High-temperature stress severely affects rice grain quality. While extensive research has been conducted at the physiological, transcriptional, and protein levels, it is still unknown how protein phosphorylation regulates seed development in high-temperature environments. Here, we explore the impact of high-temperature stress on the phosphoproteome of developing grains from two indica rice varieties, 9311 and Guangluai4 (GLA4), with different starch qualities. A total of 9994 phosphosites from 3216 phosphoproteins were identified in all endosperm samples. We identified several consensus phosphorylation motifs  induced by high-temperature treatment and revealed a core set of protein kinases, splicing factors, and regulatory factors in response to high-temperature stress, especially those involved in starch metabolism. A detailed phosphorylation scenario in the regulation of starch biosynthesis  in rice endosperm was proposed. Furthermore, the dynamic changes in phosphorylated enzymes related to starch synthesis  were confirmed by Western blot analysis, which revealed that phosphorylation might play specific roles in amylopectin biosynthesis in response to high-temperature stress. The link between phosphorylation-mediated regulation and starch metabolism will provide new insights into the mechanism underlying grain quality development in response to high-temperature stress. Rice is an agriculturally major cereal crop worldwide. The yield and quality of rice are often severely affected by heat stress, and this phenomenon is further aggravated with the intensified global warming . During 2+-dependent protein kinase [It is now well accepted that phosphorylation plays a vital role in the regulation of many intracellular processes during plant growth and development. The possible role of protein phosphorylation in the formation of a protein complex participating in starch synthesis in wheat endosperm was first proposed by Tetlow et al. ,11. Laten kinase . With thn kinase , severaln kinase ,18, despn kinase . Qiu et n kinase . Developn kinase . Hence, n kinase , an in-dHere, a label-free quantitative phosphoproteomic analysis was applied to examine the heat-induced phosphorylation change in two rice varieties with different starch qualities. As a result, a series of phosphorylation motifs, enzymes, protein kinases, splicing factors, and other potential regulators involved in seed development were revealed, particularly the phosphoproteins involved in starch metabolism. The properties of phosphorylation sites involved in starch synthesis and their change trends in response to heat stress were comprehensively explored through sequence alignment and site conservation analysis. Western blot with site-specific phosphopeptide antibodies was used to verify and explore the dynamic change in phosphorylation related to starch synthesis under high-temperature stress. Taken together, an in-depth understanding of phosphorylation-mediated regulation in rice endosperm under heat stress will shed new light on thermal signaling transduction and functional phosphosites related to starch metabolism.We sought to assess dynamic changes in protein phosphorylation in the developing rice seed under high-temperature stress. The control and high-temperature treatment groups were designed for two indica rice varieties, 9311 and Guangluai4 (GLA4). In the treatment group, heat stress was performed on the fifth DAF, which prevented the potential effects of high temperature on pollination and seed setting at the early developmental stage. In addition, the 6, 10, and 14 DAF were designed as sampling time points, corresponding to the 1, 5, and 9 days of high-temperature treatment, respectively . The aveGrain chalkiness is an indicator of abnormally developed endosperm and the most sensitive trait in response to heat stress ,3. It isThe thermal properties of the flour samples were determined by differential scanning calorimetry (DSC) . All GLAOverall, results of grain chalkiness and thermal properties indicated that high-temperature treatment for 5 days is sufficient to cause irreversible damage to rice grain quality. Accordingly, samples collected at 10 DAF with 5 days of treatment were the optimal treated group for subsequent phosphoproteomic analysis.To elucidate how heat stress influences rice endosperm at the phosphorylation level, a label-free analysis was performed to quantify phosphoproteome from two indica rice cultivars under normal and high-temperature conditions. As depicted in In all endosperm samples, our workflow led to the identification of 9994 phosphosites located on 3216 proteins, of which 7604 phosphosites were quantifiable . The majTo detect possible changes in the phosphoproteome attributable to heat stress, we then performed label-free quantification analysis on all quantifiable phosphosites within our dataset. Only 2680 common phosphosites were quantifiable for all sample groups due to reversible phosphorylation induced by high temperature a. More pp < 0.05, log2 (fold-change) >1) were screened out in 9311 and GLA4, respectively, where LFQ data was available in both conditions  analysis on all phosphosites identified within our dataset d. Where nditions . For subnditions d. The nunditions . In addinditions . When alnditions . It is pThe in vivo phosphorylation status induced by heat stress is often inseparable from protein kinase activity, which is usually regulated by upstream kinases or autophosphorylation. Up to now, few studies have examined the association between high-temperature response and protein phosphorylation involved in the signaling pathway ,26. It iBoth 9311 and GLA4 shared a number of consensus motifs  were prepared, which were identified in a previous study  and the Starch synthase IIIa (SSIIIa) has the second-highest starch synthase (SS) activity and plays a critical role in forming long B chains, most notably B2 and B3 chains ,33. ConsBranching enzymes (BEs) including BEI, BEIIa, and BEIIb, are fundamental to form a distinct fine structure of amylopectin . Given tTo gain a better understanding of the phosphorylation-mediated regulation mode of starch-synthesis-related enzymes under abiotic stress, we counted the number of significant differences for each of the phosphosites mentioned above and analyzed their relationship with site conservation . A meaniGenerally, japonica rice is used as the primary material for phosphoproteomic investigations ,24. HowePhosphorylation-mediated regulation associated with high-temperature stress has been explored in rice leaves . Researc2+ concentration in response to abiotic stress and translate these perceived signals into subsequent downstream signaling events to trigger response mechanisms/pathways [CDPKs belong to the CAMK kinase family and sense changes in the cytoplasmic Capathways . Among aIn the literature, so far, kinase–substrate networks induced by high-temperature have not been investigated in detail . Here, wRNA splicing, a form of RNA processing, removes introns and joins exons together to make the pre-mRNA transform into a mature messenger RNA (mRNA). Recent observations have suggested that post-transcriptional regulation, especially alternative splicing (AS), appears to function in plant responses to environmental stress . In our bWx gene at the proximal sites [bWx. The indica variety 9311 carrying bWx observed a down-regulated phosphorylation trend in RSZp23 induced by heat stress (Wx. Ser/Arg (SR)-rich proteins are a group of RNA-binding proteins that finely regulate alternative splicing by interacting with pre-mRNA sequences and splicing factors during spliceosome assembly . Our woral sites . It is wal sites ,42. Thert stress . In contt stress . Under ht stress . This inWaxy and temperature in that high temperature caused a significant decrease in the number of mature mRNAs [Waxy and eventually influences amylose biosynthesis. In this sense, the potentially essential differences of SR proteins between 9311 and GLA4 at the phosphorylation level may provide novel ideas for the improvement of starch quality.Previous studies have revealed the association between the splicing efficiency of re mRNAs . OverallAmylose content is one of the key factors that strongly influence rice grain quality. Regretfully, to date, few studies have been able to carry out any systematic research of amylose biosynthesis mediated by phosphorylation, particularly under abiotic stress. The potential roles of phosphorylation in regulating amylose biosynthesis were addressed in three dimensions c.bWx, modulating GBSSI expression at the post-transcriptional level.First, the