The combined use of KMnO4 and PMS (KMnO4/PMS) showed its superiority in improving sludge dewaterability over the separate use of KMnO4 or PMS. By dosing 4 mmol g−1 VSS KMnO4 and 3 mmol g−1 VSS PMS, the dewaterability of waste activated sludge (WAS) significantly enhanced as capillary suction time (CST) decreased from 73.65 s to 24.65 s while the water content of dewatered sludge cake (WC) decreased from 78.96% to 70.47%. Apart from CST and WC, the KMnO4/PMS process could also affect negative zeta potential, sludge flocs size and the concentrations of protein and polysaccharide in extracellular polymeric substances (EPS). The enhanced sludge dewaterability and changes of the physicochemical characteristics of the WAS samples during the KMnO4/PMS process were actually ascribed to sulfate radicals (SO4˙−) and hydroxyl radicals (HO˙) in situ generated via PMS activation by manganese oxides (MnOx) in the states of MnO2 and Mn3O4 transferred from KMnO4 oxidation, which was verified by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX) techniques and radical scavenging experiments. Moreover, the Fourier transform infrared spectroscopy (FTIR) analysis further confirmed that the in situ generated SO4˙− and HO˙ could improve sludge dewaterability. Thus, the KMnO4/PMS process could be considered as a promising conditioning method of sludge dewatering.Ever-increasing efforts have been made to develop rapid and practical conditioning methods of sludge dewatering. This study demonstrated an innovative combination of potassium permanganate (KMnO Ever-increasing efforts have been made to develop rapid and practical conditioning methods of sludge dewatering. WAS has a high water content in the form of free and bound water (over 90%).5 And sludge dewatering has been generally considered as a promising strategy in reducing sludge volume and cost of sludge transport and ultimate disposal.3,4 However, the sludge dewatering process is usually inhibited due to the presence of extracellular polymeric substances (EPS) that can retain a large amount of water, especially bound water.6,7 Hence, increasing efforts have been focused on development of efficient sludge dewatering techniques, which can promote the release of bound water from EPS. So far, thermal, ultrasonication, freezing and thawing, and chemical oxidation have been investigated to improve sludge dewaterability.7–12 Among all the developed methods, advanced oxidation processes (AOPs) have recently shown their superiority in sludge disintegration and dewatering.The management of large quantities of waste activated sludge (WAS) produced from activated sludge processes has posed great challenges to municipal wastewater treatment plants (WWTPs) due to great economic and environmental burden.4˙−) to disrupt WAS and degrade EPS, thereby enhancing sludge dewaterability.2,13,14 It has been reported that the Fenton (combination of H2O2 and Fe2+) and Fenton-like  processes based on HO˙ production could efficiently improve the dewaterability of WAS.1,7 However, only under strongly acidic conditions (pH = 2–5) can these processes generate sufficient amounts of HO˙ for better sludge dewaterability, which significantly hindered the sludge dewatering process and subsequent disposal.15–17 Compared to HO˙ (E0 = 1.8–2.7 V), SO4˙− owns a higher redox potential (E0 = 2.5–3.1 V), a longer life time (30–40 μs) and a wider working pH range (pH 4–9).4,18 Therefore, pretty much attention has been recently paid to the SO4˙−-based AOPs as new alternative methods of sludge conditioning. SO4˙− is usually generated through the activation of peroxydisulfate  and peroxymonosulfate  by ultraviolet (UV), heat, base and (transition) metal catalysts.19–25AOPs usually rely on the produced highly reactive radical species, such as hydroxyl radicals (HO˙) and sulfate radicals  synthesized via a facile hydrothermal method as activators of PMS for ciprofloxacin (CIP) degradation, and found that α-MnO2 nanoflowers achieved much higher degradation efficiency of CIP than the other three MnO2 , which would be attributed to its higher catalytic activity of PMS and larger quantities of SO4˙− produced from its activation of PMS.26 Interestingly, Cui et al. recently studied chemical oxidation of benzene and trichloroethylene (TCE) by an innovative combined use of permanganate (KMnO4) and PMS (KMnO4/PMS), and also found that it was mainly colloidal and amorphous MnO2in situ generated from KMnO4 oxidation that activated PMS to trigger powerful SO4˙−-mediated oxidation of both benzene and TCE.27 As known to us all, both KMnO4 and PMS have been widely used as powerful oxidants to destruct organic pollutants in water and wastewater. Recently, the two powerful oxidants were also applied to enhance the filterability of waste activated sludge.28,29 Wu et al. reported that KMnO4 could efficiently disintegrate the excess sludge with soluble chemical oxygen demand (SCOD) increasing by 3473%, and that the optimal KMnO4/sludge solid mass ratio was 0.1 with a stable disintegration degree (DDCOD) of about 34% while suitable reaction time was 30 min.28 Niu et al. found that PMS oxidation could effectively break sludge particles, and that EPS increased significantly and transferred to slime layer after PMS treatment.29 Yang et al. also reported that PMS could facilitate the disintegration of WAS and the biodegradability of organics could be enhanced after treatment by PMS.30 Although it has been proven that KMnO4 and PMS could disintegrate WAS and enhance sludge dewaterability, the synergistic effect of both oxidants on the dewaterability of WAS still remains unknown.Our recent study investigated α-MnO4 and PMS (KMnO4/PMS) for sludge dewatering. The dewaterability of WAS during the KMnO4/PMS process by the measurement of variations in capillary suction time (CST) and water content of dewatered sludge cake (WC) was initially investigated. Then, the physicochemical characteristics of WAS  at different dosages of KMnO4 and PMS were further studied. Finally, the mechanism of sludge dewatering by the KMnO4/PMS process was deeply explored.In this paper, we proposed a combined use of KMnO2.2.1.The raw sludge was collected from Qige municipal wastewater treatment plant in Hangzhou, China. A sieve with 20 mesh was then used to remove big gravels and debris from the collected sludge samples. The supernatant was finally decanted to acquire the denser sludge samples used in this study after plain sedimentation. The acquired denser sludge samples were stored at 4 °C in a refrigerator prior to use, and all the experiments were completed within 48 h. The basic characteristics of the sludge samples were measured and shown in 5·0.5 KHSO4·0.5 K2SO4, KHSO5 ≥47%) was provided by Aladdin Co., Ltd. . Permanganate , sodium hydroxide , and sulfuric acid  were supplied by Sinopharm Chemical Reagent Co., Ltd. .All the chemicals were used without further purification. Peroxymonosulfate , and was ceased after 120 min. All the experiments were duplicated at least, and the average values with standard deviations were presented.All the sludge dewatering experiments were conducted in an Erlenmeyer flask with a volume of 500 mL. In each test, 300 mL sludge sample was added and then mixed with a stoichiometric amount of PMS. When PMS was completely dissolved under thorough stirring, H2.3.2.3.1.WC. CST was measured using a CST instrument  equipped with an 18 mm diameter funnel and a standard Whatman no. 17 chromatography-grade paper. To determine WC, the sludge samples were pretreated by a vacuum filtration method. Briefly, 50 mL sludge sample was poured into a standard Buchner funnel and filtered at a constant vacuum pressure for 15 min. The water content of the vacuum-filtered sludge cake trapped by the filter paper was measured according to the standard method, and calculated according to m1 is the weight of the filter paper, m2 is the weight of the vacuum-filtered sludge cake, and m3 is the weight of the vacuum-filtered sludge cake completely dried at 105 °C.The dewaterability of WAS is commonly evaluated by CST and 2.3.2.et al.6 Briefly, 50 mL sludge sample was centrifuged at 4000g for 5 min at 4 °C to acquire the supernatant as soluble EPS (S-EPS). The sludge pellets were then diluted with a 0.05 w/v % NaCl solution that was pre-heated to 70 °C. The diluted sludge solution was immediately sheared by a vortex mixer for 1 min and centrifuged at 4000g for 10 min at 4 °C to collect the supernatant as the loosely bound EPS (LB-EPS). The residual sludge pellets were resuspended in a 0.05 w/v % NaCl solution and heated to 60 °C in a water bath for 30 min. At short notice, the resuspended sludge solution was centrifuged at 4000g for 15 min at 4 °C, and the supernatant was collected as the tightly bound EPS (TB-EPS).EPS were extracted using a heat extraction method described by Yu 2SO3 to quench the residual colored KMnO4. The pretreated EPS sample was then filtered through a 0.45 μm cellulose acetate membrane. The protein content was quantified by the modified Lowry–Folin method using bovine serum albumin as the standard.31 The polysaccharide content was determined by the phenol-sulfuric acid method using glucose as the standard.32Before the analyses of protein and polysaccharide (assumed as dominant components of EPS), the EPS samples were pretreated with an optimal amount of Na2.3.3.−1 by a Nicolet 6700 spectrometer .The zeta potential of the WAS samples was analyzed using a Zetasizer Nano . The particle size distribution was measured by a laser particle size analyzer (LAP-W2000H) using ultrapure water as the dispersant. The Fourier transform infrared spectroscopy (FTIR) analysis of sludge samples was recorded in a wavenumber range of 4000–400 cm4/PMS process to monitor whether manganese oxides (MnOx) formed after KMnO4 oxidation of the organics in EPS. Briefly, after the extracted EPS solution was treated with the KMnO4/PMS process, the reaction solution was filtered through a 0.45 μm cellulose acetate membrane to collect the brown-red solids. Then, the collected solids were rinsed alternately several times using ethanol and ultrapure water, and vacuum-dried at 60 °C overnight. Finally, the dried solid samples were scanned by transmission electron microscopy (TEM) to capture the microscale crystallization and structure and energy dispersive X-ray spectroscopy (EDX) to analyze the elemental composition.Given the difficulty in separating the formed solids from the conditioned WAS samples, the extracted EPS solution was used and treated with the KMnO3.3.1.4 alone, PMS alone, and a combination of KMnO4 and PMS (KMnO4/PMS), respectively. As observed, the CST increased from 73.65 s to 140.60 s and 278.55 s, respectively when the WAS samples were conditioned by 7 mmol g−1 VSS PMS and KMnO4 alone, respectively, which indicated that neither PMS alone nor KMnO4 alone could efficiently improve the sludge dewaterability, and that the two individuals even worsened the dewaterability. The deterioration of sludge dewaterability was likely resulted from the progressive cell lysis and further releasing of intracellular biopolymers under the influence of KMnO4 or PMS when the intracellular biopolymers could not be degraded effectively in current condition.33 Lee et al. also found that the PMS conditioning could deteriorate the dewaterability after the WAS samples were treated with different dosages of PMS at room temperature.5 Surprisingly, a combined use of 4 mmol g−1 VSS KMnO4 and 3 mmol g−1 VSS PMS instantly decreased the CST from 73.65 s to 24.65 s, and apparently showed its synergistic effect on significantly improving the sludge dewaterability. Besides, the decrease of WC (from 78.96% to 70.47%) also indicated the synergistic effect after the WAS samples were conditioned by the KMnO4/PMS process