N-glycosites (NGS) in its Fab and Fc domains may modulate its functions. However, targeted modification of glycans in multiply glycosylated proteins remains a challenge. Here, we applied an in vivo approach that allows the manipulation of IgE N-glycans, using a trastuzumab equivalent IgE (HER2-IgE) as a model. Taking advantage of plant inherent features, i.e., synthesis of largely homogeneous complex N-glycans and susceptibility to glycan engineering, we generated targeted glycoforms of HER2-IgE largely resembling those found in serum IgE. Plant-derived HER2-IgE exhibited N-glycans terminating with GlcNAc, galactose or sialic acid, lacking, or carrying core fucose and xylose. We were able to not only modulate the five NGSs naturally decorated with complex N-glycans, but to also induce targeted glycosylation at the usually unoccupied NGS6, thus increasing the overall glycosylation content of HER2-IgE. Recombinant human cell-derived HER2-IgE exhibited large N-glycan heterogeneity. All HER2-IgE variants demonstrated glycosylation-independent binding to the target antigen and the high affinity receptor FcεRI, and subsequent similar capacity to trigger mast cell degranulation. In contrast, binding to the low affinity receptor CD23 (FcεRII) was modulated by the glycan profile, with increased binding to IgE variants with glycans terminating with GlcNAc residues. Here we offer an efficient in planta approach to generate defined glycoforms on multiply glycosylated IgE, allowing the precise exploration of glycosylation-dependent activities.Human immunoglobulin E (IgE) is the most extensively glycosylated antibody isotype so glycans attached to the seven  N-glycosylation sites (NGS) and ~12% of their molecular weight made up of carbohydrates . Serum IgE is heavily sialylated, with significant N-glycan diversity between different (patho-) physiological stages , normally the least abundant antibody class in human serum, is most commonly known for its role in the allergic response. The principal mechanisms of IgE antibodies in allergic diseases are (i) recognizing allergens through their antigen-binding regions (Fab) and (ii) interacting via their Fc regions with their two cell surface receptors to induce the allergic cascade  for FcεRI binding and subsequent initiation of the allergic cascade  have become an interesting platform for glycoengineering. Paradoxically, their limited endogenous glycosylation repertoire provides an advantage for glycoengineering, as it results in the production of homogeneous N-glycans, with secreted proteins typically carrying only one or two glycoforms, i.e., GnGnXF (GlcNAc2Man3XylFucGlcNAc2) and MMXF (Man3XylFucGlcNAc2). This feature facilitates a controlled manipulation of N-glycans. We recently developed an engineering approach by combining stably engineered N. benthamiana glycosylation mutants and transient expression modules to both knock-out and knock-in targeted glycosylation genes  were not very successful, with only low amounts of sialylated forms . In addition, other structures usually not present on IgM, like incompletely processed hybrid glycans, were detected , galactose or sialic acid. In addition, HER2-IgE was produced in human embryonic kidney (Expi293F) cells  as a model. Using βγ2) FcεRI receptor, and the lymphoblastoid B cell line RPMI 8866, which highly expresses CD23, were grown in Roswell Park Memorial Institute Medium  supplemented with 10% FCS. All cells were maintained at 37°C in 5% CO2. Human embryonic kidney (HEK) cell-derived Expi293F™ cells (Thermo Fischer Scientific) were grown on a Stuart orbital shaker (model SSL1), at 125 rpm in 8% CO2 in serum-free Expi293 expression medium (Thermo Fischer Scientific).The human breast adenocarcinoma cell lines SK-BR-3  and BT-474, which naturally express the HER2 antigen, were grown in Dulbecco's Modified Eagle's Medium  supplemented with 10% Fetal Calf Serum (FCS). The HER2-expressing human breast ductal carcinoma cell line ZR-75-30 (ATCC® CRL-1504™), the rat basophilic leukemia mast cell line RBL-SX38  were described earlier  and of the Leishmania major oligosaccharyltransferase single subunit STT3D (LmSTT3D) were described elsewhere  0.05–0.1. Protein expression was monitored 3–5 days post-infiltration. The procedure was recently described in detail  pre-packed column and NIP-IgEHEK was purified using a CaptureSelect™ LC-lambda (Hu) Affinity Matrix (Thermo Fischer Scientific) pre-packed column. Antibodies were eluted with 0.2 M Glycine/HCl + 0.01% NaN3 (pH 2.3), neutralized with 1 M Tris (pH 9) and dialyzed against 1 × PBS overnight. After dialysis, HER2-IgE samples were concentrated with Amicon centrifugal filters, MWCO 10,000 kDa .HER2-IgEg for 20 min, filtered through Miracloth and clarified with pH precipitation . The supernatant was centrifuged for 20 min using an SS-34 rotor at 47,808 g, followed by successive filtration steps through an 8–12 μm filter and a 2–3 μm filter. These centrifugations and filtration steps were repeated until a clear extract was obtained. Using an ÄKTA pure chromatography system , a Protein A column  was washed with ddH2O and equilibrated with 1 × PBS. Then, the clarified total soluble protein was applied to the column at a flow rate of 2 ml/min. The column was washed with 20 CV 1 × PBS and antibodies were eluted with 0.1 M Glycine/HCl (pH 2.7), immediately neutralized with 1 M Tris (pH 9) and dialyzed against 1 × PBS overnight.For plant-produced HER2-IgE, 100 g of infiltrated leaves were harvested 4 days post-infiltration, flash-frozen in liquid nitrogen and crushed with a mortar and pestle. Total soluble proteins were extracted in 1.5 M NaCl, 45 mM Tris, 1 mM EDTA, and 40 mM ascorbic acid for 1.5 h at 4°C (2:1 buffer/fresh leave weight). The extract was then centrifuged on a Sorvall® RC 6™ Plus Superspeed Centrifuge (Thermo Scientific) using a SLA-1500 Rotor at 31,916 All IgE antibodies were subjected to preparative size exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 prep grade column  in order to separate monomeric IgE from aggregates, degradation products, and other impurities. The column was equilibrated with 1.5 CV running buffer  before loading the sample. All steps took place at a flow rate of 0.8 mL/min. The fractions corresponding to the monomeric peak were collected and concentrated with Amicon centrifugal filters, MWCO 10,000 kDa (Merck Millipore).g, 10 min, 20°C) and filtered . Experiments were performed at a flow rate of 0.75 mL/min at 25°C. The proper performance of molar mass calculation by MALS was verified by the determination of a sample of bovine serum albumin.HPLC-SEC combined with multi-angle light scattering (SEC-MALS) was performed to confirm the molar mass of the monomeric IgE resulting from preparative SEC. HPLC  was equipped with MALS , refractive index detector  and a diode array detector . The column  was equilibrated with running buffer . Prior to analysis, the IgE sample was centrifuged : NGS1 (Asn21): NIPSNATSVTL; NGS2 (Asn49): DTGSLNGTTM; NGS3 (Asn99): VAHTPSSTDWVDNK; NGS4 (Asn146): TINIT; NGS5 (Asn252): GTVNLTW; NGS6 (Asn264): ASGKPVNHSTR, and NGS7 (Asn275): NGTLTVTSTLPVGTR. NGS1 and 2 were obtained by the combined trypsin and chymotrypsin digestion; NGS3, 5, 6, and 7 are tryptic peptides, and NGS4 was obtained by digestion with proteinase K. Following appropriate treatment, the peptide mixture was analyzed using a Dionex Ultimate 3000 system directly linked to a Q-TOF instrument  equipped with the standard ESI source  5.0 L/min; dry temp 200°C) in the positive ion, data dependent acquisition mode. MS-scans were recorded . Instrument calibration was performed using an ESI calibration mixture (Agilent). For peptide separation, a Thermo BioBasic C18 separation column  was used. For the relative quantification of the different glycoforms, peak areas of EIC (Extracted Ion Chromatograms) of the first four isotopic peaks were summed. All observed charge states and ammonium adducts, as well as formylated glycopeptides, were considered. MS/MS spectra were used for the verification of the glycopeptides by detection of oxonium ions HexNAc (m/z = 204.1), Hex+HexNAc (m/z = 366.1) and the unique Y1 ion (peptide + HexNAc).For the glycan analysis of HER2-IgE, we followed the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based protocol described in Montero-Morales et al. . BrieflyN-glycosylation site occupancy was calculated from the ratio of deamidated to unmodified peptide upon N-glycan release with 0.15 mU of PNGase A (Europa Bioproducts) overnight at 37°C.2SO4 were added to stop the coloring reaction. Absorbance was read at 450 nm, 1 s. The values from eight BSA-coated wells as well as two HER2-ECD-coated wells with no antibody and two HER2-ECD-coated wells incubated with unspecific antibody were subtracted from the mean values of the three technical repeats of each sample. Data was analyzed on GraphPad Prism 7 and is presented as mean values with error bar indicating the standard deviation. EC50 was calculated using a four-parameter variable slope regression.Medium-binding microtiter plates were coated with the extracellular domain of HER2 . 1 μg/mL HER2-ECD was diluted in 0.5 M carbonate buffer and the plates were coated overnight at 4°C. Control wells were coated with 1% BSA in 0.5 M carbonate buffer. The plate was washed with 200 μL 0.05% Tween-20 in 1 × PBS (washing buffer) three times, gently shaking for 5 min each time. Blocking was performed with 200 μL washing buffer supplemented with 2% BSA (blocking buffer) for 1 h at room temperature, gently shaking. Blocking buffer was subsequently removed and serially diluted IgE-HER2 antibody  were added on the plate, in triplicate, and incubated for 1 h at room temperature, gently shaking. Following three 5 min washes, 100 μL of HRP-conjugated anti-human kappa light chain antibody  diluted 1:5,000 in blocking buffer were added to each well and incubated for 1.5 h at room temperature, gently shaking. After three 5 min washes, 100 μL HRP substrate  were added to each well and incubated for 15 min in the dark. Subsequently, 100 μL 2 M H6 cells/ml) and incubated on ice with serially diluted HER2-IgE antibody variants , or with the isotype negative control NIP-IgEHEK (used for HER2-expressing cell lines), for 30 min in 96-well round-bottom plates . Cells were subsequently washed with 1 × PBS (spinning at 500 g for 5 min) and incubated on ice for 20 min with either anti-heavy chain detection antibody, namely goat anti-human IgE–fluorescein isothiocyanate  . This was followed by another wash with 1 × PBS and final resuspension of the cells in 200 μL FACS buffer. The protocol was adapted for analyzing CD23 binding on RPMI8866 cells: 5 × 104 suspension cells were used per well (instead of 105) and were resuspended in RPMI medium + 2% FCS in place of FACS buffer, in order to have a higher Ca2+ concentration. The antibodies were also diluted in RPMI medium + 2% FCS. Samples were analyzed on a BD LSRFortessa™ , using the High Throughput Sampler (HTS) option. The results were analyzed using FlowJo software 7.6.5. Data was analyzed on GraphPad Prism 7. The trend line was calculated using a four-parameter variable slope regression.Antibody binding to the membrane-bound tumor-associated antigen HER2 and to the FcεRI and trimeric CD23 receptors was analyzed using a flow cytometric approach. HER2-expressing and FcεRI-expressing adherent cells were detached using 0.25% trypsin-EDTA, resuspended in FACS buffer  (104 cells were incubated with the labeled AlexaFluor647-Expi293F IgE (25 μg/mL) and the unlabeled IgE glycovariant , in a final volume of 100 μL. Samples were incubated for 30 min at 4°C followed by a wash with 3 mL of RPMI + 2% FBS. Samples were resuspended in RPMI + 2% FBS and acquired at FACS Canto II (BD Biosciences). The results were analyzed using FlowJo software 7.6.5 and GraphPad Prism 7.IgE produced in Expi293F cells was labeled with AlexaFluor647 (Thermo Fisher Scientific) according to manufacturer's instructions. RPMI8866 cells were harvested and resuspended in RPMI + 2% FBS. For every sample, 5 × 10in vitro using the rat basophilic mast cell line RBL-SX38, which expresses the human FcεRI receptor as a αβγ2 tetramer, the form naturally expressed on the surface of human mast cells. 