phosphorylation of regulatory factors and splicing factors may affect the expression of GBSSI c. In ricSecond, the most crucial point to note is that 11 phosphosites of GBSSI were identified in GLA4 a. In ricThird, protein targeting starch (PTST) was phosphorylated in rice endosperm c. PTST iAmylopectin is the major component of starch and serves as a key substance in rice grains. Several works on maize and wheat endosperm have emphasized the vital importance of protein phosphorylation in the formation of a starch-synthesizing protein complex ,11. DephSS, covering four isoforms, SSI, SSII, SSIII, and SSIV, has been attributed to catalyze the chain elongation reaction of α-1,4-glucosidic linkages in amylopectin synthesis . AnalysiBEs are responsible for catalyzing the formation of α-1,6-glucosidic linkages of amylopectin in rice endosperm . The phoWestern blot analysis of five residues of rice BEs  further verified that the degree of phosphorylation change is possibly significantly associated with corresponding site conservation . In otheDBE consists of isoamylase (ISA) and Pullulanase (PUL) with catalytic function for hydrolyzing α-1,6-glucosic linkages . Recent pho1 mutant and found an essential role of Pho1 during its initial stages of α-glucan biogenesis, especially under conditions of low temperature [Plastidial phosphorylase (Pho1), a temperature-dependent enzyme, is considered crucial not only during the maturation of amylopectin but also in the initiation process of starch synthesis . Satoh eperature . Hence, perature , and we Oryza sativa L.) 9311 and Guangluai4 (GLA4) were used to examine the effect of high temperature on the developing rice endosperm with different starch qualities. The detailed parameters of starch qualities are provided by Pang et al. [Two indica rice varieties  were obtained from the DSC thermogram. Three replicates were performed per sample. Duncan’s multiple-range test of ANOVA was performed in SPSS (IBM SPSS Statistic 20). Statistical significance was defined at the level of p < 0.05.Under flowing nitrogen conditions, differential scanning calorimetry (DSC) measurements were conducted using a TA instrument Q20  at a heating rate of 10 °C /min according to the method described by Bao et al. . The onsg for 40 min at 4 °C. Supernatants were gathered and kept in a freezer at −80 °C for subsequent phosphoproteomic pretreatment. The protein amount was estimated by BCA assay .For the extraction of endosperm proteins, other tissues  were removed from immature rice grains . The pro4HCO3, the protein suspensions were digested with trypsin  overnight at 37 °C. The enzymatic peptides were desalted on a Sep-Pak C18 cartridge  and subjected to TiO2-based phosphopeptide enrichment, as previously described by Pang et al. [For FASP digestion, samples were treated as previously described . SDS, DTg et al. . The enrv/v acetonitrile (B). The column was equilibrated with 95% solution A. A volume of 6 μL of phosphopeptide solution was loaded onto Acclaim PepMap100 , and separated by an EASY column  at a flow rate of 300 nL/min. Over a period of 0–2 min, the concentration of solution B rose linearly from 5% to 7%; from 2 to 162 min, it increased from 7% to 25%; from 162 to 225 min, it increased from 25% to 40%; from 225 to 230 min, it increased from 40% to 100%; and from 230 to 240 min, it was maintained at 100%. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was performed using an Easy-nLC System coupled with a Q-Exactive Plus mass spectrometer . The mobile phases consisted of 0.1% formic acid (A) and 0.1% formic acid in 84% m/z. The resolution of first-order mass spectrometry was 70,000, the AGC target was 1 × 106, and the first-order maximum IT was 50 ms. For subsequent MS2 analysis, only the top 10 precursors were selected. HCD-MS2 spectra were acquired with 1 microscan at a resolution of 17,500, and the AGC target was 1 × 105. The MS2 scan range was set from 200 to 2000 m/z, the maximum IT was 110 ms, and the isolation window was 2.0 m/z. Dynamic exclusion was employed with an exclusion duration of 60 s. Three biological replicates were performed independently for each group (The Q-Exactive Plus mass spectrometer was operated in positive ion mode over 240 min. Full-scan mass spectra were acquired over a mass range of 300–1800 ch group .Oryza sativa subsp. indica-ASM465v1). Trypsin/P was specified as the enzyme, and two missed cleavages were allowed. The precursor mass tolerance was set to 20 ppm for the first search  and to 4.5 ppm for the main search. The MS/MS mass tolerance was set to 20 ppm. Carbamidomethylation of cysteine residues was selected as a fixed modification, while protein N-terminal acetylation, methionine oxidation, and phosphorylation on serine/threonine/tyrosine were allowed as variable modifications. False discovery rate (FDR) thresholds for protein, peptide, and modification sites were specified at 1%. A minimum peptide length of seven amino acids was required. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium  via the iProX partner repository [Raw mass spectrometric data were processed with MaxQuant software (version 1.5.5.1) and compared with the indica rice protein sequence database  > 1) on phosphosites with at least two valid values in any condition. For a more comprehensive understanding of phosphosites showing significant differences in expression, phosphorylation sites that were uniquely identified in either condition and significantly regulated from the statistical test were combined and divided into four groups  and none in the other indicate a massive change in phosphorylation levels. We, therefore, opted to select those phosphosites that feature a consistent presence/absence profile in 9311 and GLA4, respectively. On the rest of the dataset, we performed Student’s t-test  with the motif-x algorithm. The conserved motif patterns were then redrawn and visualized by TBtools [http://bioinfo.cau.edu.cn/agriGO/, accessed on 6 January 2020). Cytoscape  was used to generate network visuals. KEGG pathway annotation was performed by using KEGG Automatic Annotation Server (KAAS) software . A p-value of <0.05 was used as the threshold of significant enrichment. Pfam  and InterPro  were used to identify functional domains. Protein sequences were aligned using T-coffee. The figures were annotated with Adobe Illustrator . A total of 13 amino acid (AA) sequences centered by the phosphosite were extracted, and the enriched phosphorylation motifs induced by high-temperature stress were predicted using the MoMo tool  were kindly gifted by Prof. Naoko Fujita . Mouse monoclonal antibodies against plant actin were obtained from Sigma-Aldrich . Site-specific phosphopeptide antibodies: Modification peptides of interest from phosphoproteome were selected and are listed in Protein expression of starch-synthesis-related enzymes as well as the site-specific phosphorylation intensity were examined by Western blot assay . Total pIn conclusion, this study set out to provide the first systematic investigation of the phosphoproteome induced by high-temperature stress in rice endosperm. Comparative analysis of the temperature-induced phosphorylation status revealed some interesting similarities and differences between two indica rice varieties (9311 and GLA4). On the one hand, both 9311 and GLA4 shared several consensus motifs  and were highly enriched in terms of heat response (GO) and the spliceosome pathway (KEGG). On the other hand, a dramatic difference was observed in the phosphorylation status of kinases induced by heat stress, which could reasonably explain the different phosphorylation regulatory patterns of 9311 and GLA4. More importantly, we detailed the most comprehensive starch metabolism pathway at the phosphorylation level in rice endosperm, including starch-synthesis-related enzymes , transcription factors (OsbZIP58 and FLO2), SR protein (RSZp23), and CDPK kinase (SPK). Western blot with site-specific phosphor-antibodies was used to verify and explore the dynamic change of the phosphorylation status of SSIIIa (SSIIIa-S94), BEI , and BEIIb , which might play specific roles in amylopectin biosynthesis in response to high-temperature stress. An exciting phenomenon was discovered in that conserved sites tend to reflect steady-state phosphorylation levels. However, non-conserved sites exhibit more flexible phosphorylation regulation patterns when exposed to high-temperature stress. These findings provide valuable insights into the role of phosphorylation response to high-temperature stress, adding unprecedented depth and breadth to the cereal research community.