while the WC values almost remained unchanged after the samples were treated with PMS alone and KMnO4 alone . The synergistic effect of the KMnO4/PMS process on the WAS dewaterability was interpreted with the decrease of both CST and WC, which also implied the generation of SO4˙− and HO˙ during the KMnO4/PMS process as mentioned in Section 1.3.2.3.2.1.4 and PMS. As shown in −1 VSS, the increase of KMnO4 dosage from 2 mmol g−1 VSS to 6 mmol g−1 VSS did not decrease the CST, and instead jeopardized the dewaterability, which indicated the absence of the synergistic effect of the KMnO4/PMS process at a low PMS dosage. This might be because the in situ generated SO4˙− and HO˙ during the KMnO4/PMS process was too few to improve the dewaterability of the WAS samples when PMS dosage was too low. However, increasing PMS dosage to 2 mmol g−1 VSS efficiently enhanced the dewaterability, and the CST decreased from 73.65 s to 54.30 s, 46.45 s and 63.45 s, respectively with increasing KMnO4 dosage from 2 mmol g−1 VSS to 6 mmol g−1 VSS. When PMS dosage continuously increased to 5 mmol g−1 VSS, the CST decreased to 22.45 s, 23.85 s and 22.00 s, respectively as KMnO4 dosage increased from 2 mmol g−1 VSS to 6 mmol g−1 VSS. This is because the higher PMS dosage, the more SO4˙− and HO˙ generated to achieve better sludge dewaterability.34 The similar results were also reported by Liu et al. when the Fe2+-activated PMS was used for sludge conditioning.4 Remarkably, the CST values did not decrease all the way, and instead decreased firstly and then increased with sustainably increasing KMnO4 dosage, which might be ascribed to the consumption of the generated SO4˙− and HO˙ by KMnO4 with a too high dosage.35 Liu et al. also observed the similar results when using the magnetic Fe3O4–MnO2 core–shell nanocomposites-activated PMS for 4-chlorophenol degradation.36The dewaterability of the WAS samples were further investigated by simultaneously dosing different amounts of KMnO3.2.2.WC of the WAS samples after conditioning of the KMnO4/PMS process with different KMnO4 and PMS dosages were depicted in WC changes in the WAS samples were quite similar to the CST changes discussed in Section 3.2.1. By dosing 1 mmol g−1 VSS PMS, the WC after conditioning with 2 mmol g−1 VSS, 4 mmol g−1 VSS and 6 mmol g−1 VSS KMnO4, respectively  almost remained unchanged, compared to the WC of the raw sludge (78.96%). This suggested that the synergistic effect of the KMnO4/PMS process with 1 mmol g−1 VSS PMS would not work. However, the WC of the WAS samples would continue to decrease with the ever-increasing PMS dosages, which was also attributed to more SO4˙− and HO˙ generated from the KMnO4/PMS process. The WC after conditioning could decrease to about 67.5%, and by 11.46% compared to the WC of the raw sludge, which was higher than the variation in the WC of the WAS samples conditioned by the Fenton-like process (8.7%).1The profiles of 3.2.3.4/PMS process on the WAS dewaterability, the zeta potentials of the WAS samples before and after conditioning were also measured. The unconditioned raw sludge was negatively charged with the zeta potential of −17.7 mV, which was due to the ionization of the carboxyl and amino in the protein of EPS.37 It can be obviously observed from 4 or PMS. The zeta potential tended to increase to 0 mV with increasing KMnO4 or PMS dosage. By simultaneously dosing 4 mmol g−1 VSS KMnO4 and 5 mmol g−1 VSS PMS, the zeta potential increased to −2.15 mV. The increase in the zeta potential suggested the ever-increasing sludge dewaterability, which quite matched with the changes of the CST and WC. Zhen et al. also reported that the sludge dewaterability improved as the zeta potential increased in the Fe2+-activated persulfate process.38 According to the DLVO theory, the aggregation of sludge flocs is mainly controlled by the surface charge. The decrease in the negative surface charge of sludge flocs would reduce the electrostatic repulsion and bring about the increment of interaction energy, which led to the aggregation of sludge flocs and finally enhanced sludge dewaterability. Therefore, it can be concluded that sludge flocs are easier for agglomeration and sedimentation, and thus dewatering as the zeta potential increases.39To deeply investigate the effect of the KMnO3.2.4.4/PMS process on particle size distribution of the WAS samples was also investigated. The D50 value of the unconditioned sludge flocs was determined to be 39.2 μm. When the WAS samples were conditioned with 2 mmol g−1 VSS KMnO4 and 1 mmol g−1 VSS PMS, the D50 value decreased to 32.95 μm. With PMS dosage increasing to 5 mmol g−1 VSS, the D50 value also decreased to 30.08 μm. By dosing 4 mmol g−1 VSS and 6 mmol g−1 VSS KMnO4, the D50 values of the conditioned sludge flocs decreased from 30.42 μm and 28.35 μm to 25.78 μm and 24.44 μm, respectively with increasing PMS dosage. Therefore, increasing both KMnO4 and PMS dosages could reduce the size of the conditioned sludge flocs. The decrease of the sludge flocs size suggested that the KMnO4/PMS process could efficiently break the sludge flocs to enhance the sludge dewaterability as the flocs breakage could provide more passages for the removal of water in the inner of sludge flocs.40 It was also reported by Higgins and Novak that the sludge dewaterability was adversely impacted by the flocs in the range of 1–100 μm and decreased as the quantities of flocs in this size range increased.41As depicted in 3.2.5.42 Since EPS are mainly composed of protein and polysaccharide, variations in the concentrations of protein and polysaccharide in different EPS fractions  were monitored to reveal why the KMnO4/PMS process could improve sludge dewaterability. As illustrated in −1 VSS KMnO4, the protein content in total EPS decreased from 53.46 mg g−1 VSS to 36.99 mg g−1 VSS while the protein content in S-EPS increased from 21.57 mg g−1 VSS to 29.31 mg g−1 VSS before decreasing to 21.59 mg g−1 VSS, as PMS dosage increased. Moreover, the protein content in LB-EPS decreased from 25.21 mg g−1 VSS to 9.01 mg g−1 VSS while the protein content in TB-EPS decreased from 9.68 mg g−1 VSS to 6.39 mg g−1 VSS. Similar changes of the protein content in total EPS, S-EPS, LB-EPS and TB-EPS could also be observed when 4 mmol g−1 VSS and 6 mmol g−1 VSS KMnO4 were dosed. In contrast to EPS can have significant effects on the compressibility of sludge and the ability to bind water molecules of sludge flocs, and thus the dewaterability of sludge.4˙− and HO˙ generated in the KMnO4/PMS process.29 Nevertheless, more SO4˙− and HO˙ were generated to degrade the organics in S-EPS when PMS dosage continued to increase, so the protein and polysaccharide content in S-EPS tended to decrease. In addition, the slump of the protein and polysaccharide content in S-EPS, LB-EPS and TB-EPS accounted for SO4˙− and HO˙ generation in the KMnO4/PMS process and its high reactivity toward S-EPS, LB-EPS, and even TB-EPS.4The dissolved organics (protein and polysaccharide) in S-EPS increased before decreasing because TB-EPS were dissolved and transferred to S-EPS after the sludge flocs were destructed by SO3.3.4 was deemed as an important precursor for the fabrication of manganese oxide nanomaterials through chemical reduction, including manganese dioxides, tetraoxides and oxyhydroxides.43 Wu et al. used KMnO4 as a conditioner for disintegration of excess activated sludge, and found that after sludge oxidation KMnO4 mainly transferred to the states of MnO2 and Mn3O4 while some compounds of KxMnO4 also existed in the solids.28 Due to the multivalent nature of Mn, MnO2 and Mn3O4 have been proven to possess a high catalytic activity toward PMS for SO4˙− generation.44,45 Thus, in the KMnO4/PMS process, manganese oxides (MnOx) in the state of MnO2 and Mn3O4 might be generated from the reduction of KMnO4, then MnO2 and Mn3O4 further activated PMS to produce SO4˙− and HO˙.Due to the versatility and efficiency, KMnO4/PMS process. And the samples were observed through TEM and EDX. From 4/PMS process. Moreover, the EDX analysis further confirmed that the formed solids were mainly composed of element Mn, O and K  and tert-butyl alcohol (TBA) as radical scavengers were conducted to confirm the formation of SO4˙− and HO˙ in the KMnO4/PMS process. As can be seen in −1 VSS EtOH and TBA, the CST decreased from 73.65 s to 38.50 s and 30.25 s, respectively while decreasing from 73.65 to 24.65 s without addition of any radical scavenger. Moreover, the reduction value of CST with the addition of EtOH was higher than that with the addition of TBA. Such difference in CST value drop by the two radical scavengers implied that both SO4˙− and HO˙ were generated in the KMnO4/PMS process because EtOH can effectively quench both SO4˙− and HO˙ while TBA reacts much faster with HO˙ than SO4˙−.25In order to verify the hypothesis, the formed solids were separated from the KMnO O and K , and the4 transformed into MnO2, Mn3O4 and KxMnO4 after oxidizing the organics in EPS. Then, the in situ generated MnO2 and Mn3O4 efficiently activated PMS to generate SO4˙− and HO˙ (5− (the species of PMS) initially integrated with  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>MnIV and MnIII located at the surface of MnO2 and Mn3O4 by surface hydroxyl groups  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O and C–N, and C–N stretching vibration and N–H deformation vibration at 1550–1560 cm−1.49 The typical band located at 1040–1070 cm−1 was due to the stretching vibration of OH.49 Besides, some peaks (<1000 cm−1) monitored in the “fingerprint” region could be attributed to the phosphate or sulfur functional groups, which is the functional groups for the production of nucleic acids.48 Compared with conditioning by KMnO4 or PMS alone, it can be clearly seen that the intensity of absorption bands of WAS decreased obviously after treatment by the KMnO4/PMS process, implying that SO4˙− and HO˙ generated in the KMnO4/PMS process could effectively degrade the proteins and polysaccharides in EPS and then resulted in the release of bound water and the improvement of sludge filterability.FTIR spectrometer was also used to observed the variations of WAS functional groups after conditioning in different conditions. As illustrated in 4.4 and PMS (KMnO4/PMS) for sludge dewatering. The major conclusions can be drawn as followed:This paper investigated the application of a combined use of KMnO4 or PMS, the combined use of KMnO4 and PMS (KMnO4/PMS) could significantly improve sludge dewaterability. After conditioning with the KMnO4/PMS process, CST of the WAS samples decreased from 73.65 s to 24.65 s. However, the dewaterability of WAS would be worsened by conditioning with KMnO4 alone or PMS alone because the CST value increased significantly.(1) Compared with the separate use of KMnO4/PMS process exerted significant effects on the physicochemical characteristics of the WAS samples. When the WAS samples were treated by the KMnO4/PMS process, both CST and WC would decrease, the zeta potential was less negative, and the particle size would also decrease as the sludge flocs were broken into smaller fragments. Besides, the concentrations of protein and polysaccharide in EPS were also reduced due to the synergistic effect of KMnO4 and PMS.(2) The KMnOx in the states of MnO2, Mn3O4 and KxMnO4 after KMnO4 oxidation. The radical scavenging experiments confirmed that the in situ generated MnO2 and Mn3O4 could activate PMS to generate SO4˙− and HO˙, which were responsible for significantly enhanced sludge dewaterability. Moreover, the FTIR analysis further indicated that SO4˙− and HO˙ generated in the KMnO4/PMS process could improve sludge dewaterability.(3) The combined use of TEM and EDX techniques verified the generation of MnOThere are no conflicts to declare.


