Dentifrices can augment oral hygiene by inactivating bacteria and at sub-lethal concentrations may affect bacterial metabolism, potentially inhibiting acidogenesis, the main cause of caries. Reported herein is the development of a rapid method to simultaneously measure group-specific bactericidal and acidogenesis-mitigation effects of dentifrices on oral bacteria. Saliva was incubated aerobically and anaerobically in Tryptone Soya Broth, Wilkins-Chalgren Broth with mucin, or artificial saliva and was exposed to dentifrices containing triclosan/copolymer (TD); sodium fluoride (FD); stannous fluoride and zinc lactate (SFD1); or stannous fluoride, zinc lactate and stannous chloride (SFD2). Minimum inhibitory concentrations (MIC) were determined turbidometrically whilst group-specific minimum bactericidal concentrations (MBC) were assessed using growth media and conditions selective for total aerobes, total anaerobes, streptococci and Gram-negative anaerobes. Minimum acid neutralization concentration (MNC) was defined as the lowest concentration of dentifrice at which acidification was inhibited. Differences between MIC and MNC were calculated and normalized with respect to MIC to derive the combined inhibitory and neutralizing capacity (CINC), a cumulative measure of acidogenesis-mitigation and growth inhibition. The overall rank order for growth inhibition potency (MIC) under aerobic and anaerobic conditions was: TD> SFD2> SFD1> FD. Acidogenesis-mitigation (MNC) was ordered; TD> FD> SFD2> SFD1. CINC was ordered TD> FD> SFD2> SFD1 aerobically and TD> FD> SFD1> SFD2 anaerobically. With respect to group-specific bactericidal activity, TD generally exhibited the greatest potency, particularly against total aerobes, total anaerobes and streptococci. This approach enables the rapid simultaneous evaluation of acidity mitigation, growth inhibition and specific antimicrobial activity by dentifrices. Streptococcus mutans and homofermentative lactobacilli [Dental plaque is a taxonomically diverse microbial community, which plays an important role in oral health and disease –3. The eobacilli .Streptococcus mutans, which lack FabI, triclosan may also inhibit glycolysis, leading to a reduction in the production of harmful acids [The control of dental plaque by the routine brushing with dentifrice can improve oral hygiene, reducing the incidence of oral disease ,14. Antiul acids . Zinc saul acids ,21. Thesul acids , 21. Theul acids . The mitin vitro systems, combined with differential viable counts. Investigating the functional effects of dentifrices  at sub-lethal concentrations is normally achieved (for acidogenesis inhibition) by exposing oral bacteria to diluted formulations and assessing changes in pH with a microelectrode, or by the chemical analyses of fermentation acids. Whilst such approaches have proven utility, a rapid and reproducible endpoint-based method for high-throughput testing of these variables would be a useful addition to the currently available methodology. Here we report the development of a method for assessing both acidogenesis-mitigating and antimicrobial effects of dentifrices against salivary microorganisms.Several variables are of interest in the pre-clinical assessment of oral care formulations. From a microbiological perspective these include the extent of bacteriostatic and/or bactericidal activity, which can be tested with pure or mixed cultures of oral bacteria. Assessing differential antimicrobial effects on the oral microbiota normally necessitates growing mixed oral bacteria in various ® Total® (TD), containing the actives triclosan (0.3% w/v), copolymer (2% w/v) and sodium monofluorophosphate (0.76% w/v); Colgate® Cavity Protection® (FD), containing, sodium monofluorophosphate (0.76% w/v); Crest® Pro-Health® (SFD1), containing the actives stannous fluoride (0.454% w/v) and zinc lactate (2.5% w/v) and Crest® Pro-Health Clinical® (SFD2), containing the actives stannous fluoride (0.454% w/v), zinc lactate stannous chloride (2.5% w/v). Dentifrices were suspended in sterile broth to produce slurries (50 mg ml-1). Test media included; artificial saliva (AS), Wilkins Chalgren with 2% mucin (WCM) and Tryptone Soya broth (TSB). Artificial saliva growth medium (AS) comprised (g/L) in distilled water: Mucin , 2.5; bacteriological peptone, 2.0; tryptone, 2.0; yeast extract, 1.0; NaCl, 0.35; KCl, 0.2; CaCl2, 0.2; cysteine hydrochloride, 0.1; haemin, 0.001; vitamin K1, 0.0002 [Chemicals were obtained from Sigma  and bacteriological media from Oxoid , unless otherwise stated. Bacterial growth media were sterilized at 121°C, 15 psi for 15 min prior to use. The dentifrice formulations evaluated were Colgate, 0.0002 .2, 10% CO2 and 10% H2) were generated using an MG1000 anaerobic workstation . The MIC was defined as the lowest concentration at which bacterial growth was inhibited. Growth was measured as light absorbance (496 nm) in comparison to an uninoculated well containing the equivalent concentration of dentifrice (negative control) and was detected using a microtiter plate reader .MICs were determined using the microdilution method as described previously . Briefly2, 10% CO2 and 10% H2; MG1000 anaerobic workstation; Don Whitley Scientific, Shipley, UK). The MBC was defined as the lowest concentration of dentifrice at which no visible growth occurred after 3 d of incubation.A transferable solid phase screening system  was used to aseptically transfer media (approximately 3–4 μL) from the MIC plate wells to a 96 well-plate containing 200 μL of one of the following bacteriological media; Wilkins Chalgren Agar (with and without anaerobic Gram-negative supplement) or yeast extract, cysteine, sucrose agar   was added directly to each MIC plate well after incubation to determine acidification of the growth media. Plates were incubated for a further 15 min at room temperature before determination of colour change. The MNC was defined as the lowest concentration at which no colour change (red to yellow) occurred. The pH of each dentifrice was determined at all test concentrations using a pH meter with a needle electrode .The CINC was calculated as follows; CINC = (MIC-MNC)/MIC. The CINC therefore provides a numerical value that represents the cumulative ability of a particular dentifrice to prevent bacterial acidogenesis below growth inhibitory concentrations in addition to its ability to completely inhibit bacterial growth. A higher CINC value would therefore indicate better overall acid mitigation activity and growth inhibitory activity when compared to a product with a lower CINC.Data  were analyzed using a one-way ANOVA  and post-hoc Tukey analysis. MICs were directly compared to MNCs for individual formulations using a paired student’s t-test. In both cases P<0.05 was deemed to be statistically significant.Advice was taken from the Chair of a University of Manchester Research Ethics Committee regarding the correct procedures associated with the use of human saliva samples. The committee granted exemption from formal ethics approval due to the nature of the work but as advised, informed written consent was obtained from all volunteers and all samples were collected anonymously.The bactericidal efficacy of the dentifrice formulations was assessed against total aerobes, total anaerobes, streptococci and Gram-negative anaerobes. Overall, TD showed significantly greater bactericidal activity  when compared to the other test dentifrices against total aerobes P<0.05 , while FWhen incubated aerobically, MBCs for SFD2 against streptococci ranged between 3.1 and 6.3 mg/ml whilst values for TD ranged between 3.1 and 8.3 mg/ml, both were therefore potent but there was no statistically significant difference between MBCs for these two formulations (P>0.05). Equal or lower bactericidal activity against streptococci was observed for SFD1  and FD . Under anaerobic conditions bactericidal activity against streptococci of TD  was also significantly higher (P<0.05) than that of SFD2 , FD  and SFD1 , TD (0.8–4.2 mg/ml), SFD1 (0.8–6.3 mg/ml) and FD , SFD2 (pH 7.22–7.25), FD (pH 7.27–7.24) and TD (pH 7.27–7.25). There was no significant difference in pH between the formulations at any test concentration (P<0.05). MNCs were significantly lower than MICs for both TD and FD (P<0.05) when incubated aerobically and for all dentifrices when incubated anaerobically . The ranThe difference between MIC and MNC for each test paste was calculated and normalized with respect to MIC. The resulting values reflect the combined inhibitory and neutralizing capacity (CINC) of the test dentifrices, with a high value indicating a good ability to inhibit both acid production and bacterial growth. The CINC is therefore a single numerical measure of both specific acidogenesis mitigation and the inhibitory activity of a particular dentifrice against oral bacteria, providing the means of a clear preclinical measure of the activity of each test formulation against acidogenic bacteria indicating potential anti-caries activity. The rank order for CINC was TD > FD > SFD2 > SFD1 under aerobic conditions and TD > FD > SFD1> SFD2 under anaerobic conditions . WhetherThe use of antimicrobial formulations to control the accumulation of oral bacteria and therefore to reduce plaque accumulation can markedly decrease the risk of the development of oral disease such as caries and gingivitis , 25, 26.TD exhibited greatest bactericidal activity against total aerobes and total anaerobes during MIC testing, prior to plating on selective agars. These findings are in agreement with previous studies showing the broad-spectrum and high level of antibacterial activity of triclosan , 28. TD TD exhibited greater growth inhibitory effects against bacteria than FD, SFD1 and SFD2. MNC data also indicate that the test dentifrices inhibited acidogenesis at sub-inhibitory concentrations. This may be attributed to specific inhibitory effects on bacterial metabolism and subsequent acid production. For example, fluoride has been previously shown to inhibit sucrose-induced acid production in bacteria , 31, potBoth SFD1 and SFD2 demonstrated lower values for MNC than MIC, particularly when incubated anaerobically. In addition to stannous fluoride, which has been shown to reduce acid production in cariogenic bacteria , 31, botThe capacity of a dentifrice to inhibit plaque acidogenesis is an important property of oral healthcare formulations. Objective criteria to express the minimum concentration of dentifrice required to mitigate acidogenesis in controlled conditions (MNC), and the combined inhibitory and neutralizing capacity (CINC) may therefore be used in the pre-clinical evaluation of dentifrices, enabling acidity mitigation, growth inhibition and specific antimicrobial activity to be determined simultaneously in a robust and reproducible fashion.The etiology of dental caries primarily involves acid production by oral bacteria and therefore the reduction of bacterial acidogensis; whether mediated through the inactivation of bacteria, the inhibition of bacterial growth or by specific effects on bacterial metabolism, is a desirable activity for oral health products. In the current investigation a method through which acidity mitigation, growth inhibition and specific antimicrobial activity can be determined simultaneously is described. Test formulations used to evaluate the method exhibited marked differences in their growth-inhibitory and group-specific bactericidal activities. Acidogenesis-mitigation was observed at concentrations lower than required for bacterial growth inhibition. A dentifrice containing triclosan (TD) exhibited a higher level of antimicrobial activity and acidogenesis-mitigating ability than other test dentifrices.