In the workup of a 55-year-old atopic patient with cough and viscous secretions, we diagnosed an allergic bronchopulmonary aspergillosis (ABPA) on the basis of common diagnostic criteria for adult asthma patients (Rosenberg-Patterson and ISHAM), supported by the use of IgE antibodies against the Aspergillus components Asp f 2, f 4, and f 6. Initial treatment with prednisolone and itraconazole led to remission. In the long-term follow-up, there were few relapses until 2015, which responded well to standard treatment with oral steroids, and since 2016 the patient is in stable remission. The case highlights the valuable contribution of Aspergillus IgE measurements, including the specific IgEs against the components Asp f 1, f 2, f 4, and f 6 in the diagnosis of ABPA.  A 55-year-old male patient in good general condition presented for the first time in July 2010. He described rhinitis that had been present for 2 months and cough symptoms with viscous secretions that were difficult to expectorate and were yellowish in color. In the previous few days, he had occasionally coughed up a “worm-like plug of secretion”. Occasionally, there was also discrete shortness of breath associated with the cough, so the family physician prescribed salbutamol DA as needed, but the patient used it very rarely. For many years, the patient had been known to have tree pollen allergy with seasonal rhinoconjunctivitis in March and April and oral allergy syndrome to pome fruits. However, the hay fever had improved in recent years. Otherwise, there were no relevant previous illnesses. The patient was a journalist, there was no obvious exposure to dust or molds in his professional or private life and no pets. Clinical examination on July 15, 2010, revealed a somewhat attenuated breath sound apically over both lungs with otherwise unremarkable findings. 1 3.5 L = 93%, FEV1/FVC (Tiffenau) = 66%, RAWtot 0.29 kPa/L*s = 98%). Pulmonary function revealed borderline obstructive ventilation disorder with normal airway volumes  was significantly elevated at 95 ppb (norm < 25 ppb). Aspergillus fumigatus and Alternaria alternata as well as sensitization to birch, ash, and grass pollen and house dust mites  and Alternaria alternata. In addition, there was an elevated IgG against Aspergillus fumigatus in the sense of a type-3 reaction, as well as an eosinophilia. The laboratory values  showed aChest X-ray on August 18, 2020 , showed Aspergillus fumigatus and its components Asp 2, Asp f 4, and Asp f 6 raised the suspicion of the presence of allergic bronchopulmonary aspergillosis (ABPA), although the bronchial symptoms were mild except for the coughing up of secretory granules at initial presentation, and the pulmonary function showed only borderline obstruction with, however, a marked increase in FeNO, consistent with Th-2 bronchial asthma. The strongly elevated total IgE as well as the elevated specific IgE antibodies against Aspergillus IgG, chest X-ray), a diagnosis of ABPA was finally made. Five major and two minor criteria of the Rosenberg-Patterson diagnostic criteria were present  caused by this fungus. Despite its ubiquitous occurrence, Alternaria triggers ABPM much less frequently than Aspergillus fumigatus and is also treated with systemic steroids in the same way as ABPA so that in the further course only the Aspergillus parameters were controlled. The high-level sensitization to the mold Under systemic steroid therapy with initially 40 mg prednisolone daily, which could be phased out over 5 months, as well as itraconazole 200 mg daily during the first 4 weeks, the symptoms subsided rapidly, the radiograph normalized, and the serological parameters decreased . Aspergillus IgE antibodies occurred again in October 2014 and August 2015  and Mala f 3, 4  [However, in recent years it has been shown that some er fungi , for exapodialis . In addiassezia) . Aspergillus-sensitized asthma patients and patients with ABPA in asthma [A recent work from India has now shown that the combination of IgE antibodies against Asp f 1 (at a cutoff of 4.4 kU/L) and Asp f 2 (at a cutoff of 1.3 kU/L) have a sensitivity of 100% and a specificity of 81% in differentiating between n asthma . Aspergillus components have only a limited additional benefit [For follow-up during therapy and for the detection of a relapse, the determination of total IgE is usually sufficient; here, antibodies against the  benefit . HoweverAspergillus extract. The determination of sIgE against Asp f 4 and f 6 can then be performed for confirmation due to the very good specificity of these two parameters. It would be desirable to verify the value of the two parameters Asp f 1 and f 2 in a study including European patients, since genetic differences of the patients as well as variations of the Aspergillus strains in India and Europe may well play a role [For ABPA screening in asthma patients, in the future, based on the current data from India, the excellent combined sensitivity of Asp f 1 and Asp f 2 may allow these two parameters to be used in combination with total IgE instead of specific IgE against total y a role . Aspergillus sensitization in the specific IgE or in the skin test, ABPA should then be considered. The IgE antibodies against the Aspergillus components Asp f 1, f 2, f 4, and f 6 make a valuable further contribution in the diagnosis of ABPA. ABPA may additionally be present even in mild or moderate asthma. Therefore, in any type of asthma and in cystic fibrosis, total IgE determination is useful to avoid overlooking ABPA. If total IgE values exceed 500 kU/L and there is simultaneous evidence of I thank Prof. Dr. Joachim Sennekamp for critical review of the manuscript and his valuable comments. None. The author declares no conflict of interest.


IgE antibodies elicit powerful immune responses, recruiting effector cells to tumors more efficiently and with greater cytotoxicity than IgG antibodies. Consequently, IgE antibodies are a promising alternative to conventional IgG-based therapies in oncology (AllergoOncology). As the pharmacokinetics of IgE antibodies are less well understood, we used molecular imaging in mice to compare the distribution and elimination of IgE and IgG antibodies targeting the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4).111In. CSPG4-expressing A375 human melanoma xenografts implanted in NOD-scid IL2rg-/- mice were also engrafted with human immune cells by intravenous administration. 111In-anti-CSPG4 antibodies were administered intravenously. Their distribution was determined by single-photon emission computed tomography (SPECT) and ex vivo gamma-counting over 120 h. SPECT imaging was conducted from 0 to 60 min after antibody administration to precisely measure the early phase of IgE distribution.Anti-CSPG4 IgE and IgG1 antibodies with human Fc domains were radiolabeled with 111In-labeled anti-CSPG4 IgG and IgE showed serum stability in vitro of >92% after 5 days. In A375 xenograft-bearing mice, anti-CSPG4 IgE showed much faster blood clearance and higher accumulation in the liver compared to anti-CSPG4 IgG. However, tumor-to-blood and tumor-to-muscle ratios were similar between the antibody isotypes and higher compared with a non-tumor-targeting isotype control IgE. IgE excretion was much faster than IgG. In non-tumor-bearing animals, early SPECT imaging revealed a blood clearance half-life of 10 min for IgE.Using image-based quantification, we demonstrated that the blood clearance of IgE is much faster than that of IgG while the two isotypes showed comparable tumor-to-blood ratios. Modified antibodies, or bioconjugates, can be used for targeted delivery of drugs, toxins, and diagnostic probes, such as fluorophores and radionuclides.3 Both immune and nonimmune activities depend on specific molecular recognition of the target site via the Fab regions of the antibody.4 The antibodies used until now for therapeutic or diagnostic applications almost exclusively belong to the IgG class.1Antibodies have emerged as a major class of drugs in the last two decades, with a variety of a therapeutic and diagnostic applications,5 This approach exploits the naturally high affinity of the IgE Fc region (8–10 This affinity (Ka = 1010 M−1) is two to five orders of magnitude higher than that of the IgG Fc region for its cognate receptor, FcγRI-III),12 while the affinity of the IgE Fc for its low-affinity receptor, FcϵRII/CD23 (Ka = 108 M−1), is similar to that of IgG for FcγRI.13 The resulting cross-linking of IgE with its FcϵR receptors has been shown to recruit effector cells to tumors more efficiently than IgG antibodies14–16 and consequently evoke greater cytotoxicity and class-specific immune cell-activating and pro-inflammatory signals in the tumor microenvironment,17–20 offering the potential to overcome some of the limitations of conventional IgG antibody treatment. In addition to several promising pre-clinical studies, the first clinical trial of an IgE-based immunotherapy (NCT02546921) has shown promising early safety and efficacy data.21In the search for approaches that might trigger more potent targeted cytotoxic immune responses to tumors, IgE-class antibodies, known for their involvement in powerful immune responses associated with allergic reactions, have shown promise.c region  to its Fin vivo. It is known that IgE clears from blood more quickly than IgG, but this difference has not been firmly quantified. The