104 RBL-SX38 cells in 100 μL DMEM + 10% FCS were plated in each well of a 96 well flat-bottomed tissue culture plate and incubated overnight at 37°C in a humidified CO2 incubator. The following day, cells were sensitized with the different HER2-IgE variants (and the isotype NIP-IgEHEK) diluted in culture medium at 400 ng/mL, incubated for 2 h at 37°C and washed twice with Hanks' Balanced Salt solution (HBSS) + 1% FCS (wash buffer). The negative control for the sensitization step was the incubation with medium (without HER2-IgE).We evaluated the ability of HER2-IgE variants to trigger degranulation 4 cells/well) in wash buffer at 37°C. The negative control treatment for the degranulation step was wash buffer (no crosslinker). The positive control for degranulation was wash buffer +0.1% Triton-X-100 (Tx) for total degranulation.IgE crosslinking and subsequent triggering of cell degranulation were achieved by incubating the wells for 45 min with either 100 μL of polyclonal rabbit anti-human IgE (Dako)  or HER2-expressing SK-BR-3 cells  prepared according to a standard protocol  and in N. benthamiana plants  in Expi293F cells .We have previously reported on the relative abundance of specific  al., WT  and HER2 WT  and antennarity remain constant, but terminal sugar residues  vary significantly  in the glycosylation mutant ΔXTFTGal resulted in heterogeneous profiles with only up to 21% bi-galactosylated (AA) N-glycans   being the most abundant glycoform. In total, glycans with terminal sialic acid range between 45 and 78%, depending on the NGS  and hybrid structures (21–22%).We have also produced IgE variants with GlcNAc2) . Due to  the NGS . In bothL. major OST, glycosylation profiles of HER2-IgE variants show mainly complex glycans in NGS1-5 and oligomannose structures in NGS7, independently of their expression host . Furthermore, the glycans present on NGS6 follow the pattern of NGS1-5. A pooled sample of HER2-IgE produced in ΔXTFT, ΔXTFTGal, and ΔXTFTSia plants that exhibits significant amounts of β1,4-galactose (over 50%) named HER2-IgEOST  compared to the plant-produced counterparts.Fab-mediated functions were monitored by the ability of HER2-IgE glycovariants to bind antigen, the extracellular domain of HER2 (HER2-ECD), using an ELISA-based assay. All plant-produced variants bind to the HER2-ECD similarly, with EC.5 ng/mL . HER2-IgHEK.The interaction of HER2-IgE with the target antigen was also assessed by flow cytometry. All HER2-IgE variants recognized HER2 on three different HER2-expressing breast cancer cell lines  and bound in a dose-dependent manner, with no obvious impact of their glycosylation profiles . As expeβγ2) FcεRI. All the antibody variants successfully recognized and bound to the receptor in a dose-dependent manner. A slightly reduced binding was observed in HER2-IgEOST and HER2-IgEHEK samples (To examine the Fc-mediated properties of HER2-IgE we tested the recognition of Fcε receptors (high affinity FcεRI and low affinity CD23) on human immune effector cells by flow cytometric measurements. Binding of HER2-IgE variants to the high affinity receptor, FcεRI, was studied with the rat basophilic leukemia cell line RBL-SX38, which stably expresses human tetrameric (α samples .WT and HER2-IgEXFΔ) and in those that carry mainly galactosylated structures (HER2-IgEOST and HER2-IgEHEK) , but at higher concentrations (5–50 μg/ml) its binding is reduced  . It also content . HER2-Ig reduced . These b reduced .The most relevant IgE mediated biological function is the induction of an allergic response. When immunogens crosslink IgE-FcεRI complexes on the cell surface of mast cells, they trigger the early phase of the allergic reaction, which involves mast-cell degranulation: the release of pro-inflammatory mediators such as histamine, tissue matrix-remodeling enzymes and interleukins . Significant levels of degranulation were detected upon sensitization of mast cells with HER2-IgE followed by crosslinking with either polyclonal anti-IgE or HER2-overexpressing SK-BR-3 breast cancer cells . SensitiN-glycans. Combining transgenic N. benthamiana and transient expression modules, we generate HER2-IgE antibodies glycosylated with human-like complex N-glycans at all five NGS that naturally carry complex N-glycans.In this study, we applied an efficient method for the production of targeted glycoforms of a protein carrying multiple NGS, exemplified by a monoclonal IgE antibody recognizing the breast cancer antigen HER2 (HER2-IgE). HER2-IgE expressed in human Expi293F™ cells exhibited large glycan diversity and batch-to-batch differences, unfavorable factors when studying glycosylation-dependent activities. We took advantage of the limited endogenous glycosylation repertoire of plants, which results in the synthesis of homogeneous complex XFΔ exhibits one dominant glycan, namely GnGn (up to 80%), a structure which can be elongated to generate glycoforms present on serum IgE, i.e., complex N-glycans terminating with β1,4-galactose or α2,6-sialic acid. We show the generation of efficiently sialylated HER2-IgE using XTFTSia plants. The expression of HER2-IgE in XTFTGal plants in order to generate galactosylated antibodies resulted in low levels of galactosylation and batch-to-batch differences, most probably due to the presence of β-galactosidases . In addition, overexpression of OST initiated glycosylation on NGS6, normally unglycosylated, and glycans at this site were then processed into complex-type, similar to NGS1-5.HER2-IgEIgE antibodies carry three NGSs in the Fab region, so one could expect a link between glycosylation and antigen binding. However, in our studies we do not observe any substantial difference in binding of the HER2-IgE variants to the target antigen on different HER2-overexpressing breast cancer cell lines. Similar to IgG-FcγR interactions, IgE antibodies mediate their pro-inflammatory properties through crosslinking of the FcεRI—IgE complexes on the surface of effector cells  bind to CD23 differently, with HER2-IgESia showing significantly better binding to CD23 than HER2-IgEHEK. This could be due to differences in galactosylated glycans, as HER2-IgESia has only 9–26% terminal galactose while 47–66% of HER2-IgEHEK glycans are galactosylated. However, we cannot exclude the possible effect of higher NGS occupancy on CD23 binding, since HER2-IgEOST and HER2-IgEHEK have increased NGS occupancy . In vitro studies have proposed that glycosylation of IgG may affect its binding to DC-SIGN and even CD23 , respectively, which may confer different anti-inflammatory or immune-suppressive responses depending on the immunological context (Anthony et al., Collectively, we demonstrate an unmatched glycan engineering approach to generate multiply glycosylated IgE with a targeted glycosylation pattern. Our study opens a door to more precisely exploring of glycosylation-dependent activities, which may be exploited for therapeutic purposes, e.g., in cancer settings or allergic diseases.All datasets generated for this study are included in the manuscript/LM-M, SC, AC, KI, SM, FA, and HS designed research. LM-M, DM, SC, KI, and SM performed research. LM-M, DM, SC, AC, and KI analyzed data. LM-M, AC, and HS wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.SK is founder and shareholder of IGEM Therapeutics Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous studies reported that boreal winter precipitation in southeastern China (SEC) tends to increase during El Niño. In this study, however, we find that most weak El Niño events are accompanied by below-normal precipitation in SEC, although strong El Niño events are accompanied by above-normal precipitation in SEC for both eastern Pacific El Niño and central Pacific El Niño. Both the cold SST anomalies in the western North Pacific (WNP) and the warm SST anomalies in the central tropical Pacific are important for the formation of anomalous anticyclone over the WNP, which favors above-normal precipitation over SEC by transporting more water vapor to SEC. The cold SST anomalies in the WNP only excite a weak anomalous anticyclone locally when the weak warm SST anomalies in the central tropical Pacific are accompanied by weak enhanced convection anomalies. In such condition, El Niño does not affect precipitation in SEC apparently. ENSO exerts different impacts on East Asian climate in different phases and statuses10. In boreal winter, the impacts of El Niño and La Niña on precipitation are asymmetric11; only El Niño favors above-normal precipitation over southeastern China (SEC) by exciting the western North Pacific anomalous anticyclone (WNPAC) in the lower troposphere, which transports more water vapor to SEC by the southwesterlies14. Thus, El Niño is a critical predictor for SEC precipitation in boreal winter.El Niño and Southern Oscillation (ENSO), the strongest interannual variability source, exerts significant impacts on global climate20. The two types of El Niño have different impacts on SEC precipitation in winter. The eastern Pacific (EP) El Niño causes more precipitation compared with the central Pacific (CP) El Niño because the former can excite stronger Walker circulation and WNPAC16. In addition, El Niño could cause above-normal precipitation over SEC in positive phase of the Pacific Decadal Oscillation (PDO) compared to that in negative phase of PDO, because the El Niño-like decadal SST anomalies during