Steroid receptor coactivator 3 (SRC-3) is a multifunctional protein that plays an important role in malignancy of several cancers and in regulation of bacterial LPS-induced inflammation. However, the involvement of SRC-3 in allergic response remains unclear. Herein we used passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) mouse models to assess the role of SRC-3 in allergic response.SRC-3-deficient mice exhibited more severe allergic response as demonstrated by a significant drop in body temperature and a delayed recovery period compared to wild-type mice in PSA mouse model, whereas no significant difference was observed between two kinds of mice in PCA mouse models. Mast cells play a pivotal role in IgE-mediated allergic response. Antigen-induced aggregation of IgE receptor (FcϵRI) on the surface of mast cell activates a cascade of signaling events leading to the degranulation and cytokine production in mast cells. SRC-3-deficient bone marrow derived mast cells (BMMCs) developed normally but secreted more proinflammatory cytokines such as TNF-α and IL-6 than wild-type cells after antigen stimulation, whereas there was no significant difference in degranulation between two kinds of mast cells. Further studies showed that SRC-3 inhibited the activation of nuclear factor NF-κB pathway and MAPKs including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in antigen-stimulated mast cells.Our data demonstrate that SRC-3 suppresses cytokine production in antigen-stimulated mast cells as well as PSA in mice at least in part through inhibiting NF-κB and MAPK signaling pathways. Therefore, SRC-3 plays a protective role in PSA and it may become a drug target for anaphylactic diseases. Mast cells play a pivotal role in IgE-dependent allergic diseases such as allergic rhinitis, asthma and anaphylaxis ,2. IgE a-/- mice are highly susceptible to LPS-induced lethality [E. coli-induced peritonitis [Steroid receptor coactivator-3 (SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM-1) is a member of p160 coactivator family that also includes SRC-1 and SRC-2, which interacts with nuclear receptors and other transcription factors to enhance their effects on target gene transcription . SRC-3 dethality  and are itonitis . In addiitonitis . These r-/- mice to determine the role of SRC-3 in IgE-mediated anaphylaxis. We found that SRC-3-/- mice suffered severe passive systemic anaphylaxis than wild-type mice. In addition, SRC-3 suppressed cytokine production in antigen-stimulated mast cells at least in part through inhibiting MAPK and NF-κB pathways. These results demonstrate that SRC-3 plays a protective role in passive systemic anaphylaxis.Anaphylaxis is a severe, systemic allergic reaction involving the respiratory and cardiovascular systems, usually with additional cutaneous and/or gastrointestinal features . Traditiin vivo role of SRC-3 in allergy, we examined the mast cell dependent, IgE-mediated PSA reaction, an extreme form of allergic response [-/- and wild-type mice. Passive systemic anaphylaxis was elicited by injecting of 10 μg anti-DNP IgE intravenously, 24 hrs later, mice were administrated with DNP-human serum albumin (DNP-HSA) antigen by intravenously injection, and then core body temperature was monitored at indicated time intervals. As shown in Figure -/- mice compared to wild-type mice. The recovery of body temperature began at 15 min in wild-type mice while this event occurred at 40 min in SRC-3-/- mice. These results suggest that the allergic reaction is more severe in SRC-3-/- mice compared to wild-type mice in PSA animal model.To determine the response , in SRC--/- mice were intradermally injected with anti-DNP IgE, then DNP-HSA and Evan’s blue dye were injected 24 h later. After IgE and DNP-HSA treatment, the vascular permeability increased to allow the Evan’s blue dye to leak from the blood vessels. As shown in Figure -/- and wild-type mice. However, there was no significant difference in the extent of dye leakage between these two kinds of mice.To further investigate the role of SRC-3 in anaphylaxis, we performed another allergic mouse model named passive cutaneous anaphylaxis (PCA). In PCA, local extravasation is induced by local injection of anti-DNP IgE and intravenous injection of DNP-HSA . The ear-/- and wild-type bone marrow cells with BMMC complete medium for 5 weeks. As shown in Figure -/- and wild-type BMMCs, indicating that SRC-3 deficiency does not affect the development and maturation of BMMCs.To further assess the function of SRC-3 in mast cell-mediated anaphylaxis, BMMCs were used. Mast cell progenitors in the bone marrow can be induced by IL-3 to further proliferate and differentiate into BMMCs. Mature BMMCs express several kinds of receptors, among which FcϵRI and c-kit are most well-known . Therefo-/- and wild-type mice showed no significant difference. This data suggest that enhanced anaphylaxis in SRC-3-/- mice are not due to enhanced degranulation.It has been demonstrated that IgE-mediated mast cell activation and allergic response show the features of degranulation and inflammatory mediator production . To dete-/- BMMCs produced more IL-6 and TNF-α than wild-type BMMCs after different concentrations of antigen treatment overnight (p < 0.05). Further study showed that the mRNA levels of IL-6 and TNF-α in SRC-3-/- BMMCs were higher than that in wild-type BMMCs after antigen stimulation (p < 0.05). These results indicate that SRC-3 inhibits cytokine production at both protein and mRNA levels.Upon stimulation, mast cells synthesize and secrete proinflammatory cytokines ,24. Cyto-/- and wild-type BMMCs. These results are consistent with the observation that there was no difference in degranulation between SRC-3-/- and wild-type mice.In mast cell, re-exposure to allergen triggers cross-linking of IgE/FcϵRI, which activates Src family protein tyrosine kinases such as Lyn and Fyn. Lyn initially phosphorylates Syk and then activates phospholipase (PL)Cγ1, a critical enzyme for generation of the calcium signal for degranulation . We inve-/- BMMCs produced more IL-6 and TNF-α compared with wild-type BMMCs after IgE and antigen stimulation, we explored whether SRC-3 exerts its effect on IKK-IκB-NF-κB pathway. As shown in Figure -/- BMMCs. These data demonstrate enhanced activation of IKK-IκB-NF-κB pathway in SRC-3-/- BMMCs.The cross-linking of allergen-specific IgE bound to its high-affinity receptor FcϵRI results in a series of molecular events leading to NF-κB activation and subsequent cytokine production ,27. Phos-/- BMMCs produced more cytokines including IL-6 and TNF-α than wild-type BMMCs, and activation of MAPKs such as ERK1/2, p38, and JNK are also required for cytokine production by BMMCs in response to antigen stimulation [-/- and wild-type BMMCs. As shown in Figure -/- BMMCs than that in wild-type BMMCs. These results indicate that MAPK activation is enhanced in SRC-3-/- BMMCs.Given that SRC-3mulation , We exam-/- mice suffered more severe IgE-induced passive systemic anaphylaxis but not passive cutaneous anaphylaxis compared to wild-type mice, indicating a potential role of SRC-3 in anaphylaxis. Mast cells were considered to be the important effector cells for IgE-induced anaphylaxis. The high affinity receptor for IgE (FcϵRI) is critical for mast cell development and function [In this study, we found that SRC-3function . Cross-lfunction . Given t-/- and wild-type mice and treated them with IgE/DNP-HSA. Degranulation and cytokine production were observed in both SRC-3-/- and wild-type BMMCs after stimulating with IgE/DNP-HSA. However, SRC-3-/- BMMCs released higher amounts of IL-6 and TNF-α than that from wild-type BMMCs. Therefore, more severe passive systemic anaphylaxis in SRC-3-/- mice is due to increased cytokine production in mast cells. No difference was observed in degranulation between wild-type and SRC-3-/- BMMCs, explaining no significant difference in passive cutaneous anaphylaxis between wild-type and SRC-3-/- mice.To corroborate the role of SRC-3 in IgE-dependent mast cell activation, we obtained mouse BMMCs from SRC-3+ influx, which is a key regulator for mast cell degranulation [-/- and wild-type BMMCs. Activation of NF-κB and MAPKs including ERK1/2, JNK and p38 is important for the cytokine production in mast cells [-/- BMMCs produced more cytokines than wild-type BMMCs, we examined whether SRC-3 exerts its effect on NF-κB and MAPK signaling pathways. Our results showed that the phosphorylation of IκB and NF-κB subunit p65, ERK1/2, JNK and p38 were increased in antigen-stimulated SRC-3-/- BMMCs as compared to wild-type BMMCs, suggesting that SRC-3 negatively regulates NF-κB and MAPK signaling pathways.In antigen-stimulated mast cells, Src family kinases such as Lyn and Fyn are activated and subsequently phosphorylate Syk, a central signaling molecule that can activate the downstream signaling molecules including PLCγ1 and MAPKs . Activatnulation . Our stust cells . Cross-lst cells ,27. Sincst cells , and SRCIKK is known to be responsible for the phosphorylation of IκB and p65 and the subsequent activation of NF-κB. However, the phosphorylation of IKK was not affected by SRC-3 deficiency, indicating that SRC-3 inhibits the activation of NF-κB downstream of IKK. It has been shown that SRC-3 is able to associate with IKKs to enhance or suppress the activation of NF-κB on cellular context dependent manner ,16. In oIt is interesting that SRC-3 could inhibit IgE-induced activation of MAPK pathway by blocking the phosphorylation of JNK, p38 and ERK. JNK, p38 and ERK have been shown to be able to phosphorylate multiple sites on SRC-3 , implicaRPMI 1640, DMSO, sodium pyruvate, antibody against β-actin and flag, anti-DNP IgE (clone SPE-7) were obtained from Sigma Aldrich ; DNP-human serum albumin (DNP-HSA) was purchased from Biosearch Technologies ; recombinant murine SCF and IL-3 were purchased from peprotech ; anti-mouse CD117 (c-Kit)-PE, anti-mouse FcϵRI-FITC, IL-6 ELISA kit, and TNF-α ELISA kit were obtained from eBioscence ; nonessential amino acid was purchased from Gibco ; 2-mercaptoethanol was obtained from AMRESCO ; fetal bovine serum (FBS) was obtained from Hyclone ; antibodies against IKKβ, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, p65, phospho-p65, PLCγ1, phospho-PLCγ1 and SRC-3 were purchased from Cell Signaling Technology ; antibody against IκBα and Syk was obtained from Santa Cruz Biotechnology ; antibody against phospho-Syk was purchased from ProSci .