half-life of human IgE circulating in the human serum has been estimated to be in the range up to 3 days,23 whereas for IgG a serum half-life of up to 23 days23–26 has been estimated. In mice, these values range from 1.5 to 14 h for IgE, compared to 6–10 days for IgG.27–30 Conversely, estimates of residence time of IgE in tissues (mainly skin) suggest a much longer residence time for IgE  compared with a shorter tissue retention for IgG (2–3 days). The fate and transport mechanisms of IgE after clearance from circulation have not been fully elucidated.Despite the promise of passive IgE anti-tumor therapy in animal cancer models, little is known about the trafficking and fate of IgE 7 targeting a tumor-associated antigen, CSPG4 (chondroitin sulfate proteoglycan 4): anti-CSPG4 IgG and anti-CSPG4 IgE. These antibodies, respectively, have a human IgG and IgE Fc region but share the same mouse variable region targeted to the CSPG4 antigen ,44 and as a target for IgG1- and CAR-T-based immunotherapy.45–47 We have recently demonstrated the in vivo safety of a rat IgE targeting CSPG4 in immunocompetent animals.48 By radiolabeling each of the antibodies with 111In  Act (1986), with local approval from King’s College London Research Ethics Committee (KCL-REC). Mice were maintained under specific pathogen-free conditions. Experiments using human cells received approval from KCL-REC. All donors provided written, informed consent.7 Further details of anti-CSPG4 IgE and IgG production and conjugation are provided in the Supplementary Material. Briefly, anti-CSPG4 antibodies  were incubated overnight at 4°C with a 20 × molar excess of p-SCN-CHX-A”-DTPA (Macrocyclics). The antibodies were buffer-exchanged into 0.2 M ammonium acetate buffer (pH 6) and concentrated to 2–3 mg/mL.Anti-CSPG4 IgE and IgG antibodies were engineered and produced as previously described.4OAc, pH 6) and incubated for 30 min at room temperature. Labeling efficiency was measured by radio-thin layer chromatography (radio-TLC) and high-performance liquid chromatography (radio-HPLC). Radio-TLC was performed on ITLC-SA paper strips (Varian) with a mobile phase of 0.1 M sodium citrate (pH 5) with 5 mM EDTA. The strips were analyzed using a Mini-Scan™ radioTLC linear scanner (LabLogic Systems) equipped with a gamma probe (LabLogic B-FC-3200). Radio-HPLC was performed on an Agilent 1200 system using a size-exclusion chromatography column  and phosphate-buffered saline pH 7.4 containing 0.2 mM EDTA as mobile phase (1 mL/min). Signals were detected with a G1314B UV detector (Agilent) and a gamma probe (LabLogic B-FC-3200). As radiolabeling efficiencies of >94% were found, no post-labeling purification was required.Indium-111 (30–180 MBq) in 60–300 µL of 0.1 M hydrochloric acid  was added to the DTPA-conjugated antibody (200–700 μg at 2–3 mg/mL in 0.2 M NH111In in 1 mL of human AB type serum (Sigma) at 37°C. Samples were analyzed by radio-HPLC at 0 and 120 h. Stability was calculated as the AUC of the antibody peak as a percentage of the total activity.Stability of the antibody conjugates in serum was assessed by incubating 245 μg of antibody radiolabeled with 30–31 MBq In vitro binding of anti-CSPG4 antibodies to U937 human monocytic cells (expressing Fcγ and Fcϵ receptors) and A375 human melanoma cells (expressing CSPG4) was assessed by flow cytometry, and immunofluorescence microscopy for A375 cells. Briefly, cells were incubated with non-radiolabeled DTPA-analog conjugated anti-CSPG4 IgE and IgG. The primary antibodies were detected using goat anti-human IgE-FITC or IgG-FITC (Jackson ImmunoResearch). Flow cytometry analysis was performed in FACSCalibur™ or FACSMelody™ (BD Biosciences) instruments. Immunofluorescence images were acquired on an Eclipe Ti inverted microscope (Nikon). Further details are provided in the Supplementary Material.scid IL2rg-/- mice  were used at between 6 and 10 weeks of age, with equal numbers of males and females in each group. Human PBLs were isolated and implanted as previously described.49 Briefly, approximately 5 × 105 A375 melanoma cells were injected subcutaneously into the lower left flank. Five days later, heparinized human peripheral blood (25 mL) was haemolysed using ammonium-chloride-potassium (ACK) lysis buffer. The remaining PBLs were isolated by centrifugation, washed twice in PBS and injected intravenously to the mice (1 × 107 PBL/mouse). Tumor size was measured with callipers and volume calculated using formula: V = d2 × (D/2), where d = small diameter and D = large diameter. Spleen engraftment of human CD45+ cells was confirmed by flow cytometry.34 A therapeutic study of the anti-CSPG4 IgE in this xenograft model is described in Chauhan et al.34Male and female NOD-111In-labeled anti-CSPG4 IgE or IgG  were administered intravenously at t = 0 h, immediately after starting the scan. SPECT/CT data sets were reconstructed using a Monte-Carlo-based full-3D iterative algorithm . Images were co-registered and analyzed using VivoQuant v2.50 (Invicro). Regions of interest (ROIs) were delineated for 111In activity quantification in specific organs. Uptake in each ROI was expressed as percentage of injected dose per volume (%ID/mL).SPECT/CT imaging was performed on a NanoSPECT/CT scanner  in helical scanning mode using energy windows centered around 171.3 keV and 245.4 keV (±20%). For t = 0–60 min, images were acquired in six 10-min windows using 13 s/projection. At t = 4, 24 and 120 h, images were acquired over 50–55 min using 70 s/projection. CT images were acquired using 45 kVp tube voltage and 1200 ms exposure time in 180° projections. Mice were anaesthetized with isoflurane and ex vivo biodistribution studies at t = 4 h (n = 4) and 24 h (n = 4) were administered 111In-labeled anti-CSPG4 IgE or IgG  intravenously. At t = 120 h, ex vivo biodistribution studies were performed with the mice used for imaging. Mice were culled by cervical dislocation, organs were dissected, weighed, and gamma-counted along with standards prepared from a sample of injected antibody. Antibody uptake was calculated as a percentage of injected dose per gram (%ID/g) of tissue. Blood clearance rates were calculated using a one-phase exponential decay equation.Mice for Excretion of indium-111 was calculated by deducting the sum of the radioactivity in organs, tissues, and carcass from the total injected activity, after decay correction. Excretion rates were calculated using a two-phase exponential decay equation, constrained to a final plateau value of zero and a starting value of 100%.Statistical analyses were performed in GraphPad Prism version 9. Differences between IgE, IgG, and control groups were analyzed by Student’s t-test or ANOVA testing, as appropriate.3.p-SCN-CHX-A”-DTPA, SDS-PAGE analysis revealed bands corresponding to full-size IgG and IgE6  and, under reducing conditions, corresponding to the IgG and IgE heavy and light chains.The anti-CSPG4 IgE and IgG antibodies  were pro111In-radiolabeling efficiencies of 96.9% for the IgG and 92.7% for the IgE . Further purification was judged unnecessary. Non-antibody-bound radioactivity (<8%) can be seen eluting as EDTA complexes with a retention time of 12.8 min. Similar results were obtained by radio-TLC, with the radiolabeled antibody conjugates remaining at the origin whereas free 111In was chelated by citrate ions in the mobile phase . The radio-HPLC traces after 120 h incubation in serum showed some signs of degradation of the radiolabeled antibody, with increased area under the curve (AUC) of peaks corresponding to a higher molecular weight impurity and to unchelated 111In. The degradation was more pronounced for the IgE  than the IgG .The conjugation of the chelator was successful, as evidenced by high A375 cells   and tumor-to-muscle (T/M) ratios were similar (T/B: IgE: 11.5 at 48 h at 48 h) . In contat 48 h) . These r111In-anti-CSPG4 IgG in the blood was calculated as 16.1 h  with a plateau at 3.3%ID/g, whereas 111In-anti-CSPG4 IgE was cleared from the circulation much faster with a calculated blood half-life of 7.3 h  based on samples taken between 4 h and 120 h . To capture the early distribution of the antibodies, SPECT images were acquired in blocks of 10 min over the first hour, starting immediately from antibody injection. These earlier scans revealed that the anti-CSPG4 IgE cleared more rapidly from the circulation than calculated from the previous experiments; high uptake was seen in the liver and kidneys in the first 10 min after administration and very little circulating radioactivity was observed 50–60 min after administration (t1/2 = 9.8 min (R2 = 0.92)  . In cont4.et al.,5 Chauhan et al.50), the fate and distribution of exogenous IgE-based drugs after administration remains unclear. By comparing an IgE and an IgG antibody sharing the same CSPG4-targeting variable domains, we aimed to determine the impact of these different immunoglobulin backbones on in vivo antibody distribution and uptake in CSPG4-expressing tumors. Having conjugated the antibodies to a DTPA-analogue chelator for radiolabeling with the gamma-emitting radionuclide 111In (t1/2 = 67.3 h), we used SPECT/CT imaging to perform longitudinal, noninvasive, and quantitative measurements of antibody distribution. The long half-life of 111In is appropriate to track large biomolecules with long biological half-lives. Whilst limited degradation was noted after 5 days incubation in serum, a