the positive phase of PDO enhance the magnitude of SST anomalies of El Niño, which induces a stronger WNPAC21. This study implied the impact of El Niño on SEC precipitation may be dependent on the magnitude of El Niño.In the recent years, the different features and impacts of two types of El Niño received extensive attention24. Then, the question is whether the SEC precipitation is sensitive to magnitude of El Niño. In this study, thus, we investigate the relationship between boreal winter precipitation over the SEC and El Niño, with a focus on the impacts of different types of El Niño with different intensity on precipitation anomalies.Some studies indicated that the climate impact of El Niño is sensitive to its magnitude25. However, it is worth noting that SECPI is below normal in about half of the El Niño years To further verify the importance of anomalous anticyclone over the WNP in precipitation anomalies in SEC, we composite 850-hPa wind anomalies for positive and negative anomalies of precipitation in SEC for weak El Niño events Fig. . There i6. Figure 16. These features suggest that both the central tropical Pacific warm SST and the WNP cold SST anomalies favor suppressed convection over the WNP. Cold SST anomalies in the WNP can be initiated by the surface equatorward northeasterlies excited by warm SST anomalies in the central Pacific, then amplified and maintained by local air-sea interaction in the WNP6. This feature is well presented in the composites for strong EP El Niño. However, wind anomalies over the central and western Pacific for strong CP El Niño do not support that the cold SST anomalies in the WNP are caused by the warm SST anomalies over the central tropical Pacific, as there are equatorward northwesterlies over the WNP rather than northeasterlies which are supposed to be at the northwest of a cyclonic circulation over the central tropical Pacific as a response to the warm SST anomalies.Previous studies indicated that the anomalous anticyclone over the WNP is a Rossby wave response to cold SST anomalies and adiabatic cooling of suppressed convection over the WNP, which can be amplified by remote atmospheric forcing6.As for weak EP El Niño, there are weak cold SST anomalies in the WNP and warm SST anomalies in the central and eastern tropical Pacific. The convection anomalies over the WNP and the central tropical Pacific are also weak, especially the former Fig. . In addiThe above analyses suggest that the SST anomalies can affect the magnitude of the anomalous anticyclone over the WNP by modulating the convection over the central tropical Pacific, which induces upper-tropospheric divergent winds convergence over the WNP and thus suppresses convection over there. From this perspective, the convection anomalies over the central tropical Pacific may have a closer relationship with precipitation anomalies over SEC compared to SST anomalies. Figure 26. El Niño events in NCEP CFSv2 simulations are shown in Table The sample size of El Niño events is small, especially for strong El Niño events, which may affect the robustness of the above results. To improve the robustness of the observed results, we analyze the simulations of National Centers for Environmental Prediction (NCEP) Climate Forecast System version 2 (CFSv2)Figures Southeastern China receives considerable precipitation in boreal winter. Previous studies reported that boreal winter precipitation in SEC is linked to ENSO, with asymmetric impacts of El Niño and La Niña on SEC precipitation. Both the EP and CP El Niño events tend to produce above-normal precipitation over SEC in spite of different magnitude of precipitation anomalies. In this study, we find that strong El Niño events are accompanied by above-normal precipitation in SEC for both EP and CP El Niño, but most weak El Niño events are accompanied by below-normal precipitation. The anomalous anticyclone over the WNP is the key circulation system, by which El Niño causes above-normal precipitation over SEC. Both the cold SST anomalies in the WNP and the warm SST anomalies in the central tropical Pacific are important for the formation of anomalous anticyclone over the WNP. The cold SST anomalies in the WNP only excite a weak anomalous anticyclone locally when the warm SST anomalies in the central tropical Pacific are accompanied by weak convection anomalies. The OLR anomaly over the central tropical Pacific should be considered for the impact of El Niño on precipitation anomaly in SEC.28. As there is a considerable uncertainty in the impact of El Niño on extratropical climate variability over the land29 and the precipitation in SEC is also affected by extratropical atmospheric circulation, the response of precipitation anomalies in SEC to El Niño with similar intensity shows some differences from one case to another. As the classification of intensity and type of El Niño is only based on the winter ONI and El Niño Modoki index (EMI)30, respectively, there are some differences in classification between current study and other studies, such as the 1987/88 and 2015/16 El Niño events. However, these differences in classification of El Niño events do not obviously affect the results obtained in this study (figures not shown).While the impact of El Niño with different intensity on precipitation anomaly in SEC is different, the composite precipitation anomaly in SEC is below normal for both weak and strong La Niña events (figures not shown). Warm SST anomalies are also found in the eastern tropical Indian Ocean during strong El Niño events Fig. , especiahttp://data.cma.cn/en). The quality control has been processed by NMIC, CMA31. Monthly wind at 850-hPa with horizontal resolution of 2.5° × 2.5° data are from the National Centers for Environmental Prediction-National Center for Atmospheric Research (NCEP-NCAR) Global Reanalysis 1 (NCEP-1)32. Monthly outgoing longwave radiation (OLR) with horizontal resolution of 2.5° × 2.5° for the period 1979–2016 is from NOAA33. The SST data with horizontal resolution of 2° × 2° derives from the merged Extended Reconstructed SST version 3b34. The ONI which is defined as mean SSTA in the Niño3.4 region  derives from the Climate Prediction Center  and used as an index to depict the ENSO variability in this study. Climatology represents the period from 1981 to 2010.The data of observed precipitation used in this study cover the period from December 1961 to February 2016. The 1961 winter (DJF) represents the three months mean from December 1961 to February 1962. Monthly precipitation observations of 753 stations in China are obtained from the National Meteorology Information Center, China Meteorological Administration (NMIC, CMA; 35. One is the 9-month atmosphere-ocean coupled simulations from 1982 to 2016, initialized with observed atmospheric and oceanic status in November, having 24 ensemble members. The other is atmosphere-only simulation, forced by observed SST and initialized from January 1950 with 11 different atmospheric initial conditions and ended in December 2010. The climatology of the simulations is the average over the periods of 1982–2010 and 1980–2009 for the coupled and the atmosphere-only simulation, respectively.The simulations of NCEP CFSv2 are applied in this study. Outputs from two types of simulations including atmosphere-ocean coupled and atmosphere-only by the CFSv2 are usedhttps://www.ggweather.com/enso/oni.htm. There are six strong events in the years 1972/73, 1982/83, 1991/92, 1997/98, 2009/10 and 2015/16, including three super strong El Niño events as 1982/83, 1997/98 and 2015/1636 and fourteen weak El Niño events. The EMI is used to distinguish CP type El Niño event from EP type El Niño. There are 9 El Niño events and 11 CP El Niño events identified during the study period, respectively (Table t test is used to determine the statistical significance of the correlation and composition.El Niño events are classified into the strong and weak events respectively according to DJF ONI. The strong El Niño events are identified with the DJF ONI reaching up to 1.5 °C, being about 1.3 standard deviation of ONI. Otherwise, the rest is classified as the weak El Niño events. The intensity criterion of 1.5 °C is same as that on website Supplementary Information


Escherichia coli (ETEC) is the most frequently isolated enteropathogen in diarrheal diseases. In developing countries, a very large number of people, especially children, suffer from diarrhea. To combat this problem, World Health Organization has constituted the Diarrhea Diseases Control Program which guides studies on traditional medicinal practices and preventive measures. Gusuibu, a traditional folk medicine, has been claimed to heal certain types of diarrhea. However, so far no scientific study has been carried out on the anti-diarrheal mechanism of Gusiubu. The present study was performed to examine the suppressive activities of ethanol extracts of six sources of folk medicinal ferns used as Gusuibu on heat-labile enterotoxin (LT)-induced diarrhea. Inhibitory effects of six sources were evaluated on the ETEC LT subunit B (LTB) and monosialotetrahexosylganglioside (GMI) interaction by GM1-enzyme linked immunosorbent assay and patent mouse gut assay. Our results indicated that Drynaria fortunei had no anti-diarrheal effect, while, among the remaining five folk medicinal ferns, four belonging to family Davalliaceae had significant abilities on both the blocking of LTB and GM1 interaction and the inhibition of LT-induced diarrhea. In conclusion, these findings suggested the potential application of Gusuibu as an anti-diarrheal remedy.Diarrheal disease is one of the most important worldwide health problems. Enterotoxigenic  Escherichia coli (ETEC) are the most frequently isolated enteropathogen, which is responsible for approximately 380,000 deaths annually [Diarrhea diseases constitute one of the most important worldwide health problems, particularly in developing countries with an estimated 5 billion cases and a mortality of 5 million cases per year. Children less than 5 years of age have been reported to experience higher incidence and severity of gastrointestinal symptoms . Enterotannually . Pneumonannually ,5,6.materia medica literature, several species of ferns have been mentioned as Gusuibu [Drynaria fortunei (DF) contains several flavonones, such as naringin, which present oestrogen-like protective effects in bone [Davallia divaricata (DD), and its effects on apoptosis induction in A549 lung cancer cells were reported [D. mariesii (DM) could inhibit protein kinase C [O-β-d-Gluco-pyranosyl-2,6,4′-trihydroxybenzophenone, which has been isolated from D. solida (DS), could bind to the purified C-terminal cytosolic domain of p-glycoprotein [Drynaria fortunei (DF)., Pseudodrynaria coronans (PC), Davallia divaricata (DD), D. mariesii (DM), D. solida (DS), and Humata griffithiana (HG)) [Among the vast array of remedies used in the Traditional Chinese Medicine system, many ferns (rhizomes and whole plants mostly) have been recorded to have activities against diarrhea and thus act as very useful remedies . Gusuibu Gusuibu . Only a  in bone . Davallireported . Some ofkinase C . 