-/- mice and wild-type littermates (6–8 weeks age) on a BALB/c background were used for all experiments. Animals were maintained with specific pathogen free air at a temperature between 20 and 23°C with 12-h light and dark cycles and relative humidity of 50%. Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. All animal experimental procedures were approved by Animal Care and Use Committee of Xiamen University (Protocol Number: XMULAC20120001). Every effort was made to reduce the suffering of animals.Female SRC-3BMMCs were cultured as described with modifications ,5. Bone 2, 5 mM KCl, 1.4 mM CaCl2, 5.6 mM glucose, and 1 mg/ml BSA, pH 7.4) and resuspended in Tyrode’s buffer at 2 × 105 per well in 96 wells. Then, cells were stimulated with DNP-HSA for 1 h. After induction, cells were harvested with 1% Triton X-100, and cell lysis or medium were mixed with p-nitrophenyl-N-acetyl-β-D-glucosamide (1 mM). The reaction was terminated by 100 μl of 0.2 M glycine (pH 10.7) after incubating for 1 h, and OD was read at a wavelength of 405 nm. Values are expressed as the percentage of intracellular β-hexosaminidase released into the medium.β-HEX release assay was performed as previously described with modification ,34,35. BMeasurement of IL-6 and TNF-α production by BMMCs was performed as described with modifications . BMMCs wPSA was performed as previously described with modification ,21. MicePCA was performed as previously described with modification . Mice weTotal RNA was isolated with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The cDNA was synthesized from 2 μg of total RNA using MMLV transcriptase  with random primers, Real-time PCRs were performed using SYBR Premix ExTaq . Quantification was normalized to the amount of endogenous GAPDH. Primers used for Real-time PCR are listed on Table Cells were lysed with lysis buffer (200 mM Tris–HCl (pH 7.5), 1.5 M NaCl, 10 mM EDTA, 25 mM sodium pyrophosphate,10 mM glycerolphosphate, 10 mM sodium orythovanadate, 50 mM NaF, 1 mM PMSF, in combination with protein inhibitor cocktail). Thirty micrograms of protein lysates of each sample was subjected to SDS-PAGE and transferred onto nitrocellulose membranes. Blots were incubated with the specific primary antibodies overnight at 4°C. After being washed three times for 15 min each with TBST (TBS + 0.1% Tween20), blots were incubated with horseradishperoxidase-conjugated secondary antibody  and visualized by chemiluminescence. The band density was quantified by densitometry using Scion Image software and normalized to β-actin levels.Cells were transfected with the indicated plasmids by using Calcium chloride. At 48 hours post-transfection, cells were harvested and then used for further experiments.For coimmunoprecipitation (Co-IP) assay, cells were lysed with lysis buffer. Cell lysates were immunoprecipitated by correspondent antibodies or control immunoglobulin G (IgG). After extensive washing, precipitates were analyzed by Western blotting.Data were collected from several independent experiments, with three replicates per experiment. All data were analyzed with one-way ANOVA with post-Tukey’s post test in SPSS 11.0 and p < 0.05 was considered statistically significant. Bars in the graph represent standard deviation (S.D.).The authors declare that they have no competing interests.XX and CY designed the overall study; XX, WW, PM, QC, KL, MS performed the experiments and data analysis; JX provided animals; XX and CY wrote the manuscript. All authors read and approved the final manuscript.(A) Co-IP analysis of the interaction between SRC-3 protein and IKKβ protein in 293 T cells. (B) SRC-3 interacted with IKKβ through its S/T and HAT domains.IKKβ interacts with SRC-3 through the S/T and HAT domains of SRC-3. Click here for file


Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein’s dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3'-OH of 2'-deoxyuridine-5'-monophosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function.Recent studies of  The role of protein dynamics in enzyme catalysis is one of the open questions in enzymology today. In particular, the participation of residues distal to the active site in catalyzing bond-activation at the active site is a topic of interest as it combines several enigmas: the long range communication in the enzyme, its “holistic” nature, and the different catalytic strategies applied by the same active site to catalyze different chemical conversions. Recent experimental evidence suggests that mutations distant from the active site affect chemical bond activations in numerous enzymatic studies including dihydrofolate reductase ,2,3,4, hR)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate) as a cofactor and 2'-deoxyuridine-5'-mono-phosphate (dUMP) as the substrate  to k9/k4 when [B] goes to zero. In the random mechanism (Cf goes to k9/k5 at infinite [B] and to k9/(k4 + k5) at zero [B]. Consequently, the KIEobs in the ordered mechanism vary between Tk9 at zero [B] to unity at high [B], while in the random mechanisms, KIEobs vary between two finite values.The commitment chanisms A, it is echanism B, Cf goeobs on the proton abstraction step of the Y209W mutant was studied as a function of the concentration of CH2H4folate. As apparent from obs for the WT approaches unity at higher concentrations (1 mM) of CH2H4folate, suggesting a strictly ordered binding mechanism in agreement with previous product inhibition and steady-state studies  dUMP (specific radioactivity 53 Ci/mol) and [5-3H] dUMP (specific radioactivity 13.6 Ci/mmol) were from Moravek Biochemicals. Unlabeled CH2H4folate was a generous gift from Merck and Cie . The WT and Y209W ecTSase enzymes were expressed and purified following a published procedure  dUMP was carried out by the published procedure of Wataya and Hayatsu [l-cysteine (pD = 8.8) and 1 mM [2-14C] dUMP in D2O solution (>99.96% D). The reaction mixture was incubated at 37 °C for 7 days until complete deuteration (>99.5% D) was achieved and verified by 1H NMR measurements.The synthesis of  dUMP, and 1.5 Mdpm [5-3H] dUMP. Varied concentrations of CH2H4folate (50–1000 μM) were added to the reaction mixture and preincubated at 25 °C. All the experiments were performed following the previously published procedure with a modified HPLC method as given in t∞) were obtained by adding concentrated WT TSase. Two independent time points with no enzymes (t0) were obtained as controls for the experiment. The competitive observed KIEs on the second order rate constant were calculated from the following equation  dUMP instead of [2-14C] dUMP. The intrinsic KIEs for the proton abstraction step were calculated using the Northrop method as in Equation (8) [ki is the rate constant for the reaction involving isotope I and T(V/K)Hobs and T(V/K)Dobs are the observed competitive KIE values on the second order rate constant. Although kT/kH, the reciprocal of kH/kT (intrinsic KIE) is the only unknown in Equation (8), it cannot be solved analytically. This equation was solved numerically using the program developed in our group. This program is available on our web site, http://cricket.chem.uiowa.edu/~kohen under “Tools”.To determine the intrinsic KIEs for the proton abstraction step, 200 μM CHtion (8) ,35,42: with the WT enzyme and altered anisotropic B-factors. We compared the temperature dependence of intrinsic KIEs on two consecutive steps in the enzyme’s catalytic cycle: the fast and non-rate limiting proton abstraction  indicated that other steps apart from the two C–H bond activations in question have also been altered. The commitments (Cf on V/K) of both H-transfers show a break above 25 °C, indicating an effect of Y209W that could be common to both steps. This observation would be in accordance with a step that precedes the proton transfer that is affected by the mutation. Such a step could be the release of CH2H4folate from the ternary complex, which would also be in accordance with higher KM for that substrate, and the thiols attack on the dUMP intermediately prior to hydride transfer, indicating poor folate orientation in the active site.The proton abstraction step, on the other hand, is not nearly as challenging following the Michael addition of Cys to C6 of the substrate , and affects several kinetic steps along the complex catalytic cascade of this enzyme. These results support a dynamic coupling of a remote residue to different kinetic events across the enzyme. The results further emphasize the importance of amino acid residues controlling long-range protein dynamics critical for the enzyme function.


Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP) but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP) causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity. Proteins were resolved by 12% SDS-PAGE. Incorporation of 32P was visualized by autoradiography.CBS-WT and CBS mutant immunocomplexes  were incubated at 30°C in kinase buffer , or PKA inhibitor, KT5720 (10 μM) and then treated with 8-Br-cGMP (100 μM) or d-cAMP (100μM) for 5, 15 or 30 min respectively. Next, CBS was specifically immunoprecipitated from protein extracts by using antibodies against the endogenous protein. Immunoprecipitated proteins were separated by SDS–PAGE and the presence of a phosphorylated CBS was evaluated by western blotting using anti-phospho-(Ser/Thr) .Starved T24 cells were transiently transfected with constructs encoding for HA-CBS WT or HA-CBS mutants. After transfection (24h), cells were treated or not with 100 μM of 8-Br-cGMP for 15 min. Samples were collected and WT or mutant proteins were specifically immunoprecipitated by using antibodies against the epitope HA. Immunoprecipitated proteins were separated by SDS–PAGE and the presence of the proteins and their phosphorylated form was investigated by western blotting analysis using anti-phospho-(Ser/Thr).227NPLAHYD) containing a single phosphorylated Ser at position 227 has been selected to obtain polyclonal antibodies in rabbits, namely anti-pCBSSer227 . The purified antibody was used at 1:400 dilutions for western blotting.Peptide from amininoacids 225–234  followed by Bonferroni, as needed. A p value <0.05 was considered significant.2S in basal condition (i.e. in presence of the vehicle). The incubation with L-cysteine (the substrate) significantly increases H2S production  , we chosp<0.001, , as reve-CBS S227A and HA-CBS S525A mutants are still increased as compared to their matched control  ii) the abrogation of 8-Br-cGMP-mediated increase in H2S production (p<0.001) both compared to WT  efficiently recognizes CBS protein in human urothelium and in T24 cells  the important role played by the urothelium in bladder pathophysiology ii) the availability of a well characterized human urothelial cell line such as T24 cells.2S pathway in human urothelium, comparing this profile with T24 cell line. We found that both human urothelium and T24 cells express CBS and CSE and efficiently convert L-cysteine into H2S. Having determined that both enzymes are expressed and are catalytically active, we assessed whether incubation with 8-Br-cGMP or d-cAMP increases H2S production. We have demonstrated that 8-Br-cGMP but not d-cAMP causes a significant increase in H2S production in both urothelium and T24 cells. The specificity of the effect of 8-Br-cGMP was confirmed by the reversion operated by KT5823, a selective inhibitor of PKG. These results differed from our previous findings where both 8-Br-cGMP and d-cAMP caused an increase in H2S production in full human bladder thickness (detrusor muscle plus urothelium) [versus PKG in the human bladder. Western blot analysis for PKG and PKA in human urothelium, detrusor muscle, whole bladder and T24 cells clearly show that urothelium as well as T24 cells strongly express PKG as opposite to PKA. This finding explains the lack of the effect of d-cAMP observed in human urothelium and urothelial T24 cells. Having well characterized that human urothelium and T24 cell line act similarly, we have sought to investigate CBS phosphorylation by stimulating T24 cells with 8-Br-cGMP. A clear time-dependent signal of CBS phosphorylation was observed in CBS isolated from T24 cells stimulated with 8-Br-cGMP. The phosphorylation reached the maximum effect at 15 min declining thereafter. CBS phosphorylation in presence of KT5823, a PKG inhibitor, was blunted. In agreement with the finding that both human urothelium and T24 cells have a very low level of PKA, d-cAMP did not trigger CBS phosphorylation. These data strongly indicate that CBS phosphorylation is PKG-dependent. Due to the lack of selective CBS inhibitors [2S in urothelium, we stably silenced CBS in T24 cells. CBS silencing markedly reduces H2S production, confirming the major role played by CBS in triggering the H2S production. In summary, we have demonstrated that i) T24 cell line mimics human urothelium behaviour ii) cGMP/PKG pathway activation triggers CBS phosphorylation iii) CBS-derived H2S is the major source of H2S in T24 cells. Through a computational approach, we searched for possible PKG-phosphorylation sites in CBS protein, focusing on the aminoacid sequence known as PKG target consensus, distinct from sequence predicted as PKA-phosphorylation sites. Predicted PKG-phosphorylation sites in CBS were Ser32, Ser227, and Ser525. To experimentally validate these Ser as sites of phosphorylation by PKG, we performed a phosphorylation assay by using proteins from T24 cells transiently transfected with plasmids expressing HA-CBS WT or HA-CBS mutants . The mutation of Ser227 and Ser525 but not Ser32 causes a drastic reduction of CBS phosphorylation. Furthermore, HA-CBS S227A and HA-CBS S525A mutants display a significantly reduced level of phoshorylation when incubated with 8-Br-cGMP. Taken together, these findings show that among predicted PKG-phosphorylation sites, only Ser227 and Ser525 were experimentally validated as sites of phosphorylation by PKG.As a first step we defined whether CBS and/or CSE were involved in driving the Hthelium) . In ordehibitors , in ordes to the enzyme activity and ultimately to the extent of H2S production, we transiently transfected cells lacking CBS (CBSΔT24) with HA-CBS S525A, HA-CBS S227A mutants or HA-CBS WT as a control. Treatment with 8-Br-cGMP did not induce a significant production of H2S in the S227A mutant as opposite to S525A mutant. Indeed, the production of H2S in S525A mutant is comparable to that induced by 8-Br-cGMP in CBSΔT24 cells transfected with HA-CBS WT. Therefore only the phosphorylation at Ser227 in CBS protein increased H2S production.To assess the contribution of each phosphorylation siteSer227 recognizes the CBS phosphorylated form in both urothelium and T24 cell line. Challenge of T24 cell with 8-Br-cGMP causes a clear increase in the signal of the anti-pCBSSer227 confirming and validating our previous findings. The specificity of the antibody was also confirmed by the finding that Ser227Ala mutant is not recognized by the anti-pCBSSer227. Thus the phosphorylation at Ser227 site is necessary for recognition of CBS protein by this specific antibody. Interestingly even though Ser525 is also phosphorylated in a PKG-dependent manner, mutation at this residue does not affect H2S production. Ser 525 lies within a non-consensus CBS domain that is described as a regulatory region target of the allosteric modulator S-adenosyl methionine. Therefore, it is reasonable to postulate that phosphorylation of Ser 525 may be implicated in the modulation of additional features of CBS protein not as yet determined.In order to further confirm our data we generated a specific antibody against the phosphorylated form. The anti-pCBSs that in human urothelium as well as in T24 cells CBS-dependent H2S generation involves cGMP/PKG mediated phosphorylation of CBS at Ser227.Although additional experiments will be required to understand the control of CBS function by phosphorylation, it is evident that this covalent modification increases CBS activity. Such an outcome may include conformational changes which could allow more efficient binding to substrates or allosteric modulators and regulation of potential binding partners. Herein we demonstrate2S production, we suggest that this mechanism dynamically contributes to bladder homeostasis by controlling detrusor muscle contractility [In the current literature it is widely accepted that the urothelium influences the contractile state of detrusor smooth muscle ,38. Indeactility .2S production driven by CBS, often ascribed in the relevant literature to a generic change in activity, could involve CBS phosphorylation.Finally, the results of this study may not solely be not confined to the bladder since they introduce the concept that changes in H2S production is markedly reduced thus supporting a relevant contribution of CBS in triggering H2S production in urothelial cells iii) CBS phosphorylation by PKG at Ser227 increases endogenous H2S production confirming that, CBS activity can be selectively enhanced in a cGMP/PKG-dependent manner in human urothelium.In conclusion, we have demonstrated that i) incubation of T24 cells with 8-Br-cGMP causes a time-dependent increase in CBS phosphorylation reverted by selective inhibition of PKG ii) in T24 cells stably silenced for CBS, H2S pathway, in any pathology where CBS is reputed to play a role.Finally, the antibody, directed against the phosphorylated form of CBS, may represent a useful tool to define the pathophysiological role of the H


Melosira arctica are more frequently found in the inner part of the Central Arctic pack ice, while rounded aggregates mainly formed by pennate diatoms are found closer to the ice edge, under melting sea ice. On the scale of an ice floe, the distribution of algal aggregates in late summer is mainly regulated by the topography of the ice underside, with aggregates accumulating in dome-shaped structures and at the edges of pressure ridges. The average biomass of the aggregates from our sites and season was 0.1–6.0 mg C m−2. However, depending on the approach used, differences in orders of magnitude for biomass estimates may occur. This highlights the difficulties of upscaling observations and comparing results from surveys conducted using different methods or on different spatial scales.The sea ice cover of the Arctic Ocean has changed dramatically in the last decades, and the resulting consequences for the sea-ice-associated ecosystem remain difficult to assess. Algal aggregates underneath sea ice are of great importance for the ice-associated ecosystem and the pelagic-benthic coupling. However, the frequency and distribution of their occurrence is not well quantified. During the IceArc expedition (ARK-27/3) of RV Polarstern in late summer 2012, we observed different types of algal aggregates floating underneath various ice types in the Central Arctic basins. We investigated the spatial distribution of ice algal aggregates and quantified their biomass, using under-ice image surveys obtained by an upward-looking camera on a remotely operated vehicle. On basin scale, filamentous aggregates of The online version of this article (doi:10.1007/s00300-014-1634-3) contains supplementary material, which is available to authorized users. The Arctic Ocean has changed dramatically in recent decades. Changes of physical processes in the climate system—such as decreased sea ice extent  of the German research icebreaker RV Polarstern to the Central Arctic in August and September 2012. Eight ice stations were selected along the retreating sea ice edge, as well as within the Central Arctic pack ice close to the geographic North Pole , to investigate the algae distribution underneath the sea ice. It was launched through a hole in the ice and operated from a tent directly on the ice floe several hundred meters away from the ship to avoid disturbance from the ship’s thrusters. At each ice station, the ROV achieved a diving time between 6 and 8 h. The setup and operation procedure was similar to the one used in Nicolaus and Katlein  with minAdditional sensors complemented the observations with measurements of the physical properties of the under-ice habitat. Ice draft was calculated as difference between the ROV depth and its distance to the ice measured by the upward-looking DST micron echosounder  altimeter. Spectral light transmittance (320–950 nm) was calculated from continuous synchronous measurements of downwelling irradiance using two RAMSES-ACC spectroradiometers . While one sensor was mounted on the ROV, a reference sensor was placed on a tripod on the ice close to the ROV launch hole. Data processing, calibration, and measurement uncertainties have been described previously  and species composition as described in Assmy et al. .ffmpeg. Extracting the images every 5 s from the video overcomes problems with multiple detection of the same aggregates in consecutive images in most cases. To account for inconsistent lighting at the image edges, and to mask out the overlay data display, we cropped the image to obtain undisturbed RGB images of 250 × 200 pixels. Analysis of image histograms showed that aggregates could be detected well with a threshold value, by selecting all pixels with a value between 0 and 100  in the green channel as aggregates. Thus, images were converted into binary images for further analyses.To quantify the spatial distribution of under-ice algal aggregates from the acquired ROV imagery, we applied a threshold algorithm implemented in MATLAB. We extracted still images (384 × 288 pixels) every 5 s from the videos of the upward-looking camera using the command line tool All images taken at a depth >5 m and at ROV tilts >10° were automatically discarded. Images where ice structures or other objects were detected as aggregates and images where clearly identifiable aggregates were not detected by the algorithm were manually discarded. In the case where several close-lying aggregates were mistaken for a larger one, the image was excluded from shape and size analysis. In total, 11,000 images out of 23,800 images were used for further analysis. These images were on average taken at a distance of one meter from the ice underside and resulted in a spatial resolution of 4–5 mm.Two-dimensional aggregate properties such as perimeter and area as well as minor axis and major axis of a fitted ellipse were calculated for each individual aggregate. For all aggregates covering more than 10 pixels of the image, shape parameters such as eccentricity, circularity, and the equivalent circular diameter were derived. The measurements were transformed from pixel units to real units using the distance to the ice measured by the altimeter and a laboratory calibration of the camera. This image registration to true geometric units also enabled us to determine aggregate abundance per square meter as the number of aggregates in the image divided by the area surveyed by the image. Uncertainties in the distance to the ice, ROV-tilt and lens distortion result in <15 % uncertainty in aggregate size measurements.The detection algorithm worked well in most situations, but due to its simple nature, detection was problematic in some cases, and 54 % of the images had to be excluded from the analysis. This was mostly related to inhomogeneous backlighting within the image at the transition between different ice features, where dark features in the ice were misinterpreted as aggregates. In addition, the differentiation of aggregates from small-scale ice structures and air bubbles can be ambiguous even to a trained observer verifying the detection. Finally, aggregates that are not dense enough or too small to leave a significant signature in the pixels green value remain undetected.To account for multiple sampling due to overlapping ROV tracks in some areas , all obtained parameters were gridded on a regular grid with 3 × 3 m cell size, corresponding to the maximal uncertainty in the ROV position. All available images within a grid cell were selected, and the average of all measurements from these pictures was assigned to the grid cell.To estimate aggregate biomass, the two-dimensional distribution deducted from the images was converted into three-dimensional volumetric information, including assumptions about the aggregate shape. While the assumption of a uniform algal layer thickness is more applicable to typical ice algal bottom layers in spring, we chose to represent the typically rounded aggregates by compact spheres. The diameter of the aggregate was determined as the equivalent circular diameter of the connected pixel region from the regions area.We used different methods to obtain an estimate of the aggregate volume per area. Comparison between the different approaches enables us to evaluate the accuracy of our biomass estimates and the disadvantages of individual approaches. Names given in parentheses identify the different approaches later in the text.For a first approach, aggregate volume was calculated for every detected aggregate. The single aggregate volumes were then added up and divided by the survey area (“aggregate list”). This approach does not take into account multiple sampling of some aggregates due to overlapping ROV tracks and should thus result in an overestimate of total biovolume.V was calculated using abundance a and diameter d values averaged over all images :In a second approach, the aggregate volume As sphere volume is dependent on the third power of diameter Eq. , this apTo avoid influences from multiple sampling due to overlapping ROV tracks, the same calculation was repeated with average and median values obtained after gridding of the results (“gridded mean” and “gridded median”). The “gridded median” approach was also used in Assmy et al. .As larger rounded aggregates can occur in some parts of the survey areas, they can contribute significantly to biomass that gets lost when averaging over many grid cells. Thus, in a last approach, biovolume was calculated from abundance and diameter in each grid cell separately and averaged afterwards (“raster cells”). In the case where aggregates deviate substantially from a spherical shape, this approach can significantly overestimate biovolume.−1 of two aggregates of known volume from ice station ICE-7  and in the central pack ice , the stations further down the transpolar drift towards Fram Strait  that were dominated by rounded aggregates formed by pennate diatoms.Mean properties of the detected aggregates are given together with the environmental parameters in Table mentclass2pt{minimp = 0.014). However, some important deviations between the different ice stations could be recognized. A distinct and unexpected feature was that the size distribution on ice stations 3, 5, 6, and 9 flattened out towards larger aggregates indicating enhanced buoyancy.The size distribution of algal aggregates obtained from the image analysis for all stations generally followed the expected power law Fig. . The chaEven though our analysis of upward-looking ROV images is currently the best available method for aggregate quantification on larger scales, one needs to keep in mind some limitations of the method. Firstly, the method is only able to detect macroscopic aggregates bigger than a few millimeters floating directly underneath the sea ice. This relatively high detection limit leads to an underestimation of the total algal aggregate biomass, but might be irrelevant in light of the huge range of biomass estimates caused by the different estimation algorithms. Secondly, close-lying aggregates that are detected as a single one, as well as aggregates with a strong deviation from the spherical shape, can lead to an overestimation of total aggregate volume. To reduce this effect, we excluded clumped aggregates from the analysis. This affects <5 % of the detected aggregates. Thirdly, aggregates are often located in transition zones between different ice types, which at the same time are often discarded during image processing, as the darker background of the thicker ice type gets classified as aggregates by the threshold algorithm. Overall, our method as used in this study might be rather underestimating ice algal aggregates. Future studies could thus benefit from a more sophisticated image classification technique and machine learning for automation of the detection.−2.The patchy spatial distribution of algal aggregates makes accurate, large-scale estimates of the aggregate biomass very challenging. As our results show, not only the choice and range of sampling sites, but also the method of estimating biomass from the data, may heavily impact the estimates. Consequently, small-scale surveys of the under-ice ecosystem such as diver observations are influenced by the choice of sampling sites. When comparing different surveys from ROVs and diver studies, differences of several orders of magnitude might simply arise due to differences in data processing and the size of the survey area. These differences can be even more dramatic when compared to results obtained by classical ice coring that do not usually capture algal aggregates. Larger-scale surveys tend to give a more realistic estimate , it was a better indicator of the total aggregate volume . Aggregate abundance measured using ROV observations off Greenland in June/July varied between <1 and 50 aggregates m−2 , indicating that aggregate biomass is greater within the Central Arctic basin than at the ice edge at the end of the summer. According to the shape analysis  almost 2 months later on 29 September at the end of the productive season, when light availability was strongly reduced. Assuming that the ice floe was in both cases representative for the area and that thus displacement of aggregates by advection can be neglected, we can deduce some information about the changes in the aggregate distribution during that time period. Along with the decrease in aggregate abundance from 2.8 to 0.4 aggregates m−1 POC from our field sites Below is the link to the electronic supplementary material.


Despite its prominent placement between the retina and primary visual cortex in the early visual pathway, the role of the dorsal lateral geniculate nucleus (dLGN) in molding and regulating the visual signals entering the brain is still poorly understood. A striking feature of the dLGN circuit is that relay cells (RCs) and interneurons (INs) form so-called triadic synapses, where an IN dendritic terminal can be simultaneously postsynaptic to a retinal ganglion cell (GC) input and presynaptic to an RC dendrite, allowing for so-called triadic inhibition. Taking advantage of a recently developed biophysically detailed multicompartmental model for an IN, we here investigate putative effects of these different inhibitory actions of INs, i.e., triadic inhibition and standard axonal inhibition, on the response properties of RCs. We compute and investigate so-called area-response curves, that is, trial-averaged visual spike responses vs. spot size, for circular flashing spots in a network of RCs and INs. The model parameters are grossly tuned to give results in qualitative accordance with previous in vivo data of responses to such stimuli for cat GCs and RCs. We particularly investigate how the model ingredients affect salient response properties such as the receptive-field center size of RCs and INs, maximal responses and center-surround antagonisms. For example, while triadic inhibition not involving firing of IN action potentials was found to provide only a non-linear gain control of the conversion of input spikes to output spikes by RCs, axonal inhibition was in contrast found to substantially affect the receptive-field center size: the larger the inhibition, the more the RC center size shrinks compared to the GC providing the feedforward excitation. Thus, a possible role of the different inhibitory actions from INs to RCs in the dLGN circuit is to provide separate mechanisms for overall gain control (direct triadic inhibition) and regulation of spatial resolution  of visual signals sent to cortex. While the basic receptive-field structure of cells in the dorsal lateral geniculate nucleus (dLGN), the station between retina and visual cortex in the early visual pathway, was mapped out half a century ago, the function of this nucleus in molding the visual signals is still poorly understood. One reason is that the dLGN contains enigmatic inhibitory interneurons which can act with different inhibitory action on the excitatory relay cells. In addition to standard axonal inhibition, relay cells and interneurons form so-called triadic synapses, where an interneuron dendritic terminal can be simultaneously postsynaptic to a retinal input and presynaptic to a relay-cell dendrite, opening up for so-called triadic inhibition. Taking advantage of a recently developed biophysically detailed multicompartmental model for an interneuron, we here use a network model to investigate putative effects of these inhibitory actions on the response properties of relay cells stimulated by circular flashing spots. Our results suggest a possible role of the different inhibitory actions in providing separate mechanisms for overall gain control (triadic inhibition) and regulation of spatial resolution  of visual signals sent to cortex. Here Θ(x) is the Heaviside step function, and an activity function l(L) converting luminance to firing rates has been introduced, i.e., lbkg ≡ l(Lbkg), lspot ≡ l(Lspot). The ‘0’ in the notation Gg signifies that the spot and receptive fields are concentric, i.e., a distance zero between their centers.We further assume that the total neuronal response is given as a sum of the inputs caused by the spot with luminance ulus see , the resra from the spot center  is the so-called incomplete gamma function given byn is an integer larger than zero. Note that for ra = 0, ra from the central GC cell.Four of the GC neurons driving the dLGN circuit have receptive fields that are not concentric with the spot, however. Rather, their receptive field centers are displaced a distance t center . In thisω, lbkg, lspot, a1, and a2, as well as the distance between central and peripheral GC centers ra. Here we assumed the five GC neurons providing the inputs to the dLGN circuit to have the same response properties, i.e., the same values of ω, lbkg, lspot, a1, and a2. The parameters used here were found in 27], i.e.In the present application of the model we only considered the response of the IN and the central RC.dc was determined numerically by identifying the spot diameter that produced the maximum response rc, see ds was given by the spot size diameter producing the minimum response rcs, and at the same time fulfilling ds > dc.The receptive-field center diameter From these four quantities we calculated several response measures: The ratio d tuning , 27. In rc) and the minimum response when the surround is stimulated as well (rcs) [As a measure of how much the center response is reduced by the surround (center-surround antagonism), we also calculated the normalized difference between the maximum response to center stimulation (ll (rcs) , 27:α= were repeated 10 times or more (see above) with different seeds resulting in a total of more than one million simulations.With a relatively low total segment count (177) for the multi-compartment IN model, each network instance was simulated serially in a matter of seconds at a temporal sampling rate of exp_onset_generator.The GC model was implemented in NEST  as a spiSimulations were performed on a compute cluster with Intel Xeon 2 CPUs running Linux 2.6.32 using NEURON 7.3 and NEST 2.3.r10450. Software was compiled with the GNU Compiler v. 4.7.2 and linked against the GNU Science Library v. 1.14. Trials were configured using the NeuroTools.parameters package . Data anResults were stored in HDF5 files using the PyTables package. Further analysis was performed using Pandas/NumPy and Matplotlib for visualization.Before embarking on the dLGN circuit behavior, we demonstrate in + channels, after which the potential decays from subsequent activation of K+ (and deactivation of Na+) channels. The distal-dendrite membrane potential is observed to remain at a relatively depolarized level, i.e., above –50 mV, for an extended period of time . The endured response is partly due to the activation of local T-type Ca2+ channels, as we have shown previously [When a single GC spike arrives at a distal IN synapse (panel B), the response is partly mediated by local, active ion channels. The distal dendrites undergoes a rapid, local depolarization (up to ∼0 mV) due to activation of local Naeviously . Due to A single spike arriving at a proximal synapse results in only a small depolarization of the membrane potential (panel C), i.e., too little to evoke either triadic inhibition or generate a somatic action potential which in turn would provide axonal inhibition. Further, when a single distal and a single proximal synapse positioned on the same branch are activated at the same time (panel D), the resulting soma potential is still too small to generate an action potential. However, when all five proximal synapses are activated by simultaneous spikes (panel E), a somatic axon potential is generated which next provides axonal inhibition on postsynaptic RC cells. Moreover, this axonal action potential back-propagates into the dendrites where it also activates triadic inhibition. This latter type of triadic inhibition is here denoted soma-driven triadic inhibition.The mechanism behind the two types of triadic inhibition, i.e., ‘direct’ and ‘soma-driven’, is illustrated in In soma-driven triadic inhibition a somatic action potential in the IN, induced by sufficiently synchronous excitatory GC inputs onto the proximal dendrites cf. , resultstime-locked, or simply locked inhibition in the experimental literature [terature .+ and K+ channels in mediating the local response induces an effective refractory period (the channels do not have time to reset between two input spikes). This is evident during the first 50 milliseconds or so after stimulus onset, when the firing-rate of the central GC cell is so high that not all incoming spikes result in the distal-dendrite membrane potential passing firing threshold . Direct triadic inhibition will therefore not occur at every input spike. Such a depression of triadic inhibition for high input rates was also seen experimentally [Some key features of the dynamics of the triadic circuit when stimulated by a flashing circular post, are illustrated in mentally .Panel B in wIRt = 0) in panel C, and only triadic inhibition  in panel D. For these particular model parameters we see that the peak response in the PSTH following stimulus onset is largest for the GC (∼200 s−1) and smallest for the IN (∼50 s−1). For the RC we see that both the background  and peak responses are larger for the case with axonal inhibition (panel C) than for triadic inhibition (panel D), implying that for the present choice of model parameters the triadic inhibition is more efficient than axonal inhibition in reducing RC firing.As the spiking response to individual stimulus presentations typically varies between trials, the post-stimulus time histogram (PSTH)  is commoWe now move on to compute and investigate area-summation curves, that is, the time-average of PSTHs of the type shown in In the present modeling study we in particular investigate the effects of various types of inhibition on the area-summation curves of the RC and IN neurons. Examples of such calculated area-response curves are given in (RC-i)Direct triadic inhibition: Triadic inhibition driven by GC input on same distal IN dendrite only, i.e., wIRt > 0, wGIp = 0, wIRa = 0(RC-ii)Direct & soma-driven triadic inhibition: Triadic inhibition driven both by GC input on same distal IN dendrite and back-propagated soma activation in turn stemming from proximal inputs on the IN, i.e., wIRt > 0, wGIp > 0, wIRa = 0(RC-iii)Axonal inhibition: Axonal inhibition of RC following firing of action potential in the IN, i.e., wIRt = 0, wGIp > 0, wIRa > 0.The figure also shows the resulting area-summation curve when all these three types of inhibition is included at the same time.receptive-field center, and we observe that while the central GC cell has a center diameter A first observation in For the case with direct triadic inhibition only (RC-i) we observe that while this inhibition reduces the RC firing rate by about a factor two compared to the GC input  compared to the peak response, seen for the cases where the inhibitory effects are the strongest . The reason is that the transfer ratio, i.e., the fraction of incoming GC spikes resulting in an outgoing RC spike, is already quite high even for this lowest weight. This leaves limited room for further increase in the response. Another observation is that without inhibition the RC and GC response curves always have their maxima at the same spot diameter, i.e. For reference we show in the top row of wGR (11.6 nS). Unlike in the case with no inhibition, increased excitation strength wGR is seen to increase the RC response as extra excitation will compensate for the added direct triadic inhibition. We further see that the shapes of the RC response curves are similar to the ‘no-inhibition’ curves, the main difference is a vertical shift of the response curves. Such a vertical shift implies a larger relative reduction of the full-field response compared to the center response, i.e., an increased center-surround antagonism. Thus direct triadic inhibition increases the RC center-surround antagonism αR, particularly for the lower excitatory weights. The RC receptive-field center size With direct triadic inhibition included (second row in the soma . Thus dithe soma . Since dwGIp), while different columns still correspond to different values of the retinogeniculate excitation (wGR). The area-response curve of the IN is, of course, independent of the value of wGR, so the same IN response curve is seen in the same-row panels. By comparing area-response curves for increasing values of proximal IN excitation wGIp we see, as expected, a large increase in the IN response. The increased response is also accompanied by a reduction in center-surround antagonism of the IN neuron. However, the IN receptive-field center size