Streptomyces sp. strain FB2 is an actinomycete isolated from rice rhizosphere. A whole-genome assembly of the strain FB2 comprised 7,727,571 bp . In total, 17 biosynthetic gene clusters (BGCs), including nonribosomal peptides, polyketides, terpenes, and ribosomally synthesized and posttranslationally modified peptides, were predicted. Streptomyces, the largest genus (more than 500 species) of phylum Actinobacteria, is well known for the production of bioactive molecules which effectively control various plant diseases via two distinct mechanisms of antibiosis and induced resistance  was isolated from rice rhizosphere in Telangana, India . Briefly, 1 g of soil attached tightly to the roots was collected, suspended to prepare serial dilutions, and plated onto ISP2 agar plates. After 5 days of incubation at 30°C, a small colony with a powdery appearance was selected, and an axenic culture was obtained by streaking it onto fresh plates.Taq buffer, 0.25 μM each primer,1 μL DNA template, 0.2 mM each dNTPs, 2 mM MgCl2, and 1.25 U Taq polymerase. The reaction conditions were as follows: 95°C for 5 min; 25 cycles of 94°C for 60 s, 60°C for 30 s, and 72°C for 90 s; and a final extension at 72°C for 7 min. The amplicon sequenced and compared against the EzBioCloud database (7 July 2021 update) revealed that Streptomyces sp. FB2 (GenBank accession number MZ736626) is closely related to Streptomyces longispororuber (GenBank accession number AB184440) with 98.66% sequence identity (Streptomyces sp. FB2 genomic DNA using the Illumina NextSeq 500 (2 × 150 bp) platform. Paired-end sequencing libraries prepared using TruSeq Nano DNA library kit generated 17,938,298 reads which were trimmed using a sliding window quality cutoff of Q15 with Trimmomatic (v 0.36)  assembled into 52 scaffolds. After genome assembly, a full genomic coverage at a 240× sequencing depth was obtained. The genome was annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP v 5.3)  and following the manufacturer’s protocol. For primary identification, the 16S rRNA was amplified using universal primers (27F and 1522R). The PCR reaction (25 μL) consisted of 1× Go http://omgenomics.com/circa/) of the genome and BGC annotation are shown in The biosynthetic gene cluster (BGC) repertoire potential in strain FB2 was screened using antiSMASH v 6.0  and BIGFStreptomyces sp. FB2 has been deposited in the DDBJ/ENA/GenBank database under the accession number JAKFQV000000000 .The draft genome sequence of


Heteractis crispa and Heteractis magnifica sea anemones against PD inductors. The peptide HCIQ1c9, which was obtained for the first time, inhibited trypsin less than other peptides due to unfavorable interactions of Arg17 with Lys43 in the enzyme. Its activity was reduced by up to 70% over the temperature range of 60–100 °C, while HCIQ2c1, HCIQ4c7, and HMIQ3c1 retained their conformation and stayed active up to 90–100 °C. All studied peptides inhibited paraquat- and rotenone-induced intracellular ROS formation, in particular NO, and scavenged free radicals outside the cells. The peptides did not modulate the TRPV1 channels but they affected the P2X7R, both of which are considered therapeutic targets in Parkinson’s disease. HMIQ3c1 and HCIQ4c7 almost completely inhibited the ATP-induced uptake of YO-PRO-1 dye in Neuro-2a cells through P2X7 ion channels and significantly reduced the stable calcium response in these cells. The complex formation of the peptides with the P2X7R extracellular domain was determined via SPR analysis. Thus, these peptides may be considered promising compounds to protect neuronal cells against PD inductors, which act as ROS production inhibitors and partially act as ATP-induced P2X7R activation inhibitors.Parkinson’s disease (PD) is a socially significant disease, during the development of which oxidative stress and inflammation play a significant role. Here, we studied the neuroprotective effects of four Kunitz-type peptides from  This increase was significantly reduced in the presence of 10 μM A438079  method. For this purpose, 12.3 ng/mmNeurodegenerative disorders are socially significant diseases; they are some of the most common diseases globally among the elderly, and their frequency is steadily increasing. To date, there is growing evidence that in the development of neurodegenerative diseases, along with the degeneration of dopaminergic neurons (such as in PD), oxidative stress and inflammation play a significant role . The focH. crispa and HMIQ3c1 from H. magnifica interact with several serine proteinases, including inflammatory ones, and they were also described as the first Kunitz-type peptides with neuroprotective activity i). The obtained results showed that both peptides reduced Ca2+ influx and the Ca2+ response into Neuro-2a cells, with HCIQ4c7 more effectively decreasing the calcium response than A438079. Moreover, HCIQ4c7 and HMIQ3c1 bound to the P2X7R extracellular domain with similar dK values, indicating the formation of stable complexes and excluding non-specific binding. This is direct evidence of the ability of the peptides to bind to the studied target, which may partially explain their neuroprotective activity in PD models.Cell death induced by oxidative stress is accompanied by the release of ATP, which at high concentrations activates P2X7R-associated channel opening, leading to osmotic swelling and ultimately to cell death ,23. TherOne of the important requirements for potential neuroprotective compounds is their ability to pass through the blood–brain barrier (BBB), which restricts the entry of proteins and undesirable substances to cerebral tissues. Because most drugs do not cross the BBB, few treatments are available against neurodegenerative diseases, including PD. It has been shown that the Kunitz domain in the β-amyloid precursor protein and Kunitz-type peptide from the bovine pancreas, BPTI, effectively crosses the BBB via low-density lipoprotein receptor-related protein (LRP) ,66. MoreOn the other hand, the BBB is known to become more permeable during the inflammatory process. As mentioned above, P2X7R is actively involved in the inflammatory process. The receptor associated with pannexin-1 and the P2X4 receptor are able to stimulate the ROS production induced by ATP, which leads to the activation of NLRP3  inflammasome and further release of proinflammatory cytokines IL-1β and IL-18 . The infThus, the studied peptides are able to protect neuronal cells against PD inductors via the inhibition of ROS formation and ATP-induced P2X7R activation. However, the molecular mechanism of their interaction with P2X7R and passage through the BBB remains uncertain and requires further detailed study.hciq1c9 construction was synthesized by JSC Eurogen (Russia). The Kunitz-type peptides HCIQ2c1, HCIQ4c7, and HMIQ3c1, as well as a new peptide HCIQ1c9, were obtained as described in [E. coli. The cells were cultured at 37 °C in Luria–Bertani medium containing 100 μg/mL ampicillin until reaching the optical density (OD600) of ~0.5. After induction with IPTG across a concentration range of 0.2–0.5 mM, the cells were incubated at 37 °C for 3 h. The presence of recombinant peptides was determined in 12% polyacrylamide gel by Laemmli’s SDS-PAGE method [The pET32b(+)/ribed in ,51. The E method . Cell prE method . The recHCIQ1c9 was treated using 6 M guanidine hydrochloride in 0.5 M Tris-HCI buffer, pH 8.5, containing 2 mM EDTA. Then, dithiothreitol was added and the mixture was incubated for 4 h at 40 °C. Thiol groups of cysteine residues were modified by 50% 4-vinylpyridine in isopropanol for 20 min at room temperature in the dark. The reaction mixture was separated on a Nucleosil C18 4.6 × 250 mm reverse-phase column  using a concentration gradient of acetonitrile (from 0% to 70%) with 0.1% TFA in 160 min, with a constant flow rate of 0.5 mL/min . The N-tm/z 6107 [m/z 3053.MALDI-TOF MS spectra of peptides were recorded using an Ultra Flex III MALDI-TOF/TOF mass spectrometer  with a nitrogen laser SmartBeam (355 nm), reflector, and potential LIFT tandem modes of operation. Sinapinic acid was used as the matrix. External calibration was employed using a peptide InhVJ with m/z 6107  and its 1H NMR spectra of peptides were acquired at 30 °C on a Bruker Avance III 700 MHz spectrometer  equipped with a triple-resonance z-gradient TXO probe. Peptides were dissolved in 90% H2O/10% D2O  over a concentration range of 1.5–2 mg/mL. Excitation sculpting with gradients [The radients  was applCircular dichroism (CD) spectra were recorded on a Chirascan-Plus CD spectropolarimeter  in quartz cuvettes with an optical path length of 0.1 cm for the peptide spectrum region. The peptides were dissolved in deionized water (40 ng/mL) and incubated at temperatures ranging from 25 °C to 100 °C for 20–25 min before recording the CD spectra. The secondary structure elements were calculated using the Provencher–Glockner method  using adThe trypsin-inhibitory activity of HCIQ1c9 was estimated according to the standard procedure using N-α-benzoyl-D,L-arginine p-nitroanilide (BAPNA)  as a substrate. The trypsin inhibition constant was determined using Dyxon’s method  using suThe temperature influence on the activity levels of peptides was assessed by measuring their residual inhibitory activity levels. After preincubation at temperatures ranging from 25 to 100 °C for 25 min, the peptide solutions were added to trypsin solution and the reaction mixtures were incubated for 10 min at 37 °C, then 1.2 mM BAPNA was added, followed by incubation at 37 °C for 30 min. The final concentration of the peptides was 7 μM. Substrate hydrolysis was measured at 410 nm. The residual activity was calculated based on the results of three parallel experiments according to the following equation :% = (1 −2 web server [The comparative models of complex 3D structures were generated using the (PS)b server . The pepb server ) was chob server  or UCSF b server  using thb server . The resb server , and CONb server . Visuali2 (v/v). Before the experiments, Neuro-2a cells at a concentration of 1 × 104 cells/well were dispensed into 96-well plates and incubated for 24 h in a humidified atmosphere containing 5% CO2 to allow cell attachment. The murine neuroblastoma cell line Neuro-2a was purchased from ATCC . Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum  and 1% penicillin/streptomycin  according to ATCC’s instruction. Cells were incubated at 37 °C in a humidified atmosphere containing 5% COPeptide stock solutions were prepared in deionized water at a concentration of 10 mM. All tested compounds were added to the plate wells at a volume of 20 μL and diluted in PBS to final concentrations of 0.01, 0.1, 1.0, and 10.0 μM. 2O, 17 μL 6N HCl) was added to each well followed by incubation for 4–18 h. The absorbance of the converted dye formazan was measured using a Multiskan FC microplate photometer  at a wavelength of 570 nm [Then, 20 μL of substance solution was loaded into the cells and incubated for 24 h followed by replacement of the medium with tested substances and 100 μL of fresh medium. Then, 10 μL of MTT -2,5- diphenyltetrazolium bromide)  stock solution (5 mg/mL) was added to each well and the microplate was incubated for 4 h. Next, 100 μL of SDS-HCl solution  was added. The cells incubated with or without inductors were used as positive and negative controls, correspondingly. Cell viability was measured after 24 h  or 48 h (with ATP and MPP+) using the MTT assay [At the end of the preincubation period, the cells were treated with peptides at concentrations of 0.01, 0.1, 1, and 10 μM for 1 h, after which 600 μM paraquat, 10 μM rotenone, 80 μM of 6-OHDA, 1 mM MPPTT assay ,86,87. TNeuro-2a cells were incubated with peptides at concentrations of 0.01, 0.1, and 1 µM for 1 h. Then, paraquat (600 μM) or rotenone (10 µM) was added to each well and cells were incubated for 3 or 1 h, respectively. To study the ROS formation, the 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) assay was performed according to the manufacturer’s instructions . H2DCF-DA solution was added to each well, such that the final concentration was 10 µM, then the microplate was incubated for an additional 30 min at 37 °C. ex = 485 nm and λem = 518 nm. The data were processed using MARS Data Analysis v. 3.01R2 . The results are presented as percentages of control with inductor data.To determine the NO production, a 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) assay was performed according to the manufacturer’s instructions . DAF-FM fluorescent probe solution was added to each well to a final concentration 5 μM, then the microplate was further incubated for 40 min at 37 °C. In both cases, the fluorescence intensity was measured using a PHERAstar FS high-speed plate reader  at λThe 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the peptides was tested as described in . The pepXenopus laevis oocytes, the linearized plasmids were transcribed using a T7 or SP6 mMESSAGE-mMACHINE transcription kit . The harvesting of stage V–VI oocytes from anaesthetized female X. laevis frogs was carried out as previously described [2, 2 mM MgCl2, and 5 mM HEPES pH 7.4, supplemented with 50 mg/L gentamicin sulfate.To express TRPV1 in escribed . OocytesX. laevis animals was in accordance with the license number LA1210239 of the Laboratory of Toxicology and Pharmacology, University of Leuven (Belgium). The use of X. laevis animals was approved by the Ethical Committee for Animal Experiments of the University of Leuven (P186/2019). All animal care and experimental procedures agreed with the guidelines of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes .The physiological activity in oocytes heterologously expressing the TRPV1 channels was measured using the two-electrode voltage–clamp technique, using a Geneclamp 500 amplifier  controlled by the pClamp database system . The measurements were performed at room temperature (18–22 °C). Whole-cell currents were recorded 1–4 days after the mRNA injection. The electrode resistance was 0.7–1.5 MΩ. The signal was amplified and preliminarily filtered using an amplifier-embedded Bessel filter (cutoff frequency 500 Hz) after digitization of the signal at 2000 Hz. Recordings obtained before the activation of the examined currents were used to subtract the capacitive and leakage currents. The cells were kept at a holding potential of −90 mV. TRPV1 currents were measured in ND96 solution using a protocol of −90 mV for 400 s. The recording chamber was perfused at a rate of 2 mL/min with the ND96 solution. Capsaicin (2 µM) was used as an agonist and capsazepine (10 µM) was used as an antagonist of TRPV1. Capsaicin and capsazepine were purchased from Sigma-Aldrich . The use of the 2. Then, the cells were washed once with Hanks’ balanced salt solution (HBSS)  and filled with 180 μL of the same buffer. YO-PRO-1  was loaded into the wells to a final concentration of 5 μM, the cells were incubated for 15 min at 37 °C, then ATP was added to a final concentration of 4 mM and the plates were incubated for an additional 10 min. Then, the cells were washed three times with the buffer solution and the fluorescence intensity levels were measured with a PHERAstar FS plate reader  at λex = 480 nm and λem = 520 nm. A438079 (10 μM), a standard inhibitor of P2X7R, was used as positive control. The effectiveness of the peptides was evaluated relative to the control with ATP.For the uptake measurements of the large cationic dye YO-PRO-1, the peptides at final concentrations of 0.01, 0.1, and 1.0 μM were added to the culture medium with Neuro-2a cells and further incubated for 1 h at 37 °C with 5% CO2. Then, cells were washed twice with culture medium and loaded with 5 μM Fluo-8 dye  and 1 μM Pluronic F-127  and incubated for 40 min at room temperature in HBSS saline at pH 7.4. Then, the cells were washed two times with the same solution but without the fluorescent dye and were incubated for 20–30 min at room temperature in the dark. In some experiments, Ca2+-free medium was used. Here, 140 mM NaCl, 5 mM KCl, 0.8 mM MgCl2, 10 mM glucose, 10 mM HEPES, 5 mM EGTA, pH 7.4. ATP  were added using a robotic microinjector (20 μL/well), then 10 s later the baseline recording and additional readings were taken up to 50 s at 1 s intervals. The standard P2X7R inhibitor A438079  was used as the inhibitory control. Ionomycin  was used to generate a generic calcium signal in cells. Fluo-8 was excited at 488 nm, and the emission at 520 nm was measured with a PHERAstar plate reader .Neuro-2a cells were pre-incubated with studied peptides for 1 h at 37 °C with 5% CO2 of chip surface). HBS-N   was used as a running buffer for SPR assays. Peptide solutions in the HBS-N buffer over a concentration range of 5–100 μM were passed through biosensor channels (working and reference) at a flow rate of 10 µL/min for 3 min at 25 °C. Dissociation of the formed peptide–P2X7R complexes was registered at the same flow rate for no less than 6 min from the moment of sample injection. After each biosensor cycle, analytes were removed with two injections of regenerating solution  at a flow rate of 30 μL/min for 30 s.SPR analyses were performed based on using a Biacore 3000 optical biosensor  running under the program “Biacore 3000 Control Software v.1.0”. Recombinant human P2X7R   was covalently immobilized on the carboxymethylated surface of the Biacore CM5 sensor chip , then activated by the 1:1 mixture of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.05 M N-hydroxysuccinimide (NHS) via the injection of receptor solution (15 μg/mL) in 10 mM sodium acetate (pH 5.0) for 10 min at a flow rate of 5 μL/min. The reference channel without immobilized P2X7R was used to correct the effects of the non-specific binding of peptides to the chip surface. The quantity of the immobilized hP2X7R equaled 12300 RU  using “1:1 binding (Langmuir)” and “two-state  binding” data processing models.dK is the equilibrium dissociation constant, koff1 is the dissociation rate constant, kon1 is the association rate constant, kon2 is the forward rate constant for the CP ↔ CP * transition, koff2 is the backward rate constant for transition CP ↔ CP *, C is the compound, and P is the immobilized protein.The final kinetic parameters were obtained from the model of two-state  binding. The equation describing the used model was as follows:t-test was performed using SigmaPlot 14.0  to determine statistical significance.All data were obtained from three independent replicates and calculated values were expressed as means ± standard error of the mean (SEM). Student’s


We describe the crystal structure of a fragment of human ADAR2 bound to RNA bearing a G:G pair adjacent to an editing site. The two guanosines form a Gsyn:Ganti pair solving the steric problem by flipping the 2-amino group of the guanosine adjacent to the editing site into the major groove. Also, duplexes with 2′-deoxyadenosine and 3-deaza-2′-deoxyadenosine displayed increased editing efficiency, suggesting the formation of a Gsyn:AH+anti pair. This was supported by X-ray crystallography of an ADAR complex with RNA bearing a G:3-deaza dA pair. This study shows how non-Watson–Crick pairing in duplex RNA can facilitate ADAR editing enabling the design of next generation guide strands for therapeutic RNA editing.ADARs (adenosine deaminases acting on RNA) can be directed to sites in the transcriptome by complementary guide strands allowing for the correction of disease-causing mutations at the RNA level. However, ADARs show bias against editing adenosines with a guanosine 5′ nearest neighbor (5′-GA sites), limiting the scope of this approach. Earlier studies suggested this effect arises from a clash in the RNA minor groove involving the 2-amino group of the guanosine adjacent to an editing site. Here we show that nucleosides capable of pairing with guanosine in a  In addition, we report the effect on the ADAR deamination rate of several purine analogs paired with a 5′-G in a target site from the MECP2 transcript where mutation causes Rett Syndrome. Together these results show the use of nucleosides capable of stable pairing with the 5′ G in the syn conformation enables editing within 5′-GA target sites.ADARs (adenosine deaminases that act on RNA) catalyze adenosine deamination within duplex RNA yielding inosine ,2. SinceMolecular-biology-grade bovine serum albumin (BSA) and RNase inhibitor (RNAsin) were purchased from New England BioLabs. SDS-polyacrylamide gels were visualized with a Molecular Dynamics 9400 Typhoon phosphorimager. Data were analyzed with Molecular Dynamics ImageQuant 5.2 software. All Matrix Assisted Laser Desorption/Ionization (MALDI) analyses were performed at the University of California, Davis Mass Spectrometry Facilities using a Bruker UltraFlextreme MALDI TOF/TOF mass spectrometer. Oligonucleotide masses were determined with Mongo Oligo Calculator v2.06. Oligonucleotides for sequencing and PCR were purchased from Integrated DNA Technologies or Dharmacon. All other oligonucleotides were synthesized as described below.4OH at 55°C for 12 h. The supernatant was transferred to a new screw-cap tube and evaporated under reduced pressure. For all oligonucleotides except the azaN-modified strand, desilylation was performed by treating the pellets with 250 μl of 1M TBAF–THF at room temperature overnight. For the azaN strand, desilylation was carried out in TEA•3HF as previously described  phosphoramidite which was purchased from Berry & Associates or synthesized as previously described . Nucleosescribed . To eachg for 20 min and supernatant was removed. The RNA solutions were lyophilized to dryness, resuspended in nuclease-free water and quantified by absorbance at 260 nm. Oligonucleotide mass was confirmed by MALDI-TOF. Mass spectrometry data can be found in Single-stranded RNA oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis and visualized by UV shadowing. Bands were excised from the gel, crushed and soaked overnight at 4°C in 0.5 M NaOAc, 0.1% sodium dodecyl sulfate (SDS), and 0.1 mM EDTA. Polyacrylamide fragments were removed with a 0.2 μm filter, and the RNAs were precipitated from a solution of 75% EtOH at –70°C for 12 h. The solution was centrifuged 17 000 × For