4-O-β-doprotein . As a pana (HG)) ,15 used M1, on the surface of intestinal epithelial cells [M1 by competitive GM1-ELISA it was observed that five ferns  blocked the binding of LTB to GM1, resulting in the suppression of LT-induced diarrhea. All four species belonging to family Davalliaceae were found to be more efficient compared to two species of Polypodiaceae  (both from the Polypodiaceae), Davallia divaricata, D. mariesii, D. solida, and Humata griffithiana (HG)  used as Gusuibu were collected from the counties of Hsinchu, Taichung, Nantou and Taitung in Taiwan (Rhizomes of n Taiwan . These win vacuo at 40 °C (Dried rhizome (100 g each) was macerated with 95% ethanol  for 24 h at room temperature. Filtration and collection of the extract was done three times. Then, the ethanol crude extract  was evaporated to 10 mL and then dried at 40 °C . The dryEscherichia coli LT and LTB; (ii) Patent mouse gut assay; (iii) Biotinylation of LTB and (iv) Competitive GM1-ELISA were followed as per our previous report [The methods of (i) Expression and purification of s report ,15. Mousp < 0.05 was considered statistically significant.Data were presented as mean ± standard error. Student’s t-test was used for comparisons between two experiments. A value of M1. Drynaria fortunei had no anti-diarrheal effect, while other five ferns had significant abilities on both the blocking of LTB and GM1 interaction and the inhibition of LT-induced diarrhea. Therefore, these data suggested that Pseudodrynaria coronans, Davallia divaricata, D. mariesii, D. solida, and Humata griffithiana might be candidates for the treatment of LT-induced diarrhea.We demonstrated that the ethanol extracts of six sources of folk medicinal ferns used as Gusuibu suppressed LT-induced fluid accumulation through blocking the binding of LTB to G


What does it mean to hear something that isn’t there? Hearing inner speech or sounds, not as silent thoughts but as quasi-perceptual events in the world, confounds settled distinctions between perception, memory and imagination that structure our feeling of the real. This essay shows how her capacity for complex ‘listening in’ becomes the mainspring of her brilliance as an experimental writer, cultural observer and fictional ethicist. Eclectic in her sources – including biblical, classical, Romantic, Christian and Jewish mystic and monastic traditions of meditation – Spark reworks the concept of ‘the auditory imagination’ to produce one of the most sustained and innovative self-reflexive performances in modern fiction of the human mind’s capacity to bring to presence and realise other worlds, multiple ontologies. In her writing, fictional vocalisation is the phenomenological and expressive vehicle of an embodied ontology of plural uniqueness that opposes metaphysical reduction to a universal One that invisibly eradicates difference. The Confidential Clerk, she ‘embarked’ on a book-length study for the Catholic publisher Ward and Sheed .In T. S. Eliot’s idea of the auditory imagination, the gift of a ‘feeling for syllable and rhythm, penetrating far below the conscious lives of thought and feeling’ is able to fuse ‘the old and obliterated and the trite, the current, and the new and surprising, the most ancient and the most civilized mentality’.The Comforters was, she insisted, purely pragmatic, easier to do as voices.Spark’s writerly sense of and preoccupation with voice intensified as she made the transition from poetry to fiction, but other events in her life played a crucial role. Sometime between 1953 and 1954, she came to believe that Eliot was ‘communicating’ secretly with her, sending her covert messages in his writing which, in her autobiography, she refers to as ‘ my hallucinations’.The Comforters would explore how a novel might be an extension of a poem and the entanglement of this process with her conversion to Catholicism and the hallucinatory experiences of her breakdown.poet-novelist?For Spark this ‘magic piece of dialogue’ was confirmatory of her own sense that ‘all my hallucinatory experiences, looking back on them, seemed to integrate with this idea’: A Far Cry, the novel that returns to this period of her life, with its radionics ‘box’, its organisers and operators, that are instrumental in the suicide of Wanda).Spark’s terror at this time – that she would lose her mind and be unable to communicate her life-altering experience – is conveyed in the title of the novel, which alludes to the sufferings of Job and the ineptitude of his literal-minded and ineffectual comforters in their imaginative failure to empathise with his distress or the nature of his dialogue with God. Most likely, it also references Yeats’ 1913 poem ‘The Realists’, whose Job figure is the modern poet as mythopoeic diviner, marginalised and misunderstood in a culture of fact-mongering and reductionist naturalism.Nobody can deny that I speak with my own voice as a writer now, whereas before my conversion I couldn’t do it because I was never sure what I was … I was talking and writing with other people’s voices all the time.What I will argue here, however, is that although, after her conversion, Catholicism became what she referred to as a ‘a norm’ organising her life, preemptive harmonisation is not what she had in mind. Departing from the norm was the ‘nevertheless’ principle in operation, and the discovery of her own ‘voice’ might be deemed to be the discovery of an even greater aesthetic and ethical requirement to listen and to discern, not to evacuate or repress the voices of others heard in one’s own.In ‘My Conversion’, published a few years later, Spark describes her mind at this time ‘teeming with disorder’, but shortly after came the recognition that ‘it was like getting a new gift’.The Observer newspaper at the Eichmann Trial in 1961.The Mandelbaum Gate (1965). As the novel makes clear, like Arendt’s Eichmann in Jerusalem (1963), Spark’s sense of horror was provoked by the incantatory monologism of Eichmann’s voice, a ‘computing machine’. ‘What was he talking about? The effect was the same in any language … and the actual discourse was a dead mechanical tick’: even without semantic grasp of what is being said, the sound of the voice condemns him,The Mandelbaum Gate (1965), a recognition of how much pluralisation of voice even as it performs a kind of precarious disequilibrium with conventional ideas of unity, harmony and oneness of selfhood, must become the fundamental ethical vehicle of her fiction.It was a gift whose powerful ethical import was quickly recognised and would shape her sense of the ethical possibilities of the novel and the social responsibilities of the novelist. The eschewing of an art of harmonious and empathetic identification – effectively the incorporation of the other into the self for an art of satire, ridicule and disturbance – called for in her essay of 1971 on the ‘desegregation of art’, seems to have fully materialised during her attendance for The Mandelbaum Gate is the novel where, after Caroline Rose in The Comforters, Spark most directly poses the question, ‘Who am I?’ through her character Barbara Vaughan, who replies to herself, ‘I am who I am’: all three ponder their condition of ‘Gentile Jew’ as well as Catholic convert.Curriculum Vitae, she describes herself, as a child, as ‘an avid listener’, ‘I liked to listen’.oeuvre, might take the form of inward self-meditation, aural mind-wandering, absorbed listening, or the intent eavesdropping that follows the sudden awareness of salient vocalisation in the midst of noise that psychologists refer to – no Eliot pun intended – as the ‘cocktail party’ effect . Spark’s fiction plays, comically and mischievously, with mishearing, overhearing, aural misconstrual, in order to expose the interpellating pressures of sociolectic constraint. But ‘listening in’ is also Spark’s figure for memory in its entanglement with imagination. In Loitering with Intent, the writer-protagonist, Fleur Talbot, insists that: ‘If I recall certain encounters of the past at all … back come flooding the aural images first and the visual second.’The Girls of Slender Means opens with a familiar scene of Romantic visualisation: the (wartime) fragment, the ruined building, but this makes, the narrator concedes, ‘unusual demands on the mind’s eye’.discours overwhelms histoire as the present shifts to that of narration rather than narrated, bringing with it the first hint of diegetic violation, of metaleptic complication, with her announcement, ‘I’ve got something to tell you. Do you remember … ’. In Dorothy Markham’s ‘Darling, where have you been?’, is heard the fading phatic communion of her set.Although Spark’s is not the only ‘auditory’ poetic imagination in the twentieth century – similar claims might be made for Kafka, Joyce, Bowen, Proust, Joyce, Woolf, Richardson, Conrad, Auden, Eliot himself and many more since – she is perhaps the most persistent in her claim to be characterised thus. Attention as ‘listening in’, throughout her Loitering with Intent .reductio ad absurdum of the claims of the Romantic imagination to that anamnesic power that makes the child father to the man.Voices at Play (1961), too, Spark again describes how ‘I turned my mind into a wireless set and let the characters play on my ear’.Virginia Woolf too had described the creative process as one that begins with an intent and absorbed listening in: ‘Instead of remembering here a scene and there a sound, I shall fit a plug into the wall; and listen in to the past.’Memento Mori, the acousmatic authority of the anonymous phone caller derives from its functions as a Maxwellian demon, but one who unsorts and unseparates – the imaginary and the real, the living and the dead – calling up a fragile community of souls in the call to mindfulness of ‘Remember you must die’.Penelope Jardine has suggested that ‘something in the air’ was ‘a phrase she used to explain how things come to us. That certain, or almost certain feeling of something’s existence that most artists know when creating. It must be there. How else did they think it’.A Far Cry from Kensington, cackling through the office din, ‘with her terrible voice’.The Driver’s Seat but in Not to Disturb antic, indifferent, ‘sunlight laughing on the walls’ ushering in a new world of the mediated and sensational, of technopastoral opportunism.logos as phone semantike, his insistence that man is a creature who has logos because only he speaks in language.The Comforters, or buildings, ‘bleating to high heaven’;The Abbess of Crewe, walls have ears and so do trees; in The Girls of Slender Means, radios ‘speak’ to listeners rather than transmit sounds; in The Hothouse by the East River, the glittering city comes to Elsa ‘agitating all around her ears’, like Augustine’s coming to Carthage, city of unholy loves, that ‘bubbled around me in my ears’.A Far Cry, ‘the household’ is ‘garrulous with tacit deliverance’,Spark refigures the Proustian voice in the concentration camp survivor Cathy in Throughout her writing, voices as conduits of absent presence are the instruments of ontological leakage and liberation, of what Latour names the ‘factish’, awakening the mind to the possibility of other worlds. Voices enable ‘a wisdom of passage’ from the either/or options of the modern repertoire to the thought-worlds of the pre-modern, challenging narrow conceptions of the rational and the real.In Eliot’s version of the auditory imagination, the idea of vocal inwardness as the location of deepest selfhood is still Romantically inclined. It is a version of Plato’s soul in silent communion with itself. The image recurs, playfully, in Woolf’s short story, ‘An Unwritten Novel’, as the narrator asks: ‘When the self speaks to itself who is speaking?’