The three lower rows of wIRa. The topmost curves correspond to the situation without axonal inhibition (wIRa = 0), while the three other curves corresponds to different non-zero values of wIRa (2/4/8 nS) with the lowest curve corresponding to the largest weight considered (wIRa = 8 nS). It is seen that not only does increased axonal inhibition reduce the RC response, it also reduces the RC receptive-field center size wGIp of the IN is largest.In each of the nine panels in the lower three rows in αR, αI), and maximum firing rates vary with the axonal inhibition weight (wIRa) for a set of different values of the weight of ganglion-cell activation of the RC (wGR) and of the proximal dendrites of the IN .The effects of the various model components and parameters on key response measures for the results in wGIp) and when the axonal inhibition weight from the IN to the RC (wIRa) are increased (panel A). This is as expected as both these weights determine the overall axonal inhibition of the RC providing the shrinkage of the RC receptive-field center [wGIp). This increase is quite modest, however, and most of the observed variation in the ratio between the IN and RC center sizes (A first observation is that the receptive-field center size of the RC (d center . In contαR) is seen to be almost independent of the axonal inhibition weight wIRa (panel C). This implies that the RC center-surround antagonism is little affected by axonal inhibition.The center-surround antagonism for the RC (wGR = 11.6 nS), αR does not depend much on somatic IN activity (panel C): αR is large, about 0.7, for all considered values for wGIp. However, for the two largest values of the ganglion-cell input weight to the RC  some variation with wGIp is observed: For example, for wGR = 15.6 nS, αR is seen to vary between ∼0.4 for wGIp = 0 to ∼0.6 for wGIp = 1.8 nS. As wGIp = 0 corresponds to the case with direct triadic inhibition only  this substantial increase in αR must be due to soma-driven triadic inhibition. Thus while direct triadic inhibition alone is seen to be sufficient to assure a large centre-surround inhibition when the retinogeniculate excitation wGR is weak, soma-driven triadic inhibition can provide the same when the retinogeniculate excitation is strong.For the smallest values of the weight of the ganglion-cell input to the RC (αI) is generally much lower than for the RC [wGIp .The center-surround antagonism for the IN (r the RC  and is swIRa) and increasing synaptic input onto the proximal dendrites of IN (wGIp) (solid lines in wGIp) increases .The maximum firing rate wIRt = 0. In this case where only axonal inhibition acts on the RCs, we observe as expected less reductions of RC responses, particularly for the smallest considered values of wGIp and wGR. However, as confirmed by the corresponding parameter dependence of the key response measures shown in wIRa) and increasing ganglion-cell drive onto proximal IN dendrites (wGIp). This is also the case for the maximal RC firing rate In αR, panel C) is seen to generally be lower when triadic inhibition is absent, cf. with triadic inhibition  response is reached around 100 ms after onset. This is in qualitative accordance with observations in flashing-spot experiments on cat RCs [So far we have only considered the spike-count rate pooling all spikes within the whole 500 ms time interval following stimulus onset in our simulations. As seen in  cat RCs , 35, 51.wGR than the one used in this example, cf. In Comparison of the normalized area-response curves , a feature not present for the transient response (panel C). This minimum stems from the strong activation of the INs for these spot sizes (cf. blue curve in panel D) compared to for the larger spot sizes, i.e., d∼8–10 degrees. While this feature of the IN response curve is also present for the transient response, it is slightly less so. The more prominent role of the combined triadic and axonal inhibition in modifying the receptive-field of the RC (compared to the GC) in the sustained response than in the transient response is also manifested by the slightly smaller receptive-field center size  on the visual response properties of dLGN relay cells (RCs) relaying visual information to cortex. Taking advantage of a recently developed biophysically detailed multicompartmental model for an IN, the study is the first investigating the effects of different inhibitory actions of INs, i.e., triadic inhibition due to dendrodendritic contacts between INs and RCs and standard axonal inhibition. The interplay of these two inhibitory pathways is expectedly essential for understanding the functional role of inhibition in the dLGN circuit , 53. To e X-type  and (ii)e X-type , we havecreceptive-field center size d of RCs and INs, identified as the flashing spot diameter giving the largest spike-count firing-rate reponse, (ii) the maximal firing rate (occuring for spots exactly filling the receptive-field centers), and (iii) the center-surround antagonism α measuring the suppression of large-field responses compared to the maximal responses. A particular focus of our study has been the investigation of differential effects of triadic and axonal inhibition on the response properties of RCs, and key findings are summarized in As in  we have Our model contains three distinct types of inhibition, (i) direct triadic inhibition, (ii) soma-driven triadic inhibition and (iii) axonal inhibition, each with putatively different inhibitory effects on the RCs.One of the findings is the different roles played by direct triadic and axonal inhibition in shaping the spatial receptive fields of RCs. Direct triadic inhibition was found to essentially only provide a simple non-linear gain control of the conversion of input spikes to output spikes by RCs (cf. two top rows of wGIp) and the weight of the axonal inhibition of the central RCs by the IN (wIRa) as shown in Axonal inhibition, on the other hand, was found to have a substantial effect on the size of the receptive-field center: the larger the inhibition, the more the RC center size shrinks compared to the center size of the retinal ganglion cell (GC) providing the feedforward excitation, see, e.g., panel A of As soma-driven triadic inhibition is activated by the same IN action potentials that drive axonal inhibition, this type of triadic inhibition will also contribute to the shrinking of the RC receptive-field center. This is for example observed for the example with both direct and soma-driven triadic in Thus, a possible role of the different inhibitory actions from INs to RCs in the dLGN circuit is to provide separate mechanisms for affecting gain control only (direct triadic inhibition) and regulation of receptive-field center size  of visual signals sent to cortex. A smaller receptive-field center size suggests higher spatial resolution, but see .αR measuring the dampening effect of the inhibitory surround on the RC response compared to the maximal RC response  and wIRa (8 nS) in this study. With direct triadic inhibition only , αR is at a modest value of 0.44. With soma-driven triadic inhibition included as well , αR gradually increases with increasing excitation of the IN  up to a maximum value of αR = 0.56. Interestingly, addition of axonal inhibition on top of this triadic inhibition is seen in αR, even with our maximum weight for the inhibition of the RC (wIRa = 8 nS). Thus while direct triadic inhibition alone is seen to be sufficient to assure some extra centre-surround inhibition, the addition of soma-driven triadic inhibition increases αR, substantially. Note, however, that the model example in wGR = 15.6 nS. For weaker retinogeniculate excitation, e.g., wGR = 11.6 nS, we observed in The center-surround inhibition has been observed to be larger for LGN RCs compared to the retinal GCs driving them, see  and referesponse . With axwGR, and is particularly prominent for weaker excitation. Here the maximum RC response, i.e., occurring for spots filling the receptive-field center, is shown both for the transient and sustained responses for different values of the retinogeniculate excitation wGR. Only the cases with direct triadic inhibition or axonal inhibition (cases (RC-i) and (RC-iii)) are considered. For all values of wGR less than 15.6 nS, the value used in the example in Another difference between the effects of axonal and triadic inhibitions were observed when comparing area-response curves computed for the transient phase (0–100 ms after stimulus onset) with corresponding responses in the sustained phase (400–500 ms after stimulus onset) . As founDirect triadic inhibition where time-locked dendrodendritic inhibition occurs due to the same GC spike that provides the excitatory input, was the fastest-acting of the three types of inhibition presently considered. Both soma-driven triadic and axonal inhibition follow only after the generation of IN action potentials which, in our model, required multiple simultaneously arriving GC spikes at the proximal dendrites of the IN. Following this simultaneous input volley it took in our model on the order of ten milliseconds for the action potential occur, a time lag mainly set by the time needed to activate the calcium currents involved in driving the neuron above the action-potential initiation threshold. Thus effects both from soma-driven triadic and axonal inhibition are substantially delayed compared to putative excitatory effects of the same GC input spikes. In our model the time constant of activation of the axonal inhibition was on the order of 1 ms cf. , much shFinally, it should be noted that of the three types of inhibition, soma-driven triadic inhibition is both conceptually and biophysically less understood than the two others: Although somatically elicited APs in INs are known to successfully invade distal dendrites , it has We consider the present investigation to be only the first of several applications of the present modeling approach. Until recently, dLGN circuit models lacked a key ingredient, namely an IN model incorporating the key dual-action inhibitory features of this cell type. With the arrival of the first multicompartmental dual-action IN model , we can The neurons in the dLGN receive modulatory input from other parts of the brain which, among other things, may cause the dLGN circuit to shift between drowsy and attentive cognitive states , 57. SucThe signal processing in the dLGN depends in non-trivial manners on external conditions. In , the autlagged cells, has been found to have delayed visual-response onset and an initial suppression of response until it reaches a maintained firing level [A subclass of RC cells, the so-called ng level , contrasng level . Again, In the present model application we have focused on the situation with a single dominant retinal GC input to each RC as in the analogous firing-rate based model in . RCs recFurther, the present retinal input was described by a spatiotemporally separable filter model, i.e., the spatiotemporal GC receptive field was assumed to be described by a spatial function (in this case a DOG-model ) multiplIn our model application we have also assumed a (time-modulated) Poissonian distribution of the incoming GC spikes. Experimental recordings have revealed a more regular spike-train input than Poissionian , 69, 70.Both RCs and INs receive excitatory feedback from cortex, both via ionotropic and metabotropic receptors, and inhibitory feedback from the GABAergic neurons in the thalamic reticular nucleus , 71–74. Local dendritic processing is particularly important in the electrotonically extensive INs , but recin vitro studies have demonstrated short-term synaptic plasticity throughout the circuit. The feedforward excitatory synapse from GCs to RCs has been found to be short-term depressing [The present model assumes static synapses while pressing  as has tpressing . In contpressing . This suIn the present model the IN has been assumed to receive GC input with symmetry of the ‘same sign’, i.e., ON or OFF, as the RC it inhibits. For the direct triadic input this ‘same-sign’ follows directly, but it is also possible that the proximal synaptic input to the IN which drive the generation of IN action potentials, has the opposite ‘push-pull’ symmetry , 78. If The present model is based on data from several animal species: the target RC and GC area-response curves are from cat dLGN , 27, theWith the advent of ever more sophisticated techniques for controlling gene expression in mice (accompanied by the possibility for optogenetic activation ), the mo