crystallography, the unmodified RNA guide strand was purchased from Horizon Dharmacon and purified as described above. As in previous structures, the edited strand contained the adenosine analog azaN at the editing site. Duplex RNA was hybridized in water in a 1:1 ratio by heating to 95°C for 5 min and slow cooling to 30°C.Target RNAs for deamination kinetic analyses were transcribed from DNA templates with the MEGAScript T7 Kit (ThermoFisher). DNA digestion was performed using RQ1 RNase-free DNase (Promega). DNase-treated RNA product was purified as described above.Purified guide and transcribed RNA were added in a 10:1 ratio to hybridization buffer , heated to 95°C for 5 min, and slowly cooled to room temperature.Saccharomyces cerevisiae as previously described (g for 1 h). Lysate was passed over a 3 ml Ni-NTA column, which was then washed in three steps with 20 ml lysis buffer, wash I buffer , wash II buffer , and eluted with 20 mM Tris–HCl, pH 8.0, 5% glycerol, 1 mM BME, 400 mM imidazole, 100 mM NaCl. Fractions containing the target protein were pooled and concentrated to 30–80 μM for use in biochemical assays. Protein concentrations were determined using BSA standards visualized by SYPRO orange staining of SDS-polyacrylamide gels. Purified hADAR2 WT was stored in 20 mM Tris–HCl pH 8.0, 100 mM NaCl, 20% glycerol and 1 mM BME at –70°C.Full length human ADAR2 (hADAR2) was overexpressed in escribed . PurificS. cerevisiae BCY123 cells were transformed with this plasmid and the fusion protein was overexpressed as described previously (2 using a microfluidizer. Cell lysate was clarified by centrifugation (39 000 × g for 50 min). Lysate was passed over a 2 ml NEB amylose column (pre-equilibrated with binding buffer), which was then washed in two steps with 50 ml binding buffer followed by 100 ml wash buffer  and eluted with buffer containing 50 mM Tris–HCl, pH 8.0, 10% glycerol, 5 mM BME, 500 mM KCl, 0.01% NP-40, 50 μM ZnCl2 and 20 mM maltose. Fractions containing the target protein were pooled and dialyzed against a storage buffer containing 50 mM Tris–HCl, pH 8.0, 400 mM KCl, 0.5 mM EDTA, 0.01% NP-40, 10% glycerol and 1 mM tris(2-carboxyethyl)phosphine. Dialyzed protein was concentrated to 2–50 μM and stored as aliquots at –70°C until further use in biochemical assays. Protein concentrations were determined using BSA standards visualized by SYPRO orange staining of SDS-polyacrylamide gels.MBP-tagged human ADAR1 p110 construct was cloned into a pSc vector using standard PCR techniques. The generated construct (yeast codon optimized) consisted of an N-terminal MBP-tag, a tobacco etch virus (TEV) protease cleavage site followed by the human ADAR1 p110 gene. eviously . Purific2, 160 U/ml RNAsin, 1.0 μg/ml yeast tRNA, 10 nM RNA, and 75 nM human ADAR2. Each reaction solution was incubated at 30°C for 30 min before the addition of enzyme. Reactions were then incubated at 30°C for varying times prior to quenching with 190 μl 95°C water and heating at 95°C for 5 min. Reaction products were used to generate cDNA using RT-PCR (Promega Access RT-PCR System). DNA was purified using a DNA Clean & Concentrator kit (Zymo) and subjected to Sanger Sequencing via GeneWiz (Azenta). The sequencing peak heights were quantified in SnapGene (Domatics). Data were fit to the equation P]t is percent edited at time t, [P]f is the final endpoint of editing, and obsk is the observed rate constant. Because of the slower reactions for ADAR1 p110 and lower reaction end point, data were fit to the equation obsk reported is the average of each replicate ± standard deviation (SD). Statistical significance between groups was determined by one-way Analysis of Variance (ANOVA) with Tukey's multiple comparisons test using Prism software (GraphPad). For the ADAR1 p110 enzyme, deamination reactions were performed as above with the following modifications: The final reaction solution for ADAR1 p110 contained 15 mM Tris–HCl, pH 7.0 4% glycerol, 26 mM KCl, 40 mM potassium glutamate, 1.5 mM EDTA, 0.003% NP-40, 160 U/ml RNAsin, 1.0 μg/ml yeast tRNA, 10 nM RNA and 250 nM ADAR1 p110.Deamination assays were performed under single-turnover conditions in 15 mM Tris–HCl pH 7.5 3% glycerol, 60 mM KCl, 1.5 mM EDTA, 0.003% NP-40, 3 mM MgClS. cerevisiae BCY123 cells were transformed with a pSc-ADAR construct encoding hADAR2-R2D E488Q (corresponding to residues 214–701). Cells were streaked on yeast minimal media minus uracil (CM-ura) plates. A single colony was used to inoculate a 15 ml CM-ura starter culture. After cultures were shaken at 300 rpm and 30°C overnight, 10 ml of starter culture was used to inoculate each liter of yeast growth medium. After cells reached an OD600 of 1.5 (∼20–24 h) cells were induced with 110 ml of sterile 30% galactose per liter and protein was expressed for 6 h. Cells were collected by centrifugation at 5000 × g for 10 min and stored at –80°C. Cells were lysed in 750 mM NaCl in buffer A  with a microfluidizer. Cell lysate was clarified by centrifugation (39 000 × g for 25 min). Lysate was passed over a 5 ml Ni-NTA column equilibrated with buffer A with 750 mM NaCl, which was then washed in three steps with 50 ml of lysis buffer, wash I buffer (buffer A + 300 mM NaCl), and wash II buffer (buffer A + 100 mM NaCl). Protein was eluted with a 35–300 mM imidazole gradient in wash II buffer over 80 min at a flow rate of 1 ml/min. Fractions containing target protein were pooled and further purified on a 2 ml GE Healthcare Lifesciences Hi-Trap Heparin HP column in wash II buffer without BME. The His10 fusion protein was washed with 50 ml of wash II buffer without BME and eluted with a 100–1000 mM NaCl gradient over 60 min at a flow rate of 0.8 ml/min. Fractions containing target protein were pooled and cleaved with an optimized ratio of 1 mg of His-tagged TEV protease per 1 mg of protein. Cleavage was carried out for 4 h at room temperature without agitation, followed by overnight cleavage at 4°C before the product was passed over another Ni-NTA column at a flow rate of 0.5 ml/min. The flow through and wash were collected and passed through another Ni-NTA column to remove remaining uncleaved protein. The flow through and wash were collected, dialyzed against 20 mM Tris, pH 8.0, 200 mM NaCl, 5% glycerol and 1 mM BME, followed by concentration to just under 1ml for gel filtration on a GE Healthcare HiLoad 16/600 Superdex 200 PG column. Fractions containing purified protein were pooled and concentrated to 7–9 mg/ml for crystallization trials.Protein expression and purification were carried out by modifying a previously reported protocol . S. cereGLI1 (G:G pair) RNA complex were grown at room temperature by the sitting-drop vapor-diffusion method. A solution of 1.0 μl volume containing 5.6 mg/ml protein (95 μM) and 47.5 μM GLI1-GG RNA was mixed with 1.0 μl of 50 mM MOPS pH 7.0, 200 mM NaCl, 17% PEG 4000. Crystals took about 10 days to grow. A cluster of crystals was broken apart and a single cuboid-shaped crystal approximately 100 μm in size was soaked briefly in a solution of mother liquor plus 30% ethylene glycol before flash cooling in liquid nitrogen.Crystals of the hADAR2-R2D E488Q-GLI1 (G:3-deaza-dA pair) RNA complex were grown at room temperature by the sitting-drop vapor-diffusion method. A solution of 1.0 μl volume containing 100 μM protein and 50 μM GLI1-G3dA RNA was mixed with 1.0 μl of 50 mM MOPS pH 7.0, 100 mM NaCl, and 13% PEG 4000. Flat rhomboid-shape crystals took about 8 days to grow to 100 μm. A single crystal was soaked briefly in a solution of mother liquor plus 30% glycerol before flash cooling in liquid nitrogen.Crystals of the hADAR2-R2D E488Q-GLI1 (G:G pair) complex were collected on beamline 24-ID-E to 2.7Å resolution while the ADAR2-R2D E488Q GLI1 (G:3-deaza dA pair) data were collected on the 24-ID-C beamline to 2.8Å resolution. Both data sets were processed with XDS  was used as the phasing model. The structures were refined with PHENIX including non-crystallographic symmetry (NCS) restraints at the start, but relaxed at the final stages resulting in a lower R-free. Additionally, RNA base-stacking and base-pair restraints, when appropriate, were also imposed in refinement. Table X ray diffraction data for both structures were collected at the Advanced Photon Source (APS). Diffraction data for the ADAR2-R2D E488Q with XDS  and scalwith XDS . The strwith XDS . The preAs observed in the previous ADAR2-R2D-Gli1 structure , the asyin vitro.An earlier report describing optimization of guide strands for SNAP-ADARs, where an ADAR deaminase domain is fused to a SNAP tag and covalently linked guiding oligonucleotide, showed that pairing the 5′ G in a 5′-GAG-codon with either A or G increased editing efficiency compared to a G:C or G:U pair at that site . Since tGLI1 RNA substrate containing the G:G pair. We prepared a 32 bp duplex bearing 8-azanebularine (azaN) at a known editing site for trapping an ADAR-bound complex . . 13). The Figure , C. Indee Figure  22). Th. Thsyn cructures ,26. CompA Figure . This shsyn:Ganti pair as well as the corresponding A:U pair in native GLI1 all reside in the 2′-endo conformation. This can be due to the sliding of these bases relative to ideal A-form helical structure, resulting in the widening of the major groove as seen previously . Th. Thsyn:Gns Table . The sloMECP2 R255X RNA duplex was a poor substrate for ADAR1 p110 in vitro, we were able to measure rates of deamination for this enzyme using the model substrate shown in Figure While the syn conformation with its Hoogsteen face directed toward the Watson–Crick face of the 3-deaza dA on the opposing strand. The position of the substrate strand is shifted slightly from that of the structure described above such that, in this structure, the base pair hydrogen bonding involves 3-deaza dA N1 to G N7 (2.8 Å) and 3-deaza dA 6-amino to G O6 (3.0 Å)  Figure . This ine Figure . This ornti pair    Figure . Notably5 Figure , D. The MECP2 gene associated with Rett Syndrome  is substantially higher than for adenosine (3.7), this site will more likely bear a proton available for hydrogen bonding in the 3-deaza system under the conditions of the ADAR reaction (Ka of the 7-deazaguanosine (10.3) compared to guanosine (9.5) weakens the N1H-O6 hydrogen bond in the Gsyn:Ganti base pair  and against sites with 5′ nearest neighbor G ,29,30. TSyndrome . In our Syndrome ,22,31,32Syndrome . The Gsyribozyme ,33–35. Iribozyme , Table 4U Figure . Importar Figure . The increaction . Interesase pair . Indeed,uanosine . We also complex .syn conformation at this location in the RNA without also introducing additional sterically demanding groups into the minor groove enables efficient editing at these sites.In conclusion, the combination of deamination kinetics and structural studies described here identified an approach to facilitate ADAR editing at challenging 5′-GA sites. The use of nucleosides capable of hydrogen bonding to the Hoogsteen face of the 5′ G and inducing a Atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession ID codes: 8e0f, 8e4x.gkac897_Supplemental_FileClick here for additional data file.