. Playing with the Freudian theory of repression, she goes on to suggest the interlocutor might be the ‘entombed soul’, the one ‘that took the veil and left the world’.Caroline’s experience is of a voice ‘like one person speaking in several tones at once. They speak in the past tense. They mock me’.A Far Cry  or like Lise, in thrall to a darkly one-eyed Romantic model of the imagination where the scene of composition is already unfurled in the moment of inspirational conception; they attempt to preside over worlds where the imagination seeks to bring the real into accord with its prearrangements. They live perpetually and metafictionally, like the canny sex-and-death-driven servants of Not to Disturb, in the mode of the future anterior.In metaphysical and scientific traditions, modalities of intentionality are hierarchised: perception, memory, imagination. So too are the senses. The eye is the realist and metaphysical sense par excellence, looking on evidence laid before it. Sound is ambiguously located, blurring inner and outer senses, always in movement, conveying space as temporality, fading out, surrounding or coming towards. In listening, the ear is a transduction device that transforms the vibrations of air as they hit the tympanum: the ear and the imagination are organs and modalities that disturb embodied proprioception, the sense of the body and other bodies as here or there, now or then, inside or outside; with this comes the destabilisation of metaphysical and existential certainties. In sound, the unseen speaks; a beyond reverberates past the horizon of vision.The Hothouse by the East River) that was first tested using Churchill’s voice.The Girls of Slender Means where Nicholas Farrington’s enjoyment of Joanna’s rhapsodic performance of ‘the mountains look on Marathon’ from Byron’s ‘The Isles of Greece’ is ironically ‘interfered’ with by Churchill’s reactionary and disastrously misjudged ‘Gestapo’ broadcast of 6 June 1945. This had revealed his complete failure to hear the new democratic mood at the end of the war. Electronic mediation, interference, hissing on the line and scrambling, are ways in which Spark resists the McLuhanite optimism of a ‘second orality’: rather than a medium for a new communitarian presence, the nuance of the voice in this electronic world becomes even harder to discern. Sefton Delmer too describes the operations centre of the PWE in the last years of the war as a ‘Tower of Babel’.The auditory and the vocal in her novels serve as the phenomenological foundation of an anti-metaphysical aesthetic that promotes ontological pluralism and an ethics of radical alterity, facilitated not through vision but through cultivation of the discipline of attentive listening. Recent criticsOdyssey. For the philosopher must tremble if the poet, alone of all men, has the gift of listening to and understanding the oral testimony of the Muses, daughters of Mnemosyne, uniquely able to hear and reshape her sounds into patterns of meaning discernible to the attuned layperson’s ear.Within the literary academy, however, another revolution was afoot whose rationale was an assault on voice as presence: the Derridean critique of orality as underpinning a dangerous metaphysics of presence. As Nancy/ formerly Mrs Hawkins observes, ‘we tend to notice what we want to’: for the literary critic presence and orality would soon fall out of earshot; writing was making its comeback.The Comforters has largely been read in this Derridean tradition of absence and writerly reflexivity as a prime example of early metafiction: ‘a novel about characters in a novel’ in Caroline Rose’s own words.The novel abounds with metafictional devices, particularly those diegetic violations that create disturbing effects of metaleptic instability: what world is this taking place in; what world are we, as readers, and they, as characters, experiencing? Louisa Jepp, one moment securely rooted in a second order diegetic frame of the storyworld , in the next has broken into the ‘real’ of the assumed first order historical present of narration, so reminding the reader of her other ‘reality’ as a verbal construct. She is ‘still alive’ but ‘here in her home in Sussex, now in the present tense’.The Comforters is approached as an investigation into the complex and many ways in which the presence of the real is felt in our lives as well as in our fictions? What if the text is approached less as a Derridean play of textuality, more as a phenomenological metafiction that builds on Spark’s preoccupation with hearing and listening as modes of existential presencing? Even the flaunting of character – and by the end, the text refers to ‘the character called Laurence Manders’ – as text and ethos overturns the usual assumption of the priority of persons, to suggest that treating persons as characters is not necessarily an act of colonisation, limiting and condemning them to a ‘phony plot’, but potentially a liberating challenge to assumptions concerning the metaphysical fixity of the self. The metafictional devices are used to convey the kind of phenomenological perplexity undergone in the experience of hearing distressing voices, but in order to prompt further reflection on the kinds of inner presencing associated with different modalities of intentionality, including those involved in creating and entering fictional and other kinds of worlds. As Caroline feels increasingly alienated from the Catholics of St Philomena’s and the demi-monde world of louche Bohemians of her past, she begins to experience an inwardly focused distress that turns her thoughts on themselves so that language – as in Spark’s Kabbalistic experience with Eliot’s words – begins to take on a strangely concrete presence and personifying elaboration. Even before the voices appear, Caroline increasingly objectifies her inner speech, talking to herself, then experiencing language as material, another’s thoughts and voice inserted into her own: ‘as if the person was wanting to pounce on some insignificant thought or action, in order to make it signify in a strange distorted way’.So far so postmodern. What happens though if, instead of foregrounding the metafictional elements as simply a novel about writing a novel, am in retreat’, she thinks punningly at St Philomena’s, reflecting on the banality of the obligatory phatic communion that consolidates one’s attachment to the group. In that skittish utterance alone, she liberates herself from thralldom to it.The novel’s retelling of Job’s suffering reveals the inadequacy of excluded middle thinking that proposes an either-or model of truth: one bound to epistemology, realism and objectivity, the other viewing truth as enigma. The various supernatural, black magic, quasi-scientific, moral, even conventionally religious explanations for Caroline’s suffering are revealed as failures of the empathetically disciplined imagination and serve to increase her distress: ‘this is a very remote world I’m in now’, she tells Laurence.The diegetic play with worlds relates phenomenological disturbance to questions of ontological possibility and existential threat, but Spark further liberates the resources of the novel to begin to explore more ethical, social and political issues around identity, exclusion and belonging. The invention of the arrestingly non-realist character, Mrs Hogg – ‘an implausible character’,The Mandelbaum Gate, The Hothouse by the East River, or the Kataphatic presencing of monastic traditions of prayer and meditation.The Mandelbaum Gate.That the intellectual framework of Catholicism shaped Spark’s sense of fictional form is well known; Spark’s conception of voice as a plural singularity, however, allowed her to accommodate and transfigure the many sources of the auditory in her life and, like Eliot’s, to accommodate the trite, the current and the ancient.Ruah) is central in Jewish spirituality. That Ruah as the physical breath of Elohim precedes speech means that in the beginning is not a devocalised logos but instead the vital spirit of God sounding over the face of the waters. Similarly, Qol, the sonorous self-revelation between God and the world that becomes speech in the mouth of the prophets, begins with the physicality of the singular voice.Observer Christmas prize-winning short story, ‘The Seraph and the Zambesi’, that launched her fiction writing career in 1951. Bidding farewell to her investment in the Romantic imagination, Spark calls up an idea of poetic voice that draws directly on the Jewish tradition of Ruah. The story begins with a familiar effect of Stimmung as correspondence between weather, atmosphere and mood. But this Romantic motif is associated with a stifling, torpid and enervating heat whose humid effects muffle noise, like sound waves passing through water. Temporality itself appears reversed:On the third night before Christmas I sat on the step outside my room, looking through the broken mosquito-wire network at the lightning in the distance. When an atmosphere maintains an excessive temperature for a long spell something seems to happen to the natural noises of life. Sound fails to carry in its usual quantity, but comes as if bound and gagged. That night the Christmas beetles, which fall on their backs on every step with a high tic-tac, seemed to be shock-absorbed. I saw one fall and the little bump reached my ears a fraction behind time. The noises of minor wild beasts from the bush were all hushed up, too … . Sometimes, for a moment, a shriek or cackle would hang torpidly in space, but these were unreal sounds, as if projected from a distant country, as if they were pocket-torches seen through a London fog.Cramer – a figure borrowed from Baudelaire’s tale ‘La Fanfarlo’ of 1857 and already featured in Spark’s 1952 poem ‘The Ballad of the Fanfarlo’ – is now 150 years old. As he prepares to perform the role of Seraph in a Nativity play, a real Seraph appears, a living body, but with a completed look, who orders Cramer to shut up his noise. A contretemps ensues, the building burns down, the Seraph disappears. The story ends as the narrator and friends walk away:The thunder of the Falls reached us about two miles before we reached them … We came to the cliff’s edge, where opposite us and from the same level the full weight of the river came blasting into the gorge between. There was no sign of the Seraph … Then I noticed that along the whole mile of the waterfall’s crest the spray was rising higher than usual … we watched him ride the Zambesi away from us, among the rocks that look like crocodiles and the crocodiles that look like rocks.Though baffling, the story is surely an allegorical assertion of the need for a renewal and modification of the Romantic concept of imagination through new reflection on voice. The image of the thunder roaring, heard now in advance not lagging behind, the wind foaming the waters and hovering on the face of the waves, invokes Psalm 33 in the Hebrew bible where ‘God creates with the breath of his mouth’; after the apocalyptic fire, Yahweh’s breath – Ruah – brings energising life into the enervated scene, animating and vocalising itself through those that stop to listen.They were important for Spark too; for voice  finishes with an account of the psychotic German judge, Daniel Paul Schreber, and the teeming voices chattering inside his head relieved only by the loud bellowing that seemed to give him temporary respite. Canetti’s analysis of Schreber bears close similarities to George Herbert Mead’s earlier recognition of the processes of internalisation and reflexivity that allow the voices of the crowd, as the ‘generalised other’, dialogically to enter and inflect the flow of inner speech that normally brings self – inherently therefore an interpellated self – to presence to itself. In recognising the crowd that is enfolded within the individual and then interleaved without, Mead and Canetti posit a self that is inherently reflexive, a singularity and a crowd, a me and not-me; an I that is a we.Elias Canetti’s The Girls of Slender Means and A Far Cry from Kensington, are located in rooming houses whose architectures are built almost entirely through juxtapositions and counterpoints of sounds and voices. In The Girls of Slender Means, set in 1945, class and age are the chief ordering principles of the household; in A Far Cry (set in the early to mid-50s), what appears to be a more cosmopolitan and democratic if pandemonic background hum – born of the new post-war hybrid vocality of the dislocated, the refusenik and the class refugee – is punctuated by the screams of the radically and genuinely dispossessed, the terrorised and the entirely stateless. Both novels are presented in the form of recollection or, more accurately, re-collection. Their narrators listen in to and bring the past into presence, dissolve and resurrect the boundaries between the mind’s ear in its activity of recall and the presence of the past as it is reshaped through auditory imagination into a new and meaningful soundscape.Two of her most accomplished novels, A Far Cry begins with its narrator Nancy (once the Mrs Hawkins of the story), lying in bed, listening in and recollecting the past, now thirty years far away from the turmoil of office life – ‘my job was the noisiest I’ve ever known’ – and the cacophony of her old lodging house, with its clatter, chatter, telephones, radio, televisions, hoovers, rows, banging, of different lifestyles.Can you decide to think? Yes, you can. You can put your mind to anything most of the time. You can sit peacefully in front of a blank television set, just watching nothing; and sooner or later you can make your own programme much better than the mass product.Mrs Hawkins is as serious in her task as Proust’s Marcel, highlighting memory as composition, ordering, layering, harmonising, but with an ethical orientation:listening to the silence, prefiguring the future, picking out of the past the scraps I had overlooked, those rejected events which now came to the foreground, large and important, so that the weight of destiny no longer bore on the current problems of my life.The Comforters, there is a metafictional doubling, but here Spark intuits an analogy between the novelist’s art and the specifically pre-modern, indeed medieval, Kataphatic practice of creative thinking as the modelling of memory palaces. Through careful sifting and reordering a mental inscape is built: ‘listening to the silence with my outward ears and to a crowding-in of voices with my inward ear’.inventio – bringing together invention, inventory and intention or intentio – involved the placing of memories in locations whose analogical associations provide points of metaphorical transfer for ethical meditation and reflection, ‘where memoria and invention come together in a single cognitive process’, a repudiation of the merely imitative or mechanically mimetic.collocare, gathering up, next the business of colligere, building a pattern: the material is intimately entwined with the moral.Memento Mori, the geriatric ward provides an opportunity to meditate on mortality and dying, just as the Marcia Blaine School provides the opportunity for reflection on paideia, or the ethical practice of education. Even in its title, The Girls of Slender Means plays directly on the relation between the material and the moral. Spirit is not much in evidence: inspiration is reduced to Jane Wright’s ‘brain-waves’, the ecstatic to Nicholas Farrington’s account of Freudian sublimation.Consciously and attentively listening to and reordering the past to bring out its hidden significance, she builds a house of fiction, now discerning the emergence of patterns that carry a moral weight. As in Tonos indicated both tuning in and tuning up, a process of tightening the strings, heightening the body and its affects towards a state of concentration to raise the inscape to a higher plane for moral and allegorical meditation. In this novel, tonos is provided in Joanna’s rhapsodic proclamations of poetry, mimetic performances that are seen to give ‘culture’ and raise the ‘tone’ in order to impress visiting males. Similarly, Selina’s mantra of perfect poise, balance and tone, is an aide-memoire to the bodily self-discipline needed to acquire a man and find one’s way to the good life of monied elegance.This meditative tradition is reworked in the spiritual exercises of Ignatius, where in the composition of place too the mind is tuned up or heightened to experience the presence of God. punctus’ as wounding the page with one’s pen.punctus, is the voice of the outcast, the scapegoat, those shut out of the house but who refuse to be resolved into the convenient harmony and habitus of the one, the group : Sandy Stranger, gripping the bars of her grille but haunted too by the ‘whine’ of Miss Brodie’s voice; Lise screaming in four languages as her murderer plunges in the knife; Mary Macgregor screaming hither and thither as the fire engulfs her.Just as Sandy Stranger notes the compositional economy of Teddy Lloyd’s paintings – that each portrait of the Brodie set looks also like Miss Brodie – so the principle of economy in monastic thoughtcraft – one of layering, juxtaposing, building, creating composites so that patterns emerge through the process of attentive and disciplined inner sensing – evidently informs the art of Spark’s poetic composition. And it is what is singularly missing in the activity of her many copiers, reporters, transmitters, informers, informants, impersonators, ventriloquists. For her art too is oriented to a civic responsibility to provide patterns for communal thinking, engaging affect, form, the discipline of inward attention, in order to raise and goad feeling: to pierce, scar, puncture: the idea of ‘How to Live Together, a posthumously published work based on his late lectures, Roland Barthes, in search too of a place where one might preserve singularity, creative solitude, but within a rich community – living alone together – revisited the lifestyle of the fourth century monks at Mount Athos: what they discovered, he claims, was an ‘idiorrhythm’, preserving singularity but contributing to the rhythmic harmony of the group.Experiments in Groups, where the psychoanalyst experimented with the possibility of emergence of a group mind not organised by rule but governed only by the heterorhythmic as the subversion of centralised authority and power. Even as she satirises New Age political thinking and the cacophonous anarchisms of the 1950s and 1960s, Spark uses her novels to explore ways of conceiving community based on attunement, listening, the possibilities of the idiorhythmic but also the capacity to hear dissonance, dissent, the voice that has been masked by the habitual roar and mumble. Having reviewed more or less positively Eliot’s Notes Towards the Definition of Culture (1948), his riposte to the rationalism of the post-war planners with their calculative means-end thinking, Spark makes a further response in The Girls of Slender Means.noblesse oblige underpinned by a faith in Christian redemption, won’t do either. In Eliot’s image of the auditory imagination, the new and the ancient might be fused through the feeling for syllable and rhythm. But for Spark that process must not be segregated, by class, gender or genre; art must connect with the world outside in the hope of making something better. In refashioning the auditory imagination as a place where the voices of the other might be listened to and heard, Spark recognises that the ideal community, as a place of living alone together must begin with the capacity of the imagination to create complex inner presences, to turn concepts into percepts, and to hear the ragged or jangling or whispered voice of the other in its own. In this revocalised dialogicality of thought and of thoughtfulness, one might at least challenge the monologic and the monotonous, the ontologically narrow and the bureaucratically closed, and understand that without this mode of mindfulness, the voice of the other cannot be listened to, for it would never have been heard at all.Reflecting on Muriel’s evident enjoyment of her time at Milton Bryan, working for the PWE, part of a loose community and yet, essentially, solitary, Martin Stannard notes how ‘what she liked best was the supporting structure of a communal life which left her alone to think and to write. She did not crave intimacy’.


ARs). GABAARs are heteropentamers assembled from 19 possible subunits and are the primary mediators of fast synaptic inhibition in the brain. Although the total GABAAR population is incredibly heterogeneous, only one specific GABAAR subtype—the α2-containing receptor—is enriched at the AIS. These AIS synapses are innervated by GABAergic chandelier cells, and this inhibitory signaling is thought to contribute to the tight control of AP firing. Here, we will summarize the progress made in understanding the regulation of GABAAR synapse formation, concentrating on post-translational modifications of subunits and on interactions with intracellular proteins. We will then discuss subtype-specific synapse formation, with a focus on synapses found at the AIS, and how these synapses influence neuronal excitation.The axon initial segment (AIS) is the site of action potential (AP) initiation in most neurons and is thus a critical site in the regulation of neuronal excitability. Normal function within the discrete AIS compartment requires intricate molecular machinery to ensure the proper concentration and organization of voltage-gated and ligand-gated ion channels; in humans, dysfunction at the AIS due to channel mutations is commonly associated with epileptic disorders. In this review, we will examine the molecular mechanisms underlying the formation of the only synapses found at the AIS: synapses containing γ-aminobutyric type A receptors (GABA ARs) on pyramidal neuron dendrites, soma, and axon initial segments (AISs). Thus, the construction and maintenance of GABAergic synapses are essential for normal inhibitory neurotransmission and brain function. However, relatively little is known about inhibitory synaptogenesis compared to glutamatergic synapses. To complicate the picture, there are many GABAAR subtypes composed of different subunits, which confer distinct physiological properties on the receptors. In addition, different GABAAR subtypes are selectively stabilized at different types of synapses; the AIS, for example, contains primarily one kind of GABAAR. Thus, the type of receptor present at a given synapse determines the type of inhibition that takes place. Again, little is known about how neurons direct different types of GABAARs to different synapses. The following review will briefly summarize what is known about the formation and trafficking of GABAAR subtypes and the construction of inhibitory synapses overall and specifically at the AIS.The firing of glutamatergic pyramidal cells is tightly controlled by inhibitory interneurons (INs). By precisely directing pyramidal cell activity, INs are able to regulate network activity, generate oscillations, and even terminate pathological hyperexcitability  anions  firing , β(1–3), γ(1–3), δ, ε, θ, π, and ρ, an intracellular loop domain (ICD) between TM3 and TM4, and an extracellular C-terminal domain  expression , which is still considered to be an integral structural component of the inhibitory postsynaptic domain , a guanine nucleotide exchange factor  were generated. This mutation abolished α2’s preferential interaction with CB and led to loss of α2+ synapses at the AIS. Strikingly, Gabra2–1 animals display postnatal spontaneous seizures; these seizures are often lethal, causing death around postnatal day 20 , increases the affinity of the α1 subunit for CB and leads to an increase in α1-GABAARs expression at axo-axonic synapses. These data show that residues 360–375 of the α2 subunit are sufficient for GABAAR stabilization at the AIS (Nathanson et al., ARs at inhibitory AIS synapses.Indeed, GPN is expressed at the AIS, forming co-clusters with α2-GABAARs are inserted into the extrasynaptic PM at the AIS those receptors that contain the α2 ICD motif are able to bind intracellular scaffolding proteins, such as CB, to form stable complexes that anchor the receptor at axo-axonic synapses. Receptors that do not contain the α2 ICD motif are not stabilized at synapses and diffuse back into the extrasynaptic space (see ARs at the AIS. Future experiments utilizing the Gabra1–2 and Gabra2–1 mice could provide more information about the importance of these proteins in GABAAR stabilization. In addition, the above model only describes the postsynaptic side of inhibitory synapse formation. Additional mechanisms regulate the formation of presynaptic chandelier cell boutons apposing the AIS. Most recently, a transsynaptic mechanism was described: the cell adhesion molecule L1CAM, localized to the AIS of neocortical pyramidal neurons, was found to be necessary for the targeting of chandelier cell boutons to the AIS (Tai et al., Together, these data provide a potential model for axo-axonic synapse formation: after GABApace see . Other pAR subtype-specific synapse formation. This subtype specificity is important for the maintenance of neuronal excitability. α2-GABAAR-enriched synapse formation at the AIS is an especially intriguing case, as AIS inhibition is essential for normal brain function. Better understanding axo-axonic synapse formation will not only shed light on the molecular mechanisms of subtype-specific inhibitory synapse formation but may also provide new avenues of research into treatment for neurological disorders like epilepsy, which result from pathological hyperexcitability. It appears that protein-protein interactions between the ICD of GABAAR subunits and intracellular scaffolding proteins at inhibitory synapses play an important role in this process. The make-up of the inhibitory synaptic scaffold is variable depending on cell type and subcellular domain, making such interactions good candidates for synapse-specific GABAAR subtype enrichment. Further research will need to be done to fully explore the “interactome” of each GABAAR subtype and the importance of each interaction at the many different types of synapses present in even one neuron.Despite the progress made in understanding the formation of inhibitory synapses, little is known about how neurons direct GABAAN wrote the manuscript, with input from PD and SM.SM serves as a consultant for AstraZeneca, Bain Capital, and Sage Therapeutics, relationships that are regulated by Tufts University. SM is also a shareholder of SAGE Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors SM.


Arabidopsis thaliana chloroplasts. It is required for chloroplast development during leaf growth, and particularly for the specific degradation of photo-damaged D1 protein in the photosystem II (PSII) complex to maintain photosynthesis activity. In the thylakoid membrane, the reversible phosphorylation of proteins is known to control the activity and remodeling of photosynthetic complexes, and previous studies implicate that FtsH is also phosphorylated. We therefore assessed the phosphorylation status of FtsH and its possible role in the regulatory mechanism in this study. The phosphorylation level of FtsHs that compose the FtsH heterohexameric complex was investigated by phosphate-affinity gel electrophoresis using a Phos-Tag molecule. Phos-tag SDS-PAGE of thylakoid proteins and subsequent immunoblot analysis showed that both type A (FtsH1/5) and type B (FtsH2/8) subunits were separable into phosphorylated and non-phosphorylated forms. Neither different light conditions nor the lack of two major thylakoid kinases, STN7 and STN8, resulted in any clear difference in FtsH phosphorylation, suggesting that this process is independent of the light-dependent regulation of photosynthesis-related proteins. Site-directed mutagenesis of putatively phosphorylated Ser or Thr residues into Ala demonstrated that Ser-212 may play a role in FtsH stability in the thylakoid membranes. Different phosphorylation status of FtsH oligomers analyzed by two-dimensional clear-native/Phos-tag SDS-PAGE implied that phosphorylation partially affects FtsH complex formation or its stability.FtsH is an essential ATP-dependent metalloprotease for protein quality control in the thylakoid membrane of  Escherichia coli. FtsH protein homologues have been found in other prokaryotes as well as in organelles of bacterial origin, such as mitochondria and chloroplasts. The fundamental function of FtsH protease is the quality control of membrane proteins, but its contribution to stress responses is also suggested  are located in the chloroplast  (yellow variegated1 (var1) and var2; these mutants show weak and strong leaf-variegated phenotype, respectively, whereas mutants lacking FtsH1 and FtsH8 do not show the leaf-variegated phenotype (Arabidopsis FtsH complex contains two type-A subunits and four type-B subunits in a hexameric complex (In the oroplast . Additio (FtsHi) ; four of (FtsHi) . This AA (FtsHi) , and par (FtsHi) . Much ef (FtsHi) . Of them (FtsHi) . Mutantshenotype . Both tyhenotype . Mass sp complex , but a s complex  and a re complex  suggesteE. coli  in the chloroplasts due to impairment of the repairing-capacity of photosystem II (PSII) from light-induced damage . ThylakoE. coli . The intE. coli . HoweverChlamydomonas reinhardtii  in thylakoid membranes . For exavar2 was not rescued by expressing a mutated FtsH2 protein, harboring an amino acid substitution in the predicted phosphorylation site. These results imply that FtsH phosphorylation does not regulate FtsH function but FtsH complex formation or its stability.In this study, we attempted to assess the effect of FtsH phosphorylation on this metalloprotease function. Aiming at that goal, we investigated the potential phosphorylation in FtsH by phosphate-affinity gel electrophoresis using a Phos-Tag molecule (Phos-tag SDS-PAGE) followed by immunoblot analysis. Both types