrochemical polarization (111 vs. 212 mV for MNC/I2 and 415 mV for KB/I2), suggesting the extinguished active I2 utilization and outstanding redox reaction kinetic  [2 cathode achieve higher capacity at different current density (0.3–15 A g−1) than mostly reported metal-iodine batteries, demonstrating its fast reaction kinetics and high active I2 utilization [Inspired by the physical confinement structure of MNC and chemical adsorption/catalytic of Fe SAC, the ZnǀǀFe SAC-MNC/Ius works –49. In cig. S12) , 51, 52.lization , 41, 53.2 battery displayed super-stable cycle performance and super-long lifespan at a high current density of 5 A g−1 with initial capacity retention of 80.5% (from 198.5 to 159.7 mAh g−1) even over 50,000 cycles  and lifespan . The above results confirm the excellent electrocatalyst ability of Fe SAC and strong iodine species adsorption of MNC as I2 host. In I 3d XPS high resolution spectra, two peaks clearly located at 619.2 and 630.7 eV are ascribed to I-C and I-O bond, respectively  to metal anode not only intensify capacity decay, but also generate the oxygen evolution reaction (OER) and metal anode corrosion [2, ZnǀǀMNC/I2 and ZnǀǀKB/I2 cells are visualized by SEM images. As illustrated in Fig. 2 cathode is seriously attacked by the iodine species to engender many huge holes. Utilizing MNC/I2 cathode, although the surface of Zn anode appears less holes, we still clearly observe some Zn corrosion. The loose Zn anode with disorganized holes prejudices the transportation of electron and ions [2/I− conversion, the Zn anode in ZnǀǀFe SAC-MNC/I2 cell maintains smooth surface and dense Zn deposits. The comparisons in aspect of I2 weight content, capacity, cycle number, capacity retention and energy density are conducted to elaborate the superiority of Fe SAC-MNC host for ZnǀǀI2 batteries  adding with 8 M Acetonitrile (ACN) is selected to activate I0/I+ redox [2, ZnǀǀMNC/I2 and ZnǀǀKB/I2 batteries, representing the I0/I+ and I0/I− redox reactions  and ZnǀǀKB/I2 (136.06 mV dec−1) . For a more direct observation, the Charge/ discharge voltage profiles of three cathodes at 0.5 A g−1 are distinguished in Fig. 2 battery located at 1.28 and 1.71 V correspond to the I−/I0 and I0/I+ redox reactions, respectively. The ZnǀǀFe SAC-MNC/I2 battery delivers highest discharge capacity of 230 mAh g−1 at 0.5 A g−1 and 109.49 mAh g−1 at high current density of 10 A g−1, much higher than that of ZnǀǀMNC/I2 battery (48.18 mAh g−1 at 10 A g−1), demonstrating the distinguish electrocatalytic capability of Fe SAC-MNC/I2 cathode to realize fast four electrons redox reactions Below is the link to the electronic supplementary material.


The complex anion of the title compound reveals clathrochelate topology and includes an FeIV metal centre. In the crystal, the complex anions are connected through two Li cations into dimers, which are connected by Li—O bonds, forming infinite chains along the b-axis direction.The title compound was obtained as a result of a template reaction between oxalohydrazide, formaldehyde and iron(III) chloride in the presence of atmospheric O 2(C12H12N12O6)(H2O)4]·4H2O, consists of iron complex anions, lithium cations and water molecules. The complex anion shows a clathrochelate topology. The coordination geometry of the FeIV centre is intermediate between a trigonal prism and a trigonal antiprism. In the crystal, the complex anions are connected through two Li cations into dimers, which are connected by Li—O bonds, forming infinite chains along the b-axis direction.The title compound, [FeLi Further studies showed that these compounds are promising redox catalysts for photochemical water splitting  complexes was described -1,3,4,7,8,10,12,13,16,17,19,22- dodecaazatetracyclotetracosane-5,6,14,15,20,21-hexaone) (1) obtained as a result of a template reaction between oxalohydrazide, formaldehyde and iron(III) chloride in the presence of atmospheric oxygen   Å. The bite angle α  is equal to 40.53 (4)°, the chelate N—Fe—N′ angles being in the range 80.29 (6)–80.87 (6)°. Thus, all geometric parameters of the FeIV coordination polyhedron are close to those of the earlier published FeIV clathrochelates –1.9572 (15) Å Table 1. The N⋯N3.L]2− complex anion is chiral. Both stereoisomers of the complex cation are included in the crystal packing, thus, 1 is a racemate. In the crystal, both chiral isomers are connected through two Li cations , forming a racemic dimer {Li2[FeL]2}2−. Such dimers are connected by Li2⋯O5 interactions, forming continuous chains along the b-axis direction  from red  through white to blue . The Hirshfeld surface of the title compound mapped over dnorm is shown in Fig. 5B⋯O4, O13—H00R⋯O3, O11—H00O⋯O3, O14—H00M⋯O6, O10—H00A⋯O6).The Hirshfeld surface analysis  pair with the full fingerprint plot outlined in grey. Fig. 6A fingerprint plot delineated into specific interatomic contacts contains information related to specific intermolecular interactions. The blue colour refers to the frequency of occurrence of the . The MOFs reveal a 1D coordination polymer topology: the FeIV clathrochelate complex anions being connected by Mn2+  were added dropwise. Then an aqueous formaldehyde solution  was added. The reaction mixture was stirred for 2 h under slight warming (∼313 K), filtered off, and the solvent removed on a rotary evaporator. The crude product was dissolved in 5 ml of water and left for crystallization by slow diffusion of tetrahydrofuran vapour. Single crystals suitable for X-ray analysis were obtained after one month. Yield 0.124g (22%). IR : 3409 (O—H), 2942 (C—H), 1648 (C=O amide I). Analysis calculated for C12H28FeLi2N12O14: C 22.73, H 4.45, N 26.51. Found: C 22.79, H 4.36, N 26.73.To a mixture of 0.354 g oxalodihydrazide (3 mmol) and 0.144 g LiOH (6 mmol), 10 ml of FeCl7.Uiso(H)= 1.2Ueq(parent atom).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023008587/tx2074sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023008587/tx2074Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023008587/tx2074Isup3.cdxSupporting information file. DOI: 2298136CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: