Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides, respectively, and therefore play essential roles in the synthesis and decomposition of ester bonds in the pharmaceutical and food industries. Many SGNH family esterases share high similarity in sequences. However, they have distinct enzymatic activities toward the same substrates. Due to a lack of structural information, the detailed catalytic mechanisms of these esterases remain barely investigated.2+ might reduce enzymatic activity by blocking proton transfer or substrate binding. CrmE10 and AlinE4 showed distinctly different electrostatic surface potentials, despite the similar atomic architectures and a similar swap catalytic mechanism. When five negatively charged residues (Asp or Glu) were mutated to residue Lys, CrmE10 obtained elevated alkaline adaptability and significantly increased the enzymatic activity from 0 to 20% at pH 10.5. Also, CrmE10 mutants exhibited dramatic change for enzymatic properties when compared with the wide-type enzyme.In this study, we identified two SGNH family esterases, CrmE10 and AlinE4, from marine bacteria with significantly different preferences for pH, temperature, metal ion, and organic solvent tolerance despite high sequence similarity. The crystal structures of these two esterases, including wild type and mutants, were determined to high resolutions ranging from 1.18 Å to 2.24 Å. Both CrmE10 and AlinE4 were composed of five β-strands and nine α-helices, which formed one compact N-terminal α/β globular domain and one extended C-terminal domain. The aspartic residues (D178 in CrmE10/D162 in AlinE4) destabilized the conformations of the catalytic triad (Ser-Asp-His) in both esterases, and the metal ion CdThese findings offer a perspective for understanding the catalytic mechanism of different esterases and might facilitate the industrial biocatalytic applications. The SGNH-hydrolase family, a superfamily that includes the bacterial lipolytic enzyme GDSL family , consistCroceicoccus marinus E4A9T was previously isolated from a deep-sea sediment sample [Altererythrobacter indicus DSM 18604T was isolated from mangrove-associated wild rice [Erytrobacteraceae family. According to in silico analysis of whole-genome sequences [crme10 and aline4, were screened from C. marinus and A. indicus, respectively. Here, we report the enzymatic characterizations and crystal structures of CrmE10 and AlinE4. These esterases exhibited similar atomic architectures and a similar swap catalytic mechanism; however, their electrostatic surface potentials and enzymatic properties were distinctly different. Structural comparison and structure-based protein engineering showed that mutants with five catalytic reaction-related residues could significantly change enzymatic features. These findings provide new insights into the catalytic mechanism of SGNH-hydrolase family enzymes and might benefit our understanding of their potential uses as biocatalysts.The enzymes isolated from marine organisms usually have many specific features, including temperature adaption, alkaline adaption, salt tolerance, and metal tolerance, which might offer the potential for industrial application. t sample , and Altild rice . Both stequences , 28, twoC. marinus E4A9T and A. indicus DSM 18604T, respectively. CrmE10 and AlinE4 have similar amino acid sequences . To reveal the relationship between CrmE10 and AlinE4, we performed a phylogenetic analysis with other known lipolytic enzymes using MEGA software [To compare the enzymatic activities of different esterases, the open reading frames of CrmE10 (Accession number: ARU15426.1) and AlinE4 (Accession number: WP_160739227) were identified from the whole-genomes of strains software . The resEscherichia coli. CrmE10 exhibited the highest activity (approximately 29.4 U/mg) toward p-nitrophenyl (p-NP) hexanoate (C6) at pH 7.5 and 20 °C   at pH 7.5 and 40 °C   cells as described previously [CrmE10 and AlinE4 were expressed in eviously . They we10-helices) and 5 β-strands. The N-terminal region had a compact architecture that was mainly composed of five predominant β-strands (β1–β5) surrounded by eight α-helices (α1–α8). The C-terminal region was a long α-helix, α9 (Ala184–Asp203), that extended away from the subunit core and wrapped in the other chain. Thus, CrmE10 formed an intertwined dimer though the swapped C-terminal domain  and 5 predominant β-strands. AlinE4 had a similar topology with CrmE10, as well as the α9 (Ala168-Ala185) wrapped in other chains and formed a symmetric dimer through the swapped C-terminal domain  and 8010 Å2 (AlinE4), respectively. This suggested CrmE10 and AlinE4 were a dimer in crystal structure.AlinE4 structure was determined using esterase TesA (PDB code: 4jgg) as an initial search model, which revealed similar topology with CrmE10 Fig. b. AlinE4ain Fig. c and d. CrmE10 shared 28.42% (89% coverage), 32.04% (88% coverage), and 34.76% (80% coverage) sequence