of FtsH subunits were separated into phosphorylated and non-phosphorylated forms. We found that phosphorylation of thylakoid FtsH was neither dependent on light exposure nor the presence of the major thylakoid protein kinases, STN7 and STN8. Instead, we found that the phosphorylation status of FtsH varied with FtsH oligomerization degree. Furthermore, the leaf-variegated phenotype of Arabidopsis  Columbia ecotype was used as wild type. The mutant lines used in this study, stn7-1, stn8-1, and var2-1, were described previously (–2 s–1), at a constant temperature of 22°C. The potential phosphorylation sites in FtsH2 were searched in the Arabidopsis Protein Phosphorylation Site Database .eviously . Plants var2-1 background, vector constructions were prepared as follows, using the primers shown in VAR2 cDNA by PCR-based site-directed mutagenesis, using the appropriate set of primers . The resulting PCR fragments were cloned into the BamHI-SacI site of a binary vector pBI121 using the In-fusion HD cloning kit . Final constructs were each subjected to sequencing, to confirm the accurate mutations inserted in the corresponding region. The constructs were transformed into Agrobacterium tumefaciens strain LBA4404 cells, and the positive LBA4404 strains were used to transform var2-1 plants. Transgenic plants were selected by kanamycin resistance, and confirmed by PCR analysis  containing EDTA-free complete protease inhibitor (Roche). Homogenates filtered through miracloth (Merck Millipore) were centrifuged at 2,380 × g for 10 min. The pellet was resuspended in the same buffer and centrifuged at 300 × g for 1 min. The supernatant was again centrifuged at 2,380 × g for 10 min. The pellet was resuspended in 1 x SDS-PAGE sample buffer and used for Phos-tag SDS-PAGE analyses. For phosphatase treatment, the thylakoid membranes were resuspended in 1 x Takara buffer for CIAP (supplied with CIAP) with cOmplete™ EDTA-free protease inhibitor (Merck) to a final chlorophyll concentration of 1 µg/µl. Resuspended thylakoid membranes were incubated with or without CIAP phosphatase for 2 h at 37°C. Phosphorylation of thylakoid membrane proteins was analyzed by Phos-tag SDS-PAGE. Phos-Tag™ was purchased from FUJIFILM Wako Pure Chemical Corporation and used according to the manufacturer’s protocol. In brief, gels for Phos-tag SDS-PAGE consisted of a separating gel  and a stacking gel . Electrophoresis was performed at a constant current of ≤20 mA/gel with the running buffer , to which 5 mM of sodium bisulfite was added. For immunoblot analysis, gels were washed in wash buffer  for 10 min three times to remove metal ions, followed by one wash in transfer buffer  for 10 min. Then, the proteins were electroblotted to the polyvinylidene difluoride (PVDF) membrane (ATTO Corporation).Isolated thylakoid membranes were used for Phos-tag SDS-PAGE. Harvested 4-week-old seedlings were ground in a blender with ice-cold homogenization buffer  was added to the supernatant according to the manufacturer’s instructions, and samples were loaded onto a NativePAGE™ 4–16% Bis–Tris Protein gel (Thermo Fisher Scientific Inc.) Electrophoresis was performed at 4°C overnight at 50 V. The gel lane was then excised from the gel and incubated in equilibration buffer  for 30 min at 37°C. Then proteins were separated using Phos-tag SDS-PAGE or conventional SDS-PAGE.Purified thylakoid membranes were used for two-dimensional Phos-tag SDS-PAGE. Purified thylakoid membranes were resuspended in buffer  to a concentration of 2 mg/ml chlorophyll. To solubilize thylakoid membranes, an equal volume of n-dodecyl ß-D-maltoside was added to a final concentration of 1% (w/v). After centrifugation at 14,000 × Prior to immunoreaction, transferred membranes were blocked with 1% (w/v) bovine serum albumin (BSA) in 50 mM sodium phosphate buffer, pH 7.5, containing 155 mM NaCl and 0.05% (v/v) Tween 20 (PBST buffer) for 1 h. The membranes were then incubated with anti-D1 , anti-VAR2 , anti-VAR1 , anti-CP43 , anti-CP47 , and anti-Lhcb1 . After two washes with PBST buffer, the membranes were incubated with secondary antibodies Amersham ECL Rabbit IgG, HRP-linked F(ab)2 fragment from donkey . Luminata Forte Western HRP Substrate (EMD Millipore Corp.) was used to develop blots, and chemiluminescence was detected on ChemiDoc XRS+ System .–2 s–1). Before measurement, leaves were dark-adapted for 10 min to oxidize plastoquinone. Initial fluorescence yield of PSII (Fo) and maximal fluorescence yield of PSII (FM) were measured. Maximal PSII quantum yield (FV/FM) was calculated as (FM–Fo)/FM.Chlorophyll fluorescence was measured in mature leaves of 6-week-old plants using FluorCam800MF (Photon Systems Instruments). To induce photodamage, leaves were incubated for 4 h under high-light conditions  and a light-harvesting antenna protein (Lhcb1) were properly resolved and separated from their unphosphorylated forms , growth- (100 µmol photons m–2 s–1), or high-light (800 µmol photons m–2 s–1) for 2 h were subjected to Phos-tag SDS-PAGE and immunoblotting analysis. The results demonstrated that the phosphorylation level of PSII core protein CP43 increased in a light-dependent manner , the major phosphorylation band of Lhcb1 in stn7-mutant thylakoids was drastically decreased below the detection level . Phosphorylation of the PSII core protein D1 was still observed in this experimental condition, suggesting that the aforementioned phenomenon of overlapping activity of these two major thylakoid kinases effectively operated under this light intensity. However, under high-light condition (800 µmol photons m–2 s–1), the phosphorylation level of D1 proteins in the stn8-mutant was remarkably reduced  and anti-VAR1 (for Type A) antibodies. Conventional 2D-CN-SDS-PAGE showed that FtsH proteins migrated broadly from the higher molecular weight position, that was around PSII dimer, approximately 650 kDa, to the lower molecular weight size in a range of less than 140 kDa, estimated by the size of LHCII trimer complex  (FtsH2 constructs (driven by CaMV 35S promoter) in which the sequences corresponding to each Ser or Thr residue were replaced with Ala, and the mutanted FtsHs. The resulting constructs were introduced into a FtsH2-knockout mutant var2-1. Transgenic lines were designated as var2 (S212A), var2 (T337A), var2 (S380A), and var2 (S393A), respectively. Our previous work indicated that overexpression of FtsH2 restores leaf variegation successfully and accumulates FtsH levels comparable to the wild type (var2 (T337A), var2 (S380A), and var2 (S393S), rescued leaf variegated phenotype in var2-1, demonstrating that the corresponding amino-acid substitutions are tolerated to restore the defective activity of FtsH. However, we found that leaf variegation persisted in var2 (S212A), suggesting that Ser-212 is important to maintain its activity (In the database of hAt 4.0) , four SehAt 4.0) Table 1.ild type , suggestvar2 (T337A), var2 (S380A), and var2 (S393A) lines. By contrast, accumulation of FtsH in var2 (S212A) was reduced considerably, whose levels appeared to be comparable to those in the original var2 mutant. Since anti-VAR2 antibodies recognize type B FtsH, the remaining bands in var2 samples were assumed to correspond to another type B protein, FtsH8. Therefore, it is likely that the lack of recovery from variegation in var2 (S212A) was due to the impaired accumulation of FtsH2 in this line. On the other hand, Phos-tag SDS-PAGE demonstrated phosphorylation of FtsH in the three transgenic lines, var2 (T337A), var2 (S380A), and var2 (S393S); their phosphorylation levels were comparable to that of control. To further evaluate whether these amino-acid substitutions affected FtsH activity in the thylakoid membrane, high-light sensitivity of PSII activity was measured using chlorophyll fluorescence analysis. As expected, an increased PSII photosensitivity was observed in var2 (S212A), similar to that of var2 mutant , var2 (S380A), and var2 (S393S) lines slightly decreased with respect to that of the wild type during high-light irradiation, but these differences were not statistically significant. These results using transgenic plants were thus unable to confirm that phosphorylation of the predicted target residues plays a critical role in FtsH phosphorylation. Nevertheless, our data demonstrated that S212, localized at the stromal edge of transmembrane domain connecting the ATPase domain, is important for FtsH stability.To examine if mutated FtsH2 proteins accumulated in the transgenic lines, thylakoid membrane proteins from 4-week-old seedlings were subjected to immunoblot analysis , showed that Ser-212 is important for FtsH accumulation, whereas these residues did not critically affect the phosphorylation state of FtsH assessed by Phos-tag analysis , designed for Ser-212 amino acid substitution, this condition itself could have influenced the translation process or the stability of FtsH protein, cannot be ruled out.Amino acid substitutions carried out in FtsH2, based on information provided at the analysis Figure 6In thylakoid membranes, the FtsH functional complex is likely a temporary complex formed when the proteolysis is executed in the grana margin region . Thus, FAll datasets generated for this study are included in the manuscript/YK and WS conceived the study and wrote the manuscript. YK performed the experiments. All authors reviewed the results and approved the final version of the manuscript.This work was supported by KAKENHI grants from Japan Society for the Promotion of Science  and from the Oohara Foundation (to WS and YK).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.