These results provide valuable insights into further rational development of SST analogue drugs targeting SSTR2.The endogenous cyclic tetradecapeptide SST14 was reported to stimulate all five somatostatin receptors (SSTR1–5) for hormone release, neurotransmission, cell growth arrest and cancer suppression. Two SST14-derived short cyclic SST analogues (lanreotide or octreotide) with improved stability and longer lifetime were developed as drugs to preferentially activate SSTR2 and treat acromegalia and neuroendocrine tumors. Here, cryo-EM structures of the human SSTR2–Gi complex bound with SST14, octreotide or lanreotide were determined at resolutions of 2.85 Å, 2.97 Å, and 2.87 Å, respectively. Structural and functional analysis revealed that interactions between β-turn residues in SST analogues and transmembrane SSTR2 residues in the ligand-binding pocket are crucial for receptor binding and functional stimulation of the two SST14-derived cyclic octapeptides. Additionally, Q102 SSTR1–5 are class A G protein-coupled receptors (GPCRs), and they can couple with inhibitory G proteins (Gi or Go) upon binding SST analogues3. These SSTRs are widely distributed in the central nervous system (CNS), peripheral tissues, pancreas, gut, and associated cancer cells, playing crucial roles in hormone release, neurotransmission, cell growth arrest, and cancer suppression6. The endogenous peptides SST14 and SST28 exhibit nonspecific binding to all five SSTRs. According to the analysis of sequence homology and ligand binding specificity, SSTRs can be divided into two categories: SSTRs 2, 3, 5, and SSTRs 1, 47. Each SSTR subtype has specific pharmacological and physiological properties. For example, adrenocorticotropic hormone (ACTH) and thyroid-stimulating hormone (TSH) secretion are downregulated by SSTR2 and SSTR5. Glucagon secretion is principally suppressed by SSTR2. Both insulin and prolactin (PRL) are inhibited by SSTR59. It is necessary to develop a variety of synthetic SST analogues that selectively block the release of certain hormones through different SSTR subtypes with good receptor subtype selectivity and stability. Among the five SSTRs, SSTR2 is aberrantly expressed in many cancer cells and tumor blood vessels, and it suppresses cancer growth and promoted cell apoptosis15. Accordingly, SSTR2 is an important drug target for the treatment of multiple diseases, such as neuroendocrine tumors (NETs), thyrotropinoma, and cancer18.Somatostatin (SST) is a cyclic hormone release-inhibiting peptide with two similar physiologically functional subforms, namely, SST14 and SST28 (extension of SST14 at the N-terminus), that negatively regulates cell proliferation, the release of multiple hormones and cancer suppression via activation of five cognate SST receptors (SSTR1–5)3 and Cys14. The residues Phe7, Trp8, Lys9, and Thr10, which constitute a flexible β-turn, play an essential role in determining the binding affinity and biological activity of the peptide19. Although SST14 has the potential to modulate many pathological diseases, it has several shortcomings that have hindered its pharmacological application, including a short half-life (< 3 min) and nonselectivity 20. The development of long-acting SST analogues with greater receptor subtype selectivity to regulate unique hormone release is particularly important. By using SST14 as the template, two well-known eight-residue short cyclic SST analogues (lanreotide and octreotide) were developed as commercial drugs to treat acromegalia and NETs22. The two SST analogues showed preferential binding to SSTR2, with moderate affinity to SSTR3 and SSTR5 and no affinity to SSTR1 and SSTR425. Octreotide lacks 6 amino acids of the SST14 macrocycle but retains the three essential pharmacophore residues  to form β-turns. A D-amino acid (D-Trp4) was introduced into octreotide to replace the natural L-Trp8 of the core β-turn residues in SST14 to increase metabolic stability. The resulting octreotide exhibited many advantages compared with SST14, including a long half-life (2 h), reduced blood clearance, more stable conformation and improved selectivity for type 1 SSTRs 26. Another SST analogue drug, lanreotide , was also developed, containing the core β-turn residues  which are also present in octreotide. Moreover, two residue differences were observed in lanreotide  versus octreotide  or SST14   and thyrotrophic adenoma with TSH secretion41. However, the structural basis for the natural biological activity of the modified SST analogues (octreotide or lanreotide) with preferential binding to SSTR2 remains unknown.The endogenous peptide SST14 is a cyclic tetradecapeptide with a loop formed by a disulfide bridge between Cys10) Fig. 27. Both To understand the role of the two cyclic octapeptide SST analogue drugs developed from SST14 and their subtype selectivity, we herein determined the cryo-EM structures of SSTR2 in complex with inhibitory G proteins, with binding of endogenous SST14 and the two cyclic octapeptide SST analogue drugs (octreotide or lanreotide) at resolutions of 2.85 Å, 2.97 Å, and 2.87 Å, respectively. Combinatorial structural analysis and Gi-based functional data provide structural insights into the specific binding of SST analogues to SSTR2.7, Trp8, Lys9, and Thr10 in SST14)  signal peptide was added to aid in the detection of the surface expression of SSTR2. A dominant-negative form of human Gαi1 (DNGi1) with two mutations 43 was coexpressed with Gβ1, Gγ2, and SSTR2 in insect cells. Unless otherwise specified, Gi refers to the engineered G protein, which was used for further structure determination. A single-chain antibody, ScFv16, was added to stabilize the SSTR2–Gi complex, allowing efficient assembly of the SSTR2–Gi complex. All three complexes were imaged under a Titan Krios microscope equipped with a K3 summit direct detector, and the structures of SSTR2–Gi bound with SST14, octreotide, and lanreotide were determined at global resolutions of 2.85 Å, 2.97 Å, and 2.87 Å, respectively  Fig. . To inveely Fig. .Dβnal and Thr-NH2 at the N- and C-termini of lanreotide could not be built  contributed to the interactions with β-turn residues of the SST analogues albeit with different detailed interaction specifications.Comparisons between structures of SSTR2–Gi complexes bound with the three different SST analogues  reveal the detailed specific interactions between residues in the ligand-binding pockets of SSTR2 and the β-turn residues of the SST analogues, leading to structural insights into peptide ligand optimization for SSTR2 receptors with pharmacological significance. In general, the three structures all showed that the SSTR2 residues Q1029 in SST14, Lys5 in octreotide or lanreotide) was observed to form a strong electrostatic interaction with D1223.32, cation–π interaction with Y3027.43, and hydrogen bond interaction with Q1263.36 in SSTR2  or octreotide (from 10−7.7 to 10−7 M) binding, while the function of the same mutant was abolished upon lanreotide binding. These observations indicate that the interactions between β-turn Lys5 of lanreotide and Q1263.36 are essential, which is consistent with the observed closer distance between the side-chain carbonyl C(O) of Q126 and the N-H of β-loop Lys5 in lanreotide (3.1 Å) versus the N-H of β-loop Lys9 in SST14 (4.0 Å) or Lys5 in octreotide (3.5 Å).First, the essential β-turn Lys residue  was observed to form π–π stacking or hydrophobic interactions between the indole ring of Trp and the benzyl rings of F2085.38 and F2726.51. The change from L-Trp to D-Trp enhanced the hydrophobic interaction of this residue with F2085.38 in the structure of octreotide- or lanreotide-bound SSTR2. The change from L-Trp8 in SST14 to D-Trp4 in octreotide or lanreotide also affected the interaction between the backbone carbonyl oxygen of the β-turn Trp and N2766.55 of SSTR2  surrounding Q1022.63 in SSTR2 , but only have minor effect on SST14 function. The function data provide a clue regarding the interaction between Thr6 of octreotide and Q1022.63 of SSTR2  of SSTR2 are essential for interactions with SST analogues.Structural and functional analysis of the interactions between the β-turn residues in SST analogues and ligand-binding pocket residues of SSTR2 showed that the β-turn Lys residue is the most essential residue and plays major roles in SSTR2 binding and receptor function stimulation. Changing L-Trp to D-Trp in the β-turn of the SST analogues not only enhanced peptide stability but also enhanced the interactions between octreotide or lanreotide and SSTR2 compared to those with SST14. Replacing the β-turn Thr to Val in lanreotide strongly changed the interactions between SST analogues and SSTR2. On the other hand, the residues in the ligand-binding pocket  of SSTR2 moved downwards upon binding of octreotide or lanreotide compared with the binding of SST14   of SSTR2 to transmit the activation signal. Hydrophobic interactions between D-Trp.48 Fig.  and the tif Fig. . The obsTMD Fig. . Our stri are similar to those of other class A GPCR complexes. Of the three cryo-EM structures of the SSTR2–Gi/SST analogue complexes, two conventional interfaces between the transmembrane helices of SSTR2 and the α5 helix of Gi protein and between intracellular loop 2 (ICL2) of SSTR2 and the α5 and GαN helices were observed to stabilize the SSTR2 and Gi complex. Moreover, two more interaction interfaces were observed to contribute to the stability of the SSTR2–Gi complex in the presence of the SST analogue lanreotide.The interactions between SSTR2 and Gα6.33 and V2586.37 of TM6. Additional hydrophobic interactions between I344 and L348 of α5 helix and V2355.65 on TM5 and V1443.54 on TM3 were observed to further stabilize the interface between the α5 helix and TM3 and TM5. Furthermore, the electrostatic interaction between R1403.50 and Y2285.58 could further stabilize the conformation of TM3 and TM5 of SSTR2.The first conventional interface was observed in the α5 helix of the Gi protein, which was inserted into the central cytoplasmic cavity of the transmembrane bundle formed by TM3, TM5 and TM6 Fig. . The hydICL2 of SSTR2 and E28 of the GαN helix and the charge interaction between R154ICL2 of SSTR2 and N347 of the α5 helix were observed to stabilize the coupling of ICL2 to the Gi protein. Moreover, I148 of ICL2 was observed to form an extensive hydrophobic interaction network with I343 and F336 on α5 helix and L194 on the β2 sheet of the Gi protein.The second conventional interface was observed to contain the ICL2 of SSTR2. The ICL2 helix was observed to tightly interact with the Gi protein, mainly through interactions with the α5 helix and GαN helix Fig. . The eleAdditionally, interactions between intracellular loop 3 (ICL3) of SSTR2 and the Gi protein were also observed Fig. ; these i2 of N318 and the backbone carbonyl oxygens of K349 could further stabilize the SSTR2–Gi complex , it has been reported that SST-mediated effects are dependent on receptor subtypes. Glucagon secretion and immune responses are primarily affected by SSTR2, whereas growth hormone secretion is inhibited by SSTR1, SSTR2, and SSTR5. The endogenous cyclic tetradecapeptide SST14 does not exhibit subtype selectivity for SSTRs, and it can bind and stimulate all five SSTRs. It was reported that the SST14-derived cyclic octapeptides octreotide and lanreotide do not activate SSTR1/4 but selectively activate SSTR2/3/5, predominantly SSTR2.2.63, D1223.32, Q1263.36, F2085.38, F2726.51, N2766.55 and Y3027.43) around the ligand-binding pockets of SSTR2 are essential for interaction with the β-turn residues of SST analogues. The sequence alignment of SSTR subtypes revealed sequence conservation and diversity in SSTR1–5  in TM2, TM6 or TM7 showed distinct properties among the two SSTR types (type 1: SSTR2/3/5 or type 2: SSTR1/4).Structural analysis of the SSTR2–Gi complex bound with SST