identities with its homologous EstA (PDB code: 3HP4) , TAP  blocking proton transfer and (ii) protecting substrates from nucleophile attack.The mechanism of SGNH-hydrolase family esterase activity involves a two-step reaction (acylation and deacylation) similar to those proposed for lipolytic enzymes and serine proteases . In thisCrmE10 and AlinE4 shared similar atomic architectures  properties, including substrate specificity , 42, act2+ reduced enzymatic activity by blocking proton transfer and substrate binding. Our current findings offer a perspective for understanding the catalytic mechanism of esterases and could facilitate industrial biocatalytic applications of these esterases.Here, we reported the enzymatic characterizations and crystal structures of marine bacterial esterases CrmE10 and AlinE4. The two enzymes shared high sequence similarity and similar atomic architectures; however, they had significantly different enzymatic activities. Despite sharing a similar swapping catalytic mechanism, CrmE10 and AlinE4 had distinctly different electrostatic surface potentials. Structure-based mutation showed that CrmE10 obtained elevated alkaline adaptability and had a significant increase in enzymatic activity from 0 to 20% at pH 10.5 when five acidic residues were mutated to the corresponding basic residues in AlnE4. In addition, one key residue (Asp162 in AlinE4/Asp178 in CrmE10) was found to be able to stabilize the conformation of both esterases and the metal ion CdCroceicoccus marinus E4A9T was previously isolated from a deep-sea sediment sample [Altererythrobacter indicus DSM 18604T was isolated from mangrove-associated wild rice [crme10 and aline4 were cloned into plasmid pSMT3 to produce the N-terminal His-Sumo tagged fusion proteins from the genomic DNA of C. marinus E4A9T and A. indicus DSM 18604T, respectively. Point mutants were generated by site-directed mutagenesis using wild-type plasmids as templates for PCR. After digested with DpnI enzyme, the products were transformed into E. coli DH5α cells and determined by DNA sequencing. CrmE10-W1 contains wild-type CrmE10, ribosomal binding site (RBS) and CrmE10-D178A in the pSMT3 plasmid, whereas the polycistronic CrmE10-W2 plasmid contains wild-type CrmE10, ribosome binding sites, and CrmE10-D178A-S29A. The wide-type and mutated proteins were expressed in E. coli BL21 (DE3) cells. The cells were cultured in LB broth medium induced by adding 0.5 mM isopropyl-β-d-thiogalactoside (IPTG), when OD600 came to 0.6–0.8. After cultivation at 16 °C for 20 h, cells were harvested by centrifugation at 6000 rpm for 15 min at 4 °C, resuspended in starting buffer , and disrupted by French press homogenizer . Cell debris was removed by centrifugation and the supernatant was incubated with Ni Sepharose  for 1 h. After washing with buffer 1 , the recombinant protein was eluted with buffer 2 . Subsequently, the His-Sumo tag was removed by overnight digestion with the ULP1 enzyme. The recombinant target proteins were further purified by gel-filtration using the Superdex 200 10/300 column  in a buffer . The fractions of elution were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Furthermore, the protein concentrations were determined by the Bradford method [t sample  and was ild rice  and was d method  with bovhttp://espript.ibcp.fr/ESPript/ESPript/) [Multiple alignments of amino acid sequences were performed using ClustalX v.2 program . SecondaSPript/) . The phySPript/) .MALS analysis was performed in the National Center for Protein Science Shanghai (NCPSS). 20 μl of 1 mg/ml purified target protein was subjected to SEC-MALS using a WTC-030S5 size-exclusion column  with elution buffer  and passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument  and an optilab refractometer . Data collection and analysis were performed with Astra 6 software .p-nitrophenyl (p-NP) hexanoate , or p-NP butyrate . The enzymatic activity was determined at 20 °C (for CrmE10 and its mutants) or 40 °C (for AlinE4 and its mutants) by measuring the amount of releasing p-nitrophenol using Beckman Coulter DU 800 UV/Visible spectrophotometer  at 405 nm. All values were measured in triplicates and corrected for the autohydrolysis of the substrates. One unit of enzymatic activity was defined as the amount of enzyme required for releasing 1 μmol of p-nitrophenol per minute from the p-nitrophenyl ester. The kinetic parameters (Km and Vmax) were calculated from enzymatic activity measurements with p-NP hexanoate (for CrmE10) or p-NP butyrate (for AlinE4) ranging from 0.05 mM to 2 mM. Initial reaction velocities measured at various concentrations were fitted to the Lineweaver–Burk transformation of the Michaelis–Menten equation [Esterase activity assays were performed using a spectrophotometric method with the appropriate amount of purified enzyme in standard reaction buffer containing 100 mM Tris–HCl (pH 7.5), enzyme (concentration at 1–5 μg/mL) and 1 mM