large majority of the 111In remained bound to the antibodies, an encouraging sign that the 111In signal observed in vivo would primarily arise from the antibody conjugates or their metabolites rather than from “free”, de- or trans-chelated 111In.Although the use of IgE-class antibodies in anti-cancer therapy has been the focus of many pre-clinical studies  per day, with FCR values for human IgE ranging from 20% to >90% per day.55 In comparison, serum half-lives of 23 days and FCR of 4–8% have been found for human IgG.23–26 Considering our present results, we suggest that methodological issues (lack of very early sampling) in some of these studies may have led to significant overestimations of IgE half-life and the real values are likely even shorter than has been reported. For example, in studies performed in the late 1970s, patients were infused with IgE-rich serum over 4 h and the initial sampling performed 1 h after infusion was taken as the peak IgE serum concentration.56 In some studies in mice, the explicit assumption was made that the entire injected dose of 125I-labeled IgE was still in the serum 10 min after injection.23 In rats, Tada et al. first sampled 15 min after injection of IgE and then at 12 h intervals.32 Vieira and Rajewski found an IgE half-life in serum of 7–16 h, however, their initial sampling appears to have been performed 6 h after administration.29 In the study by Hirano et al., the initial sampling was performed either 15 min or 1 h after administration, resulting in both cases in estimated half-lives of around 12 h. Finally, Haba et al. usefully distinguished between an earlier phase and a later phase for their t1/2 calculations, with an apparent half-life of around 1.5–2 h in the first six hours and 5–8 h over the following days.28 In our initial experiment in tumor-bearing mice, primarily aimed at comparing antibody uptake in the tumors, we initially sampled 4 h after administration and found an apparent blood half-life of 7.3 h for the anti-CSPG4 IgE, in line with the aforementioned studies. The SPECT images of tumor-bearing mice, however, qualitatively suggested a much more rapid clearance of the administered IgE from the circulation. To focus on the early pharmacokinetics of IgE, we repeated the experiments in non-tumor-bearing mice with an imaging protocol refined by adding several early time points, allowing us to determine that anti-CSPG4 IgE had in fact an initial blood half-life of less than 10 min, which is much shorter than previously reported.As the prospect nears of using engineered IgE antibodies in cancer therapy, a better understanding of the pharmacokinetic behavior of this antibody class becomes increasingly necessary. A striking aspect of IgE antibodies is their rapid clearance from circulation compared to IgG. Published estimates of their half-life in circulation range from 1–2 h to 5–16 h  in mice,22 Using 125I- and 131I-labeled IgE, they later demonstrated that the high catabolic rate of IgE was in significant part due to an extravascular pathway specific to this antibody class, and suggested that it occurred through membrane-bound proteases on the surface of FcϵRI-bearing cells, such as basophils and mast cells.23 While we observed some early presence of 111In in the kidneys, which could be due to the presence of unchelated 111In, the high and rapid uptake of the IgE in the liver suggests that instead, the liver is the main location for IgE catabolism, followed by fecal excretion of radioactive metabolites as visible on SPECT images. By using total body radioactivity measurements, we also determined that 111In-anti-CSPG4 IgE (and its radioactive metabolites) was eliminated from the body much faster than its IgG counterpart. This may be due to differences in glycosylation patterns between IgE and IgG. Indeed, human IgE carries 7 N-glycosylation sites per heavy chain, characterized by the presence of terminal oligomannose and galactose residues, compared to a single N-glycosylation site on human IgG.57 These residues enable the recognition of IgE by sugar-binding receptors such as the mannose receptor and asialoglycoprotein receptors, which are expressed on liver cells and mediate the rapid clearance of mannosylated and galactosylated proteins.58 Furthermore, recent studies have shown that in the human PBL engraftment model we used, human CD11b+  and CD11c+ cells  are found in relatively high numbers in the liver and spleen,59 and that human FcϵRI expressed on dendritic cells and monocytes are responsible for the internalization, degradation, and rapid clearance of human IgE in humanized mice.60 Our results are consistent with these previous studies. In contrast to IgE, the anti-CSPG4 IgG showed a distribution and clearance rate typical of labeled monoclonal IgG antibodies, with long persistence in the circulation and accumulation in the spleen, which can be explained by the binding of the IgG to Fcγ receptors expressed on lymphocytes engrafted in the spleen. The scarcity of signal originating from the kidneys is in accordance with the high stability shown by the IgG in serum. Despite inconsistencies in literature reports of sampling times, amounts, and purity of IgE administered  and differences in antigen specificity, models, and assay techniques, there is a consensus that the short serum half-life is an intrinsic property of IgE. Lack of a domain for recognition of the neonatal Fc receptor (FcRn) on the IgE structure61 and differences in the glycosylation patterns of IgE compared to IgG5 may provide a potential explanation for this phenomenon.Imaging the animals from the point of antibody administration revealed that even in the first 10 min, a large fraction of the administered IgE had already cleared from the circulation and was found in the liver and kidneys, contrary to previous assumptions that early blood sampling represented the entire administered dose. Quantitative whole-body imaging conveniently allows measurement of the accumulation and clearance of the administered immunoglobulin on an organ-by-organ basis, which was not possible in previously reported studies of IgE pharmacokinetics. On the other hand, the SPECT signal represents the radionuclide and cannot distinguish the parent antibody from its metabolites, whereas techniques such as the radioimmunoassays used in earlier studies can quantify a relatively intact antibody. Waldmann, Iio and colleagues suggested the existence of two catabolic pathways, one common to all immunoglobulins and one specific to IgE.in vivo compared to a nonspecific isotype control IgE.34 IgE immunotherapy was associated with enhanced tumor infiltration and activation of monocytes and macrophages with pro-inflammatory phenotype characteristics. Although in our study the radiolabeled antibodies were administered at doses (2.5 mg/kg) well below those required to confer therapeutic effects (10 mg/kg) in these models, our findings point to a scenario where a relatively small proportion of administered IgE reaching the tumor might be sufficient to activate immune effector cells and potentially confer anti-tumor effects. This is consistent with known properties of IgE-immune complexes to trigger strong effector cell activation when bound to cell surface FcεRs at levels well below saturation. Early data from the first clinical trial of MOv18 IgE corroborate our results, as both rapid plasma clearance of the IgE and reduction in the tumor biomarker CA-125 were observed.21 As a practical consequence, blood sampling in patients may not be an ideal guide for dosing IgE-based therapies, and typical immunotherapy dosing schedules consisting of a loading dose followed by maintenance doses may not be appropriate. Instead, determining the amount of drug reaching the tumors of human subjects, for which nuclear imaging is a powerful tool,62 may be a preferable approach. Future imaging studies should be performed in the context of clinically relevant IgE doses, as the presence of a liver sink for the IgE means that IgE biodistribution and tumor uptake may be different if the Fc receptors are saturated. Considering the potent immune responses triggered by IgE antibodies, imaging biomarkers of immune cell recruitment and activation status in the tumor micro-environment may also be more appropriate indicators of the pharmacodynamic attributes of this therapeutic class.We report that despite the fast clearance of IgE from the blood, the tumor-to-blood ratios of anti-CSPG4 IgE were much higher than those of a non-tumor targeted IgE, suggesting that anti-CSPG4 IgE could reach its target antigen in tumors. Using the same model of A375 tumors, we recently demonstrated that anti-CSPG4 IgE restricted tumor growth In summary, by radiolabeling both an IgE and an IgG targeting the tumor-associated antigen CSPG4, we have demonstrated that the blood half-life of IgE antibodies is, at around 10 min, much shorter than previously published and that the radioactivity delivered by IgE antibodies is mostly eliminated through the liver followed by excretion via the intestinal tract. In spite of rapid clearance, IgE does reach and is retained in tumors and the tumor-to-blood ratios for IgE and IgG antibodies are comparable. These marked pharmacokinetic differences between IgE and IgG point to differential mechanisms by which each antibody isotype may operate in the context of cancer therapy.Click here for additional data file.