analogues  revealed that the transmembrane residues , which is conserved in SSTR1/4, completely abolished the activation of SSTR2 by octreotide and lanreotide . These five receptors share common signaling pathways but are related to different physiological or pathological processes. The endogenous SST peptides SST14 and SST28 could bind and activate SSTRs with nanomolar affinity57, together with structural analysis of the SSTR2–Gi complexes bound with SST14, octreotide or lanreotide, provided a great example for further rational optimization of SST analogues with increased selectivity for and higher metabolic stability with other SSTR members. Herein, detailed interaction analysis between transmembrane residues in ligand-binding pockets and the β-turn residues in SST analogues revealed the essential roles of the Lys  and Trp  residues. A parallel preprint works at bioRxiv also demonstrated the conserved interactions between Lys-Trp and SSTR58. Structural and functional analysis showed that transmembrane residues  around the ligand-binding pocket of SSTR2 are responsible for binding SST analogues for further SSTR2 activation. Compared to an inactive μ-OR, a series of conformational changes were observed for the SSTR2 receptor, from peptide–ligand interactions to transmembrane helix movements, upon its activation by SST analogues . Furthermore, it was demonstrated that the interfaces between SSTR2 and Gi protein upon binding of SST14, octreotide or lanreotide were highly conserved. Stable interactions between SSTR2 and Gi protein were contributed not only by the conventional hydrophobic interaction network formed between the TM4/5/6 of SSTR2 and the α5 helix, and between ICL2 of SSTR2 and α5/αN helices, but also by electrostatic interactions or hydrophilic hydrogen bonds between ICL3, the TM7–TM8 loop of SSTR2 and the residues in Gi protein. Additionally, Q1022.63, N2766.55 and F2947.35 enabled selective binding of octreotide or lanreotide to SSTR2/3/5 by stabilizing the interactions between the SST analogues and type-1 SSTR -specific residues.Successful development of cyclic octapeptide SST analogue drugs (octreotide or lanreotide) and their commercial application in hormone release arrest or cancer suppressionTaken together, our structures provide a framework for better understanding the activation of SSTR2 by the endogenous cyclic tetradecapeptide SST14 and its derivative shorter cyclic octapeptide analogues (octreotide and lanreotide). Combined structural, mutagenesis and functional studies of SSTR2 and SST analogues provide insights into the subtype-selective activation of cyclic octapeptide SST analogues, which will be very valuable and helpful for further engineering of SST analogues and for drug design.SSTR2A gene was cloned into pFastbac1 vector with the N-terminal HA signal peptide followed by a Flag tag, 10× His tag and TEV protease cleavage site. We truncated the C-terminus of SSTR2A by 10 amino acids to facilitate the better expression of the receptor. To obtain stable SSTR2–Gi1 complex, we used NanoBit tethering strategy, in which the C-terminus of truncated SSTR2A was attached to LgBit subunit and rat Gβ1 was followed by HiBit at its C-terminus. A dominant-negative human Gαi1 (DNGi1) with two mutations , Gβ1 and bovine Gγ2 were also cloned into pFastbac1 vector.The human 2. The purified scFv16 was flash-frozen by liquid nitrogen and stored at −80 °C until use.ScFv16 with a His8 tag at C-terminus was expressed in Sf9 insect cells and purified as follows. In detail, the cells infected with scFv16 virus for 48 h were removed by centrifugation . The supernatant was purified with nickel affinity chromatography. Precipitates was resuspended in HEPES buffer , and then the cells were disrupted and the supernatant obtained after high-speed centrifugation  was also purified with nickel affinity chromatography. The Superdex 200 Increase 10/300 GL column  was used to separate the monomeric fractions of scFv16 with running buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl1, scFv16, Gβ1 and Gγ2 at the ratio of 2:1:1:1:1:1 for 48 h at 27 °C. Cells were centrifuged , resuspended in buffer , and lysed by dounce homogenization after addition of 10 μM peptide, 0.025 U/mL Apyrase, EDTA-free protease inhibitor cocktail, 0.1 mM TCEP and 10 μg/mL scFv16. After lysis, the homogenate was incubated for 1.5 h at room temperature and then 0.5% (w/v) n-dodecyl-β-D-maltopyranoside (DDM) and 0.05% (w/v) cholesteryl hemisuccinate (CHS) were used for solubilization at 4 °C for 3 h. Supernatant was collected after centrifugation  and incubated with Flag resin at 4 °C for 1 h. The resin was loaded onto a gravity flow column and washed with 10 column volumes of wash buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 5 mM CaCl2, 10% glycerol, 10 μM ligand, 0.1 mM TCEP, 0.05% (w/v) DDM and 0.005% (w/v) CHS. Then the detergent was changed to 0.05% (w/v) LMNG and 0.01% (w/v) CHS and the column was washed with ten column volumes of wash buffer. The complex was eluted by 10 column volumes of elution buffer  LMNG/CHS, 0.01% (w/v) GDN/CHS). The flow-through was loaded onto the Superdex 200 10/300 GL column or the Superose 6 Increase 10/300 GL column  with running buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 0.00075% LMNG/CHS, 0.00025% GDN/CHS, 0.1 mM TCEP, 10 μM ligand. The peak of complex was concentrated to 0.8 mg/mL for cryo-EM sample preparation.Sf9 cells were infected with virus of SSTR2, DNGiFor the preparation of cryo-EM grids, 3 μL of the purified SSTR2–Gi complexes at 0.8 mg/mL were applied onto a freshly plasma-cleaned holey carbon grid . Grids were blotted for 10 s and plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher Scientific) at 4 °C and with 100% humidity.Cryo-EM imaging was performed on a Titan Krios electron microscope at 300 kV accelerating voltage using a Gatan K3 Summit direct electron detector with a Gatan energy filter in the Center of Cryo-Electron Microscopy, University of Science and Technology of China . A total of 4189 movies for SSTR2–Gi/SST14 complex, 4837 movies of SSTR2–Gi/octreotide complex, and 7377 movies of SSTR2–Gi/lanreotide complex were collected with a nominal magnification of 81,000×, corresponding to a pixel size of 1.07 Å using the EPU software. Each movies stack was recorded for a total of 3.5 s and 30 frames per micrograph with a defocus range of −1.2 μm to −2.2 μm.59 and contrast transfer function (CTF) estimation was performed with patch CTF estimation. Following CTF estimation, auto-picking particles were extracted by four-time downscaling resulting in the pixel size of 4.28 Å. After two rounds of 2D classification, the particles from well-defined 2D averages were extracted with a pixel size of 2.14 Å for further ab initio reconstruction and heterogeneous refinement. After several rounds of heterogeneous refinement, the selected subset of particles from heterogeneous refinement were extracted with a pixel size of 1.07 Å. The following homogeneous refinement and nonuniform refinement yielded a density map at 2.85 Å for SSTR2–Gi/SST14 complex, 2.97 Å for SSTR2–Gi/octreotide complex, and 2.87 Å for SSTR2–Gi/lanreotide complex.Dose-fractionated image stacks were subjected to beam-induced motion correction using MotionCor2 (v1.0.6). The processed images were transferred to CryoSPARC (v3.0)60. The DNGi1βγ heterotrimer was derived from μ-OR–Gi1 complex (PDB code: 6DDF). The N-terminal residues 1–40 and C-terminal residues 327–398 of SSTR receptor were not built due to the lack of corresponding densities. Structure refinement and model validation were performed using phenix.real_space_refine module in PHENIX61. The final model was subjected to refinement and validation in PHENIX. Figures were prepared using UCSF Chimera62 or UCSF Chimera X63.The initial model for the human SSTR2 receptor was derived from inactive state SSTR2, which was simulated by AlphaFold followed by extensive remodeling using COOTi9 cloned in pcDNA3.1 vector were co-transfected into 293 T cells at a ratio of 2:1 with Lipo3000. After 24 h, the cells were plated in poly-D-lysine-coated 96-well plates. After cells were washed with calcium buffer , Fluo4 AM was added and incubated for 50 min in a 37 °C incubator. After washing with calcium buffer, 50 μL calcium buffer was added to each well. Fluorescence intensities were recorded simultaneously from all the wells by a FLIPR. Plates were illuminated at 488 nm and fluorescence emission was recorded at 540 nm. The fluorescence intensity was measured at 1-s intervals for 410 s. This procedure was followed by the addition of 50 μL agonist. Data were normalized to the baseline response of the ligand. Data were analyzed using nonlinear regression in GraphPad Prism software 9.0.SSTR2 (wild-type or mutant) and Gq5 cells in 2 mL growth medium and transiently transfected with either the wild-type SSTR2 or mutants for 24 h. After transfection, cells were centrifuged at 400× g for 4 min and washed with 500 μL PBS. The cells were blocked with 3% BSA in PBS at room temperature for 10 min and then incubated with anti-Flag antibody  at room temperature for 45 min. The cells were washed 3 times, centrifuged at 400× g for 4 min and resuspended in PBS. Cell surface expression was determined by quantifying PE fluorescence when gating on the live cell population using forward and side scatter. Expression levels as measured by mean fluorescence were normalized to the expression level of wild-type SSTR2. Each construct was analyzed in three independent experiments.Cell surface expression was determined by flow cytometry (CytoFLEX). HEK-293T cells were seeded in six-well plates 24 h before transfection at a density of 5 × 10Supplementary information


The structure and functional group content of LTCFs and LTHFs were analyzed by FTIR, 1H-NMR, 19F-NMR and chemical titration. The results showed that –C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C– and carboxyl groups of LTCFs were reduced efficiently, the reduction rate reached 92% under optimum reaction conditions. Compared with other frequently-used metal chlorides, SmCl3 with a high coordination number could increase the reduction activity of NaBH4 more effectively and the reduction mechanism was explored.Using a simple one-pot method, the reduction of liquid terminated-carboxyl fluoroelastomers (LTCFs) by sodium borohydride and samarium chloride  in excellent yields and provides an attractive potential scheme for the reduction of other carboxyl organic compounds.A facile method using NaBH The stabilized carbon–fluorine single bond (C–F 485 kJ mol−1) and the shielded effect of fluorine atoms on the main chain4,5 make the chemical properties of fluoroelastomers more stable than other rubbers. Curable liquid fluoroelastomers with lower molecular weight have attracted much attention using solvent-free sealant and adhesive formulators because of their liquidity and plasticity. Low molecular weight LTCFs with Mn ranging from 500 to 10 000 were prepared by oxidative degradation of fluoroelastomers.6,7 However, due to the properties of carboxyl groups, the LTCFs are difficult to react with cross-linking agents at low temperature and have low heat resistance stability,8 which will inevitably affect their comprehensive properties and limit their application fields. Therefore, it is particularly important to convert the carboxyl groups into hydroxyl groups to improve the thermal stability and reduce the curing temperature of liquid fluoroelastomers.As an important polymer material, fluoroelastomers have been widely utilized in the field of chemical engineering, machinery and aerospace9–12 Because in contrast to ketones,13 aldehydes14 and esters,15 which easily engage in reactions with reductive agents, carboxylic acids are generally unreactive.16 On the other hand, although lithium aluminium hydride was widely used as a