Here we investigate in-situ neutron diffraction measurements and density functional theory calculations to revisit the structure and stability of ice XV. We find that none of the completely ordered configurations are particular favored; instead, partially ordered states are established as a mixture of ordered domains in disordered ice VI. This scenario in which several kinds of ordered configuration coexist dispels the contradictions in previous studies. It means that the order–disorder pairs in ice polymorphs are not one-to-one correspondent pairs but rather have one-to-n correspondence, where there are n possible configurations at finite temperature.Most ice polymorphs have order–disorder “pairs” in terms of hydrogen positions, which contributes to the rich variety of ice polymorphs; in fact, three recently discovered polymorphs— ices XIII, XIV, and XV—are ordered counter forms to already identified disordered phases. Despite the considerable effort to understand order–disorder transition in ice crystals, there is an inconsistency among the various experiments and calculations for ice XV, the ordered counter form of ice VI,  Consequently, most ice phases except ice X are found in a narrow p–T region, which could hinder the understanding of the relative stability relations of respective ice phases.Ice crystals show tremendous structural variety—with the recent discovery of ice XVI2et al.ΔV) accompanying the transition was sufficiently small such that the phase boundary could be flat in terms of pressure based on the Clausius–Clapeyron relation (dT/dP = ΔV/ΔS). Note that the ice VI-XV phase boundary under pressure was not experimentally confirmed in their study and could still be controversial because the entropy change (ΔS) would also be rather small; it should be less than the Pauling entropyR ln (3/2) ≅ 3.37 J mol−1 K−1) as the hydrogen ordering is incomplete and only occurs partiallyIce XV, the ordered form of ice VI, was identified by Salzmann et al., the phenomena suggesting hydrogen ordering in ice VI had been reported in a number of studies using neutron diffraction11et al. noted in their review paper4111416PcT, high-p in-situ neutron results have been reported, probably because of technical difficulties or insufficient data quality; (ii) all possible ordered configurations satisfying the Bernal–Fowler ice rules  were not fully considered or sometimes confused Prior to the identification of ice XV by Salzmann c-axis, as reported by Salzmann et al.d, f), additional peaks and lattice elongation were observed  correspond closely to changes in c/a . The Bragg peaks of lead, which were included in the sample room as a pressure marker, were not broadened as much as those of ice, and the Δd/d of ice constantly increased with decreasing pressure  The peak broadening is an intrinsic behavior of ice rather than an external deviatoric stress owing to downloading, or (ii) the peak broadening is caused by deviatoric stress, and only the difference of compressibility between ice and lead differentiates the broadness. As the actual behavior could not reflect just one of these two extremes but must be to some extent a mixture, we plotted (Δd/dice -Δd/dPb) as a function of pressure in order to reduce the component caused by the external deviatoric stress , whereas it may have been much faster in the previous studies, wherein the samples were directly soaked in liquid nitrogen, with the quick quench allowing DCl to be solved into D2O ice crystals as solid solutions. If the solubility of DCl into D2O ice Ih is on a similar order to that of HCl into H2O ice Ih, then it could be on the order of 10−7–10−5 mol/mol at equilibrium−4 mol/mol). Thus, in this study the DCl component would be expelled from ice crystals owing to the slower cooling rate. In other words, the effects of the relatively insignificant doping could be negligible.The results of a comparative neutron diffraction experiment using a sample not doped with deuterium chloride (DCl) also showed an increase of the pressure . The ratThere are 45 symmetry-distinct configurations for completely ordered forms of ice VI (see Methods), when the unit cell size is unchanged for the transition to ice XV. Among the 45 configurations, there are 12 pairs having a common Patterson function or a symmetrically equivalent Patterson function . Thus, the remaining 33 configurations are experimentally distinguishable by neutron powder diffraction methods see . The LanUiso revealed that four configurations belonging to the space groups (number of configurations), P2111 (40), P1211 (41), and P11n (42) have slightly lower R factors and more phase fractions for the ordered phase  z, (ii) z +1/2, (iii) −z, and (iv) −z + 1/2. Nz will be the same, irrespective of which D1, D2 or D4 site is chosen. The 45 configurations can be classified into three groups in terms of the value of Nz, as listed in Nz = 2, whereas other configurations have Nz = 0 or 1 (see l (l = odd) reflection. For most of the configurations except those from 43 to 45, Nz is equivalent to a number of “C-type” networks, one of three  symmetrically distinct configurations for single H-bonding networks defined in previous works413Nz = 1 or 2. Further, note that the configurations from 43 to 45 have two C-type networks in their structures but have Nz = 0, so they do not contribute to the intensity of 003.where or 1 see , and thePc (#38)1112et al., the surface correction term could be significant when a ferroelectric domain nucleates in a para-electric medium. The Pc (#38) configuration was particularly affected by the surface correction term (P1211 (#36) would have the least energy in cases wherein the surface correction term is relatively large . Importantly, our calculations revealed that the relative energies for all the 45 configurations are within just 0.10 eV per 10 molecules, which corresponds with the reduced temperature of 39 K. As the energy difference between several favored configurations is even lower—within a few K—the least energy configuration may be altered depending on calculation method details and the surrounding conditions.The DFT calculations for the 45 configurations revealed that the ion term , and theWe would at least indicate that none of the single configurations has a particular dominance over others in terms of both the Rietveld refinements and the DFT calculations, but several configurations have similar likelihood at the temperatures we investigated. Our DFT calculations showed that several configurations may appear even at 20 K . Thus, thhl:l = 2n and 00l:l = 2n, for the P42/nmc space group of ice VI. On the other hand, the other reflection conditions of the space group, hk0:h + k = 2n and h00:h = 2n, are still fulfilled. It is also confirmed that the transition occurs without losing translational symmetry , because all additional peaks can be indexed as integers. The obtained reflection conditions suggest Pmmn as the space group with the highest symmetry in the possible subgroups of P42/nmc meeting the condition k = 0. Note that we could not exclude the possibility that lower symmetry space groups such as Pm21n or P21mn fulfil the conditions mentioned above. It should also be mentioned that the space group Pmmn has previously been proposed as an ordered form of ice VI as ice VI’ in the first neutron diffraction study by KambIn the previous section, the completely ordered 45 configurations deduced from the disordered ice VI model were compared with the observed data. Here we inductively constrain the ordered model based on the observed diffraction pattern. The additionally appearing Bragg peaks included 003, 221, and 113 reflections , which cPmmn space group and the ice rules constrain hydrogen occupancies such that the only single parameter α, as defined in Handa et al.Pmmn model with varying atomic coordinates, Uiso, and the parameter α converging stably yield α = 0.33 –XIII (P21/a) and ice XII (P212121), the condition that space groups for lower symmetry phases are derived from single irreducible representations of the parent space groups is not actually obeyed. Although Salzmann et al.The partially ordered h Nz = 2 . In addiPmmn is a common supergroup of the space groups for the four configurations with Nz = 2 , P1211 (#41), and P11n (#42)). We also realized that these four configurations could be adjacent each other without breaking ice rules (ΔH = 0.000626 eV/10 molecule), thereby implying that ice XV comprises a mixture of micro domains, with each domain having locally ordered motifs. The existence of domain structures is supported by the observation that the position of 003 reflection, the only clearly observable 00l (l = odd) reflection, slightly deviates between calculated and observed patterns ”, to differentiate from each other.In terms of nomenclature for ice polymorphs, we would finally suggest the term for any ordered form of ice VI as ice XV without using a new Roman number, even if another completely or partially ordered form of ice VI is observed under electric fields, using other dopants, and/or other 2O (0.01 mol/L) made from pure D2O  and DCl solution  and a piece of lead (0.03–0.05 g) as a pressure calibrant were loaded into encapsulating TiZr null metal gaskets sandwiched by a pair of tungsten carbide or alumina-toughened-zirconia anvilsp–T path is drawn in p–T paths were dependent on the respective runs, which are described in detail with the respective results in the Results section. Neutron diffraction patterns were measured on the BL11 beamline (PLANET)p–T control, data reduction, and refinements are described elsewhere20Liquid DCl-doped DUZP = 1/2 Σnħωn) to the enthalpy obtained from the DFT calculations. An additional surface correction term, which was discussed in detail in previous studies444546et al.Quantum Espresso33394041M is the dipole moment, V is the simulation cell volume, and f is (2ε+1) when the system is embedded in a spherical cavity of a medium of dielectric constant, ε. The values f = 391 and 180 were chosen, which were the same as in the previous studyith configuration (pi), assuming the Boltzmann distribution on the calculated energy (Ei), is given by48where wi is number of the symmetry equivalent configurations  we did deal with larger unit cells in this study. Most of the 576 configurations are symmetrically identical to others, so we need to consider only symmetrically distinct configurations. If an ordered configuration  is symmetrically equivalent to another (H2), H1 and H2 belong to the same space group (H), which should be a subgroup of the space group of ice VI . The operation (g) to convert H1 to H2 should be one of the 16 symmetry operations of G, and g should not belong to H:The structure of ice VI consists of two hydrogen-bonding (H-bonding) networks. The two networks are isolated from each other in terms of H-bond and each network has a unit comprising five water molecules  as labeled 1–5 in g in G can convert an ith configuration Hi to a nonidentical jth configuration Hj or to Hi itself if g also belongs to H:In other words, any symmetry operation g from a configuration obeying the ice rules will also obey the ice rules. As a result, 576 configurations can be converted by 16 symmetry operations to other configurations (or themselves) within the 576, and this screening gives a full list of symmetry relations between the respective configurations. Finally, 45 symmetry distinct configurations, which are explicitly tabulated in Because symmetry operations are distance preserving and bijective, any configuration converted by i.e., these regarded as “A-”, “B-”, and “C-type” networks in previous works)1314Among the 16 symmetry operations in G, eight operations do not exchange two H-bonding networks, whereas the other eight do. Thus, if we focus on the symmetry of a single H-bonding network, each configuration has eight symmetrically equivalents, yielding three =24/8) configurations for a single network that could be chosen as symmetrically inequivalent configurations .