Design and fabricationof functional materials for anti-icing anddeicing attract great attention from both the academic research andindustry. Among them, the study of fish-scale-like materials has provedthat enabling sequential rupture is an effective approach for weakeningthe intrinsic interface adhesion. Here, graphene platelets were utilizedto construct fish-scale-like surfaces for easy ice detachment. Usinga biomimicking arrangement of the graphene platelets, the surfaceswere able to alter their structural morphology for the sequentialrupture in response to external forces. With different packing densitiesof graphene platelets, all the surfaces showed universally at least50% reduction in atomistic tensile ice adhesion strength. Becauseof the effect of sequential rupture, stronger ice–surface interactionsdid not lead to an obvious increase in ice adhesion. Interestingly,the high packing density of graphene platelets resulted in stableand reversible surface morphology in cyclic tensile and shearing tests,and subsequently high reproducibility of the sequential rupture mode.The fish-scale-like surfaces built and tested, together with the nanoscaledeicing results, provided a close view of ice adhesion mechanics,which can promote future bioinspired, stress-responsive, anti-icingsurface designs. Ice accretion is also a lethal hazard to aircraft6 due to its icing effects on the handling and performance of thewings. Icing combined with wind could cause damage and power outageson power networks. Highly relevant to our daily life during winteror in cold regions, icing could blot out the visual field from thewindshield, causing inconvenience to drivers or passengers. Many applicationsof anti-icing or deicing have been used to prevent or minimize icingeffects, aiming at lifetime extension, energy saving, and cost reduction.12 Materials with super-low ice adhesion strength are highly desiredin addressing the icing problem and are under active development today.15 After identifying the determinants of ice adhesion, it is recognizedthat intrinsic ice adhesion is a key factor for the firm attachmentof ice on different surfaces.17 Specifically, for a seemingly ice-covered area on a rough surfaceon the macroscale, termed apparent adhesion, only the truly effectivecontacting points or areas and interlockings on the nanoscale, termedintrinsic adhesion, are responsible for the observed ice adhesionstrength.1 Seeking low intrinsic ice adhesionstrength can rely not only on physical chemistry level atomistic interactions,8 for instance, using superhydrophobicmaterials, but also on the design of the stress-responsive rupturemode of atomistic ice–substrate interactions.20 Considering the full detachment of an intrinsic contacting areaas depicted in 19Unwanted icing is one of the major challenges to infrastructureand human activities in environments below water freezing temperature.22 to designing materials with low intrinsic ice adhesion, naturalsurfaces can provide inspiring guidelines. There are many naturalsurfaces with topography for tailored mechanical functions, especiallythe ones that are able to respond to external stress in wetting andadhesion.23 Two outstanding examples inthis regard are the hierarchical surfaces of water strider legs andgecko toes.25 These two nature-designed surfacesconsist of hierarchical structures of small, flexible, and organizedunits for realizing tailored properties. Specifically, the uniquelyoriented needle-like microsetae on water strider legs enable superiorwater repellence, and well-organized setae on gecko toes enable fastswitching between strong attachment and easy detachment. The microsetaeon water strider legs are superhydrophobic, namely having super-lowadhesion to water.26 In comparison, thesetae on gecko toes can on the one hand, apply strong van der Waalsforces on different surfaces,28 and, on the other hand,can easily detach from solid surfaces by the rolling of the geckofeet, namely by sequential rupture of the setae–substrate interactions.29 The ordered oriented microsetae and setae on the two surfaces aremade for sequential rupture of any adhesion, which is demonstratedin the detachment process of gecko toes from different surfaces. Mostimportantly, the weak adhesion of gecko toes to solid substrates ishighly reusable, which is enabled by the optimized packing of thesetae in the surface hierarchical structures. Mimicking the organizationand the mechanical functions of such natural surfaces by featuringtheir surface topography can serve as a practical approach for loweringintrinsic ice adhesion.19Inspiring by the application of biomaterials19 Usinggraphene platelets for constructing a fish-scale-like surface as shownin Formerstudies have illustrated that sequential rupturing of atomisticinteractions can lead to weaker adhesion, which was also applied tolow intrinsic ice adhesion strength.Given the role of the sequential rupture mode in lowering ice adhesion,an interesting question that awaits an answer is how the arrangementor packing of graphene platelets in the fish-scale-like surface affectsthe intrinsic ice adhesion in attaching-and-detaching cycles. Addressingthis question can further verify the fish-scale-like surface for successfulanti-icing and guide the pattern design. As such, this work modeledand systematically compared ice adhesion and friction on fish-scale-likesurfaces with varied packing densities of graphene platelets. Thisstudy aims at icephobic surface design for low intrinsic ice adhesion,and it also serves as a reference for the nanoscale interface tribologyof snow and ice on solid surfaces.2The focus of atomistic modeling is to construct fish-scale-likesurfaces with a low to high density of graphene platelets. The resultingsurfaces are subjected to molecular dynamics (MD) simulations to probethe ice adhesion strength and the morphological evolution of the hierarchicalsurface structure after repeating icing and deicing cycles.2.119 graphene plateletsof the same size and geometry were used in constructing the fish-scale-likesurfaces here, as shown in 2. In an area of 10.4 × 10.4 nm2 on the XZ-plane, the graphene platelets were arrangedto feature fish scales of different coverage densities as shown in X- and Z-direction , as shown in Following the modeling procedurein the previous study,Ih),30 was used for testing ice adhesionon the three fish-scale-like surfaces. Same as in the previous study,19 a periodic ice layer on the XZ-plane with a thickness of ∼2 nm was placed onto each fish-scale-likesurface, with the basal face (0 0 0 1) of the ice facing the surfaces.The ice and the surfaces had no atomic overlap initially in orderto avoid high energy spots disturbing the stability of the system.At the same time, the ice and the surfaces were adjacent in the interactioncutoff distance, enabling a fast adhesion process in the followingMD simulations.The most commonly observedice in the biosphere, hexagonal ice , with asimulation time step of 2 fs. In order to maintain the stable icestructure, a temperature of 180 K was chosen for all the systems,as in the previous studies.37 The Nosé–Hoovercoupling method with a coupling time of 0.4 ps was used to controlthe temperature of the simulation systems.39 Equilibration simulations with a length of 100 ns were then performedfor the ice to adhere onto the three fish-scale-like surfaces andfor the graphene platelets to adjust their position on the surfacesupon ice adhering. The final system snapshots of the equilibrationsimulations were then taken for nanoscale cyclic deicing tests ofpulling and shearing.Theatomistic structures of the systems were first energy minimized usingthe steepest descent algorithm before carrying out ice adhesion anddeicing simulations. All the MD simulations were carried out in thecanonical 2 was tethered to the center of the mass (COM)of the ice, similar to the previous study.40 For generating tensile force, the spring was set to move at a constantspeed of 0.5 nm/ns along with the Y-axis direction,also vertically away from the fish-scale-like surfaces. To enableshearing, the moving direction of the spring was set to be along oragainst the Z-axis direction. The tensile and shearingforces were then generated with the increasing distance between theCOM of the ice and the spring position, which was recorded every 5ps. The ice adhering tensile stress (σ) was calculated usingthe tensile force normalized by the cross-sectional area of the simulationbox on the XZ-plane (A), namely the apparent areaof the surfaces, as shown in A), as shown in 19 two pulling speeds of 0.2 and 1 nm/ns were also used toprobe the deviation in the results. As a continuous work of the previousstudy,20 this work aims to investigatethe reversibility of these fish-scale-like materials and explore thepackage density’s effect on the ice adhesion, which is expectedto supply the guidelines for anti-icing or deicing materials’design and fabrication.Nanoscale cyclic tests were conductedby first detaching fullyequilibrated ice adhered to the fish-scale-like surfaces, and thenletting the ice re-adhere back onto the surfaces for 100 ns of equilibrationagain, and finally detaching once more. As shown in 33.119 Because of the different packing densities ofthe graphene platelets, the surface area contacting the ice in thethree systems varied, as shown