strong reductant in carboxyl reduction,17 its poor chemical selectivity, flammability and explosive nature are unfavorable for large-scale production. Kamochi and Kudo reported the reduction of aryl carboxylic acid derivatives and some aliphatic carboxylic acids using SmI2,18,19 however this strategy was low yielding and limited in scope for aliphatic carboxylic acids. Szostak et al.20 developed a method for efficient electron transfer reduction of carboxylic acids using SmI2–H2O–Et3N and achieved excellent yield, the protocol was not suitable for large-scale production because SmI2 was costly and difficult to store.While significant advances had been made in reduction reactions including for carbonyl compounds, these processes often did not reduce carboxyl groups directly.4 has attracted much attention due to its applications in reduction in organic chemistry. Compared with lithium aluminium hydride and SmI2, NaBH4, with moderate cost, mild reaction conditions and excellent chemical selectivity, is conducive to safe production. Nonetheless, NaBH4 does not directly reduce the carboxylic acids by reason of its relatively weak reductive activity. To reduce carboxylic acid compounds effectively, many Lewis acids have been developed to improve the reduction activity of NaBH4 including I2,21 ZnCl2,22 LiCl,23 AlCl3,24 CoCl2 . Hydrogen peroxide (H2O2), potassium biphthalate (C8H5KO4), phenolphthalein, bromothymol blue, potassium hydroxide (KOH), acetone and benzyltriethylammonium chloride (BTEAC) were purchased from Alfa Aesar (China). NaBH4, AlCl3, CoCl2, ZnCl2, CaCl2 and SmCl3 were purchased from Aladdin Industrial Corporation . Tetrahydrofuran (THF), 0.1 mol L−1 hydrochloric acid (HCl) and diglyme were purchased from Kermel Reagent .Vinylidene fluoride-hexafluoropropylene copolymer (VDF-HFP) with VDF/HFP = 3/1 and 2.22O2 aqueous solution (60 g), 45 wt% KOH aqueous solution (43 g) were added sequently. The reaction mixture was then warmed up to 24 °C and stirred for 7 h. After the reaction, the organic and inorganic phases were separated by filtration. HCl was used to acidify the organic phases and then the mixture was washed by excess deionized water for 3 times. Finally, the deposition was dried under 60 °C for 48 h in a vacuum drying chamber to obtain LTCFs.LTCFs were prepared through chemically degradation of high molecular weight solid VDF-HFP copolymer. High molecular weight VDF-HFP (100 g) and acetone (300 mL) were added into a 2000 mL flask. The mixture was stirred at room temperature for 24 h till the copolymer was completely dissolved. Under stirring at 0 °C, BTEAC (6.5 g), 30 wt% H2.34 was dissolved into the solution under stirring for 1 h at 0 °C. Subsequently, 1.506 g SmCl3 was added into the system and temperature rose to 90 °C. After 6 h, the inorganic impurities were dissolved by 30 mL 0.1 mol L−1 HCl and the mixture was washed by excess deionized water for 3 times. Finally, the deposition was dried under 60 °C for 48 h in a vacuum drying chamber to obtain LTHFs.5.000 g LTCFs (2.66% carboxyl content) were dissolved in a mixture of THF (15 mL) and diglyme (15 mL) for 1 h, then 0.447 g NaBH2.4−1 in the range 650–4000 cm−1. The NMR spectra were analyzed by Bruker AC 80 spectrometers  at room temperature, using acetone-d6 as the solvent and TMS as internal standard the references for 1H (or 19F) nuclei. Chemical shifts are reported in ppm.ATR-FTIR measurement was performed using a PerkinElmer Instruments Spectrum One. FTIR spectra were obtained at the resolution 4 cm2.52.5.1−1 KOH/C2H5OH solution was prepared in volumetric bottle according to the China National Standards GB/T 603-2002 and GB/T 601-2002. 0.2 g of potassium biphthalate and 40 μL phenolphthalein solution (10 g L−1) was dissolved in water (50 mL). KOH/C2H5OH solution was added dropwise into the potassium biphthalate and phenolphthalein aqueous solution and when the aqueous solution changed from colorless to pink, it was the end point of titration. Blank experiment as the comparison was done. The concentration of KOH/C2H5OH standard titration solution was calculated by formula V1: volume of KOH/C2H5OH solution consumed by the potassium biphthalate and phenolphthalein solution (mL), V2: volume of KOH/C2H5OH solution consumed by blank sample (mL), m: weight of potassium biphthalate (g), M: 204.22 g mol−1.0.1 mol L2.5.2−1) was dissolved in acetone (40 mL). KOH/C2H5OH solution was used for titration and the LTCFs and bromothymol blue solution changed from light yellow to green, which was the end point of titration. Carboxyl content of LTCFs was calculated by formula V: volume of KOH/C2H5OH solution consumed by the LTCFs and bromothymol blue solution (mL), C: the concentration of KOH/C2H5OH standard titration solution (mol L−1), m: weight of LTCFs (g).0.75 g of LTCFs and 40 μL bromothymol blue . When molar ratio of R0COOH/NaBH4/SmCl3 further increases to 1/5/2.5, the reduction rate is decreasing due to the coating of the NaBH4 particles with the excess SmCl3, which likely impedes the efficient solubility of NaBH4 (No. 7). Furthermore, as shown in 3 addition amount is investigated. When the molar ratio of NaBH4/SmCl3 changes from 4/0 to 4/2, the reductive rate of LTCFs increases with increasing amount of SmCl3. Nevertheless, when the molar ratio is 4/3, the excess SmCl3 make the reductive rate decrease because the coating of the NaBH4 particles with the excess SmCl3.3.34/SmCl3 system in THF readily reduces LTCFs to their corresponding LTHFs, it has a lower reduction rate. The low reduction rate is possibly due to the weak of solubility of NaBH4 in THF. To improve the solubility of NaBH4 in THF, varying amounts of diglyme, a known excellent solvent for NaBH4, are added. After some optimizations, the 1/1 volume ratio of THF/diglyme mixed solvent is adequate to solubilize NaBH4 . In addition, the effect of mixed solvent amount on reduction rate is also investigated . The results show that the maximum reduction rate is obtained as the amount of mixed solvent is 30 mL. The lower solvent amount will lead to the decrease solubility of NaBH4 and the excessive use will affect the complexation of NaBH4/SmCl3 with carboxyl group. Consequently, 30 mL of THF/diglyme (1/1 volume ratio) mixed solvent is selected to investigate the reduction of LTCFs.As shown in 0COOH/NaBH4/SmCl3 is 1/4/2 and the THF/diglyme volume ratio is 1/1. Under the optimal reaction conditions the LTCFs are converted to LTHFs in a reductive rate about 92%. For the scale-up, when we expand the experiment by 30 times, the reduction rate do not decrease significantly. Therefore, the method can be expected in industrial utility.From above experimental results we can conclude that the optimal reaction conditions are as follows: the reaction temperature is 90 °C, the reaction time is 6 h, the molar ratio of the Rx) are also evaluated. Through the optimization studies, the optimal reaction conditions of NaBH4/MClx are consistent with NaBH4/SmCl3. As shown in 4/SmCl3 gives excellent reductive rate of LTCFs (No. 1). It is concluded that the unique electronic structure and high coordination number of samarium ions provide a new method for efficient reduction of LTCFs and a potential scheme for the reduction of other carboxyl organic compounds.As comparison, several other metal chlorides  indicating that the structure of –CC– is reduced significantly but only little –CF2COOH is reduced via single NaBH4 without SmCl3. 4/SmCl3. From C– and –CF2COOH are weakened ascribed to effective reduction of NaBH4/SmCl3. LTHFs exhibits absorption peaks at 1398, 1183 and 879 cm−1 ascribed to stretching vibration of –FCH2–, –CF2–, and –CF3, respectively, which clearly shows that LTHFs have the same backbone structure as LTCFs.FTIR spectra of LTCFs is shown in 1H-NMR spectra of LTCFs the multiple peaks at 3.51–2.86 ppm, the peaks at 1.55 ppm, 4.68 ppm and 7.50–7.70 ppm are assigned to the structures of –CH2CF2–, –CFC(CF3)CH2–, –(CF3)CCH– and –CHCF–,32,33 Respectively. C– peaks at 1.55 ppm, 4.68 ppm and 7.5–7.70 ppm, respectively, indicating that –CC– converted to –C–C–. In general, single NaBH4 does not reduce –CC– and it is commonly necessary to add Lewis acids to improve the reduction activity.34,35 However, –CC– of LTCFs can be directly reduced by single NaBH4 ascribed to effect of F atom, which increases the activity of –CC– through the electronic induction effect. Compared with the LTCFs, as seen from 2OH,30 it clearly confirms the formation of hydroxyl groups.As seen from 19F-NMR spectra were used further to characterize. 19F-NMR spectra of (a) LTCFs, (b) reduction product of single NaBH4 reductant (No. 2) and (c) LTHFs (No. 6) are shown in 2COOH. The peaks at −73.71 ppm, 80.66 ppm and 81.30 ppm are ascribed to the fluorine atoms on the structure of –(CF3)CCH–, –CHCF– and –CFC(CF3)CH2– for LTCFs36–38 respectively. C– peaks at −73.71 ppm, 80.66 ppm and 81.30 ppm disappear by reductive transformation using NaBH4, this result is similar to 2CH2OH.30 Therefore, the above results of 19F-NMR spectra, 1H-NMR and FTIR spectra consistent with each other and LTHFs is prepared successfully. 19F-NMR spectra of LTHFs in different reaction times is shown in 3.54 and Lewis acid has predominantly considered that borane (BH3) was formed by the reaction of NaBH4 with Lewis acid initially, and then BH3 reduced the substrates.21,39 To investigate the applicability of this reduction mechanism in our experiment for LTCFs, the sequence of reagent addition is investigated first . When SmCl3 and LTCFs are first added into the reactor under stirring for 1 h, then, NaBH4 is added and the reduction rate is 48% (No. 2). The above two addition sequence experiments can ensure the reaction of NaBH4 with SmCl3 firstly and have low reduction rate indicating the mechanism is not suitable for LTCFs. Nevertheless, when NaBH4 and LTCFs are first added into the reactor under stirring for 1 h, then SmCl3 is added (No. 3), the reduction rate increase dramatically to 92%. Hence, according to our experimental results and literatures,20,40 the reduction mechanism of LTCFs is proposed, as shown in C– of LTCFs is directly reduced by NaBH4 and R0COOH react with NaBH4 to form (R1COO)4BNa and release H2. Secondly, the complexation of Sm3+ with carbonyl oxygen of LTCFs makes the electrons of carbonyl group move to oxygen and increase the electrophilicity of carbonyl carbon. Electrophilicity of carbonyl carbon is facilely attacked by the borohydride anion (BH4−) of NaBH4.40 Thirdly, with the departure of Sm3+ and the other oxygen atom, aldehyde group is formed.20 Aldehyde group is then reduced by NaBH4/SmCl3 and the LTCFs is successfully reduced to the final formation of LTHFs. In contrast to carboxyl group, aldehyde group is readily reactive. The generated aldehyde group in the reaction could be immediately reduced by the strong reduction system of NaBH4/SmCl3. Therefore, no byproduct of liquid terminated-aldehyde fluoroelastomers is generated.The reported literatures on reduction mechanism of NaBHed first . When Na4.4/SmCl3 provided a method for the selective reduction of LTCFs in THF/diglyme and the 1/4/2 molar ratio of R0COOH/NaBH4/SmCl3 was suitable for LTCFs reduction at 90 °C. LTCFs were reduced to their corresponding LTHFs in excellent reduction rate (92%). FTIR spectra and NMR spectra analysis showed that –CC– and –CF2COOH of LTCFs were effectively reduced to –C–C– and –CF2CH2OH. We have proposed the reduction mechanism that Sm3+ complex with the carbonyl group increase the electroaffinity of the carbon of carbonyl and make carbon of carbonyl more receptive to the hydride moiety transfer from the borohydride anion. Application of this method to reduction of other carboxylic acid derivatives will be presented in our future work.In summary, the combination of affordable and readily available NaBHThe authors declare no competing financial interests.