in Figure S1. Specifically, the C4 and C5 surfaces with a low packing densityshowed a larger surface area than the C8 surface with a high packingdensity, as shown by Supporting Information Figure S2. Correspondingly, the equilibrated ice adhering interfacesalso differed on the three surfaces, namely the large rough ridgesobserved on the equilibrated ice interface on C4 and C5 but smalltips on C8, as shown in Supporting Information Figure S2. By comparing the interaction potential between theice and the three surfaces, the C5 surface had the strongest interactionthanks to a large amount of water/ice molecules trapped in the groovesof the surface, while the C8 had the weakest, as depicted in Supporting Information Figure S2. Thus, the C5surface exhibited the best complementing matching with the ice layer,which is highly likely to lead to strong interlocking if the graphenesurface was positionally fixed.The most interesting mechanical properties of thefish-scale-like surfaces are their ability to enable the sequentialrupture for the purpose of weakening adhesion strength.36 As the representativeice detaching event given in Supporting Information Movie (pulling-process.mp4), the ice layer was first slowlylifted from the surface under the increasing pulling force and finallydetached from the substrate, as shown in r, the ice layerwas fully detached from the surfaces, resulting in a sudden drop atthe end of the tensile stress curve. Under the concurrent and sequentialrupture modes, the fish-scale-like surfaces reacted to external pullingstress in remarkably different manners. Because all the graphene plateletswere fixed, the three surfaces showed no structural change throughoutthe deicing process in concurrent rupture. In contrast, the averagethickness of the graphene platelet layer in the sequential rupturemode first increased due to the opening of the graphene plateletsand then decreased after ice detachment, as indicated also in Supporting Information Figures S2 and S3). Betteraccommodation of water/ice molecules in the surface roughness groovesand the resulting stronger interfacial interaction between the iceand the surface have resulted in higher ice adhesion strength, withC5 showing the strongest ice–surface interaction and ice adhesion.Strikingly, the sequential rupture mode in cyclic deicing tests onthe three surfaces resulted in around a 50% reduction in σr as shown in r further confirmedthe effect of the sequential rupture in lowering ice adhesion.19 The σr by the sequential rupturemode obtained in cyclic deicing tests on each surface was stable,as demonstrated in r values monitored in thefirst and second rounds of deicing on each of the three surfaces.Although the local structure of the ice–substrate interfacehad changed after the first detaching event and the subsequent re-adhesionof ice (see below), the σr by sequential rupturewas not significantly affected. The key determinant of σr by sequential rupture was the rupture mode of detachmentrather than the local structure of the ice–substrate interface.The atomistic ice adhesion strengthof the three fish-scale-like surfaces was first compared in a cyclicdeicing test in order to verify the effect of sequential rupture.To do so, the tensile detaching process of the ice layer from thesurfaces was carried out using the same procedure as in the formerstudies.25 For these natural surfaces, the arrangement of the surface units,especially the pattern and packing density, is key to the surfacedurability. It is critical for these natural surfaces to resist anymechanical damage and, in the worst scenario, to recover rapidly fromdamage. Mimicking the abilities of damage resistance is an importantaim of the fish-scale-like surfaces. The reversibility of the hierarchicalstructure of the three fish-scale-like surfaces in the cyclic deicingtests was put together for comparison, as shown in XZ-plane, which indicatedan ability to produce a sequential rupture mode. In contrast, thesurface morphology of C5 and C4 was significantly altered in the cyclicdeicing test. The C5 surface was already partially damaged after thefirst round of deicing, as shown in 42 which can greatly weaken theanti-icing properties of the surfaces. Here, the open areas did contributeto the low ice adhesion strength in the second round of the deicingtest observed on the C4 and C5 because there were no atomistic interactionsbetween the ice and the surface. The low ice adhesion resulting fromthe sequential rupture on the C4 and C5 surfaces thus was not sustainable,given the surface structures easily destroyed by external forces.The reversibility ofthe hierarchical morphologies on biologicalsurfaces such as gecko toes and water strider legs is crucial forthe reproducibility of their special surface mechanical properties.31 The loading rate thus influences the deicing behavior and morphologyreversibility of the surfaces. For comparison, two extra tensile pullingspeeds of 0.2 and 1 nm/ns were chosen for the deicing test on theC5 surface. As the surface snapshots after first-round deicing areshown in 43 a much higher pulling loading rate is neededto generate an obvious difference in rupture stress, which is beyondthe scope of this work. The increase in rupture stress was ∼6%from the lowest to the highest pulling rate (from 161.74 to 170.96MPa). In accordance with the rupture stress, the rupture work neededfor complete deicing also increased with the pulling rate, as depictedin It is known that a higher loading rate, determined by the springtensile pulling speed, can lead to higher rupture stress.3.219 Furthermore, the different packing densities of grapheneplatelets on the three surfaces led to significant variation in thelocal structure of the ice surface and interaction potential, as shownin Supporting Information Figures S2 and S3, which also contributed to the differences in the shearing of theice on the three surfaces. For a detailed comparison, ice was shearedon all the three surfaces along and against the graphene plateletorientation, under both the concurrent mode and sequential mode. Incontrast to the no structural changes of the surfaces under the concurrentmode, the morphology of graphene platelets can be opened and overthrownin a different direction by external shearing force in the sequentialrupture mode. As shown in Supporting Information Movie (shearing-process.mp4), the sequentialrupture mode was able to keep the hierarchical structure of the surfaces.Because of the anisotropic structure of the fish-scale-like surfaces,shear stress was expected to exhibit different features along andagainst the graphene platelet orientation direction.19 Specifically, high shear stress values were observed ifthe ice was sheared against the graphene platelet orientation, owingto the intercalating ice adhering interface with the fish-scale-likesurface. Otherwise, the shear stress was relatively low with saw-teeth-likefluctuations if the ice was sheared along with the graphene plateletorientation. The saw-teeth-like stress pattern was caused by the iceattaching/detaching at the fish-scale-like surface. Because the equilibratedice adhesion led to the matching of the ice with the periodic lowand high repeated surface topography, low shear resistance along thegraphene platelet orientation facilitated sliding of the whole icelayer and repeated re-matching between the ice and the surface. Thepeak value of the shear stress was in the range of 60–80 MPa,as depicted in The anisotropic surfaces, namely the uniform arrangement of thegraphene platelet orientation, resulted in anisotropy in the observedice shear stress. The patterns of ice shear stress profiles were similarto the results reported in the previous study, as shown in Given that the graphene platelets of all the three surfaces werefixed in the concurrent mode, the interface matching of the ice andthe surfaces became strong interlocking against shearing, especiallyon the C5 surface. As highlighted in 4In summary, three fish-scale-like surfaces were built by assemblinggraphene platelets in a uniform ordered orientation. By using cyclictensile and shearing deicing tests, surfaces with a high packing densityof graphene platelets exhibited stable and reversible surface morphologyfor the reproducibility of sequential ice rupture and the subsequentlow atomistic ice adhesion strength. Despite varied ice–substrateinteractions resulting from different graphene platelet packing densities,all the surfaces showed a 50% reduction in ice adhesion strength.The low tensile ice adhesion strength on different fish-scale-likesurfaces was in a similar range, which signified that the sequentialrupture mode was the dominating factor for the reduction of ice adhesion.The high packing density of graphene platelets was key to the fulland reversible coverage of the surface, which remained the integralityof the effective structure for deicing. Furthermore, the high packingdensity was crucial for maintaining a uniform graphene platelet orientationunder shearing stress. Motivated by natural surface examples, thiswork supplied a low intrinsic ice adhesion surface design strategyof deicing, which further verified the sequential rupture as an effectiveapproach for lowering atomistic ice adhesion and at the same timeshed light on new icephobic materials that are responsive to externalstimuli.


Nyssorhynchus darlingi, which, combined with high human density and mobility, add to the local malaria burden.This study reports entomological profile of immature and adult Ny. darlingi at three sites in Mâncio Lima, Acre, during the rainy and dry season . From 63 fishponds, 10,859 larvae were collected, including 5,512 first-instar Anophelinae larvae and 4,927 second, third and fourth-instars, of which 8.5% (n = 420) were Ny. darlingi. This species was most abundant in not-abandoned fishponds and in the presence of emerging aquatic vegetation. Seasonal analysis of immatures in urban landscapes found no significant difference in the numbers of Ny. darlingi, corresponding to equivalent population density during the rainy to dry transition period. However, in the rural landscape, significantly higher numbers of Ny. darlingi larvae were collected in August  and September  (dry season), compared to February (rainy season), suggesting important role of fishponds for vector population maintenance during the seasonal transition in this landscape type. Adult sampling detected mainly Ny. darlingi (~93%), with similar outdoor feeding behavior, but different abundance according to landscape profile: urban site 1 showed higher peaks of human biting rate in May (46 bites/person/hour), than February (4) and September (15), while rural site 3 shows similar HBR during the same sampling period . This study contributes to a better understanding of the larvae biology of the main malaria vector in the Vale do Rio Juruá region and, ultimately will support vector control efforts.Vale do Rio Juruá in western Acre, Brazil, is a persistent malaria transmission hotspot partly due to fish farming development that was encouraged to improve local standards of living. Fish ponds can be productive breeding sites for Amazonian malaria vector species, including  Plasmodium sp. that circulate in the Amazon region  and for September [6.62 (95% C.I.:1.29–33.89)]. Multivariate and univariate regression showed comparable IRR values in the three sites. For periodicity and abandoned characteristics, permanent condition was significant for Anophelinae L1 group in rural Site 3, for univariate analysis only for the Anophelinae L2—L4 group, whereas urban Site 2 shows an increase in larval number for both univariate and multivariate regression. The non-abandoned condition for Ny. darlingi shows an increase in larval number in urban Site 1, for both univariate and multivariate regression. Emerging associated vegetation shows an increase of larval number in the overall regression for all three Anophelinae groups. Presence of Culex sp. and fish was significant for Anophelinae L1 and L2, L3 and L4, in the overall regression. Urban Site 1 showed a similar association for both groups, however only the Anophelinae L2—L4 group maintains this value in multivariate analysis. The presence of amphibians was positively associated with Anophelinae L1 and L2- L4 groups in urban Site 2 only in the multivariate regression. The Ny. darlingi group showed a positive association with amphibian presence only in urban Site 2, for both univariate and multivariate regressions.Considering the February baseline value and a statistical significance at 95% C.I., seasonality differences were not detected in the overall regression for the three identified Anophelinae larval groups. For each site, Anophelinae L1 shows a particular pattern in the univariate regression: a decrease in counts for August and September in urban Site 1, and April and May in rural Site 3; and an increase in counts for urban Site 2 in August. The IRR values observed in the multivariate analysis were maintained relative to the univariate IRR values, indicating that these results were not influenced by possible confounding factors. The Anophelinae L2—L4 group also shows a unique pattern for each site, however, in this case the IRR values were substantially different between the univariate and multivariate analysis. For the r = 0.89), and a low correlation between carbonate hardness and pH (r = 0.38), and carbonate hardness and dissolved chlorine (r = 0.4). Physical-chemistry variables for continuous values  were not statistically associated at 95% C.I. with the abundance of any of the three larval groups. Turbidity shows a significant negative association for the Anophelinae L1 only in the univariate regression [0.98 (IC95%:.97-.99)]. Ny. darlingi L2—L4 shows a significant positive association with turbidity in multivariate analysis only . Shading reduction shows a significant negative association with the abundance of both Anophelinae L1, and Anophelinae L2—L4 in both univariate and multivariate regressions, but for the Ny. darlingi group, the univariate was not significant at the 0.05 level, however, it was near the limit [0.96 (IC95%:.93–1.00) with p = 0.052], whereas in the multivariate analysis shading was significant .Ny. darlingi group. Nitrites were not significantly associated with larval counts, and excluded from the multivariate regression analyses. For carbonated hardness, whereas both Anophelinae L1 and L2, L3 and L4 groups show a highly significant negative association , the Ny. darlingi group did not show statistical significance for any range, except a decrease in larval numbers observed at a range of 3 KH in the multivariate analysis [0.24 (IC95%:.09-.63)]. Dissolved chlorine showed a significant positive association at a range of 1.5 mg/L for the Anophelinae L1 in multivariate analysis [4.23 (IC95%:1.58–11.36)], and the Ny. darlingi for both univariate [2.21 (IC95%:1.17–4.19)] and multivariate analysis [3.41 (IC95%:1.51–7.68)].For ordinal physical-chemistry variables, increased pH values were associated with decreased in larval counts in all three groups, for both univariate and multivariate regression. Similarly, the highest nitrate level (40 mg/L) was associated with decreased larval counts for all three larval groups, and this was maintained in multivariate analysis for the Ny. darlingi. Ny. darlingi among the 3 sites , with a higher abundance in the peridomestic area. The proportion indoors was higher in Site 3 (~25%) than in Site 1 (~12%) or Site 2 (~11%). In Site 1, May showed a higher number of Ny. darlingi in all night captures (21/173 indoor/outdoor) than February (3/13 indoor/outdoor) or September (5/29 indoor/outdoor). Site 2 showed the lowest adult collections: February (3/10 indoor/outdoor), May (0/8 indoor/outdoor), September (0/7 indoor/outdoor). In Site 3, mosquito numbers were consistently high for outdoor collections, and increased for indoor captures in the last two months: February , May (51/101 indoor/outdoor), September .A total of 692 Anophelinae specimens was collected and identified as Ny. darlingi from Site 3 showed higher outdoors peaks during the first part of the night , also demonstrating, besides low values, some indoor peaks that exceeded outdoors ones, in May  and September .Regarding HBR per hour, Site 1 shows more activity in May between 19:00–20:00 (HBR = 46), while February presents low numbers between 19:00–20:00 (HBR = 4), as does September (18:00–19:00 = 13). Site 2 also presents low numbers (with peaks reaching at maximum of four mosquitoes/hr). In contrast, seringal), rural settlements and indigenous populations. For such heterogeneity, the design and the application of specific control methodologies according to each eco-epidemiologic profile is needed https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  1 Mar 2021To the PLOS One Editors,Below are our responses to the reviewers of the manuscript we submitted: [PONE-D-21-00873] - [EMID:61ecbed848a59a53], title: Population dynamics of the primary malaria vector Nyssorhynchus darlingi in a high transmission setting dominated by fish farming in western Amazonian Brazil. 1. 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