equation .The optimum pH of esterases CrmE10 and AlinE4 was determined over the pH range from 3.0 to 9.5. The buffers included citrate buffer , phosphate buffer , Tris–HCl buffer , and CHES–NaOH buffer . The enzymatic activity was measured under 348 nm. The effects of temperature on esterases CrmE10 and AlinE4 were measured over a range of 15–60 °C. For investigation of the thermostability, the residual activity of CrmE10 and AlinE4 was determined after incubation at various temperatures ranging from 10 °C to 100 °C for 1 h. And the thermostability of AlinE4 was further determined after incubation at 90 °C, 95 °C and 100 °C for 0–2.5 h.p-NP esters, including p-NP acetate (C2), p-NP butyrate (C4), p-NP hexanoate (C6) , p-NP octanoate (C8), p-NP decanoate (C10), p-NP dodecanoate (C12), myristate (C14), and p-NP palmitate (C16)  were added into the reaction buffer with final concentration of 1 mM for determining substrate specificity.Various chain lengths of 2+, Sr2+, Ni2+, Mn2+, Mg2+, Co2+, Ca2+, and Ba2+, at final concentration of 10 mM. The effect of the chelating agent ethylenediaminetetraacetic acid (EDTA) was determined at a final concentration of 10 mM. The effects of organic solvents were determined using acetone, acetonitrile, ethanol, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), glycerol, isopropanol, and methanol, at a final concentration of 15% (v/v).The effects of NaCl on CrmE10 and AlinE4 activity were evaluated by adding 0–5 M NaCl to the assay mixture. The effects metal ions were measured using various divalent cations, namely ZnSeveral crystals of AlinE4 were washed in a Tris buffer without heavy metal ions for several seconds and transferred into a micro-centrifuge tube. Then the crystals were dissolved completely with the Tris buffer. The protein concentration was measured by the Bradford method . The 8844. All X-ray diffraction datasets were collected at BL17U1 [CrmE10 and AlinE4 were applied to crystallization trials carried out at 20 °C by hanging- and sitting-drop vapor-diffusion methods by mixing 30 mg/ml protein with an equal volume reservoir solution. The crystals of native CrmE10 were grown in a reservoir solution containing 150 mM calcium acetate, 100 mM imidazole–HCl (pH 8.0), and 10% PEG 8000. The crystals were briefly soaked in 25% (v/v) glycerol dissolved in their reservoir solution, as a cryoprotectant solution, before being flash-frozen directly in liquid nitrogen. The CrmE10-D178A crystals were grown in the condition of 250 mM calcium acetate, 100 mM imidazole–HCl (pH 8.5), 5% PEG 1000, and 3% 1,6-Hexanediol. The cryoprotectant solution of the CrmE10-D178A crystals was 20% (v/v) PEG 400. The crystals of native AlinE4, AlinE4-S13A, and AlinE4-D178A were grown in 1 M NaAc, 100 mM HEPES, and 50 mM CdSOt BL17U1 , BL18U1,t BL17U1  beamlinet BL17U1 .https://blast.ncbi.nlm.nih.gov/Blast.cgi). Substrate docking studies were performed using the AutoDockTools4 program [The crystal structures of wild-type (WT) CrmE10 and AlinE4 were determined by molecular replacement using esterase TesA (PDB code: 4jgg)  as the s program . All theAdditional file 1. Additional figures and tables.


The data emerged from various analyses testified to the successful construction of the desired nano-scaled hybrid photocatalyst. Tauc plot gave the band gap of In2O3/MoS2/Fe3O4 to be ~ 2.15 eV. Synergistic effects of the integrant components enabled efficacious photocatalytic performances of the nanocomposite. The nanohybrid photocatalyst In2O3/MoS2/Fe3O4 showed photodecomposition up to ~ 92.92% within 50 min. The current work realizes its objective of constructing metal oxide based hybrid nano-photocatalyst supported on MoS2 sheets for activity in the visible spectrum, which displayed remarkable capacity of disintegrating emerging persistent organic contaminants and are magnetically recoverable.The current work involves synthesis of hybrid nanomaterial of In Water decontamination is currently among the most exhilarating and pioneering fields of science research. For there is a gradual uptick of contamination of water by emerging menaces ranging from dyes to personal care products to pharmaceutical compounds. These emerging, non-biodegradable organic pollutants reach water bodies due to their uncontrolled release from point sources such as factories, plants industries and hospital sewage as well from non-point sources such as runoff. The release of toxic waste filled water into the biosphere brings on its deterioration and undesirable alterations in ecological balance. Among the prominent emerging water pollutants, pharmaceuticals have attracted strong attention of researchers in the field of water purification.3. Therefore, the annihilation of esomeprazole in wastewater is of paramount significance.Pharmaceuticals reach water sources through sewage systems carrying the excreta of individuals who have consumed them and from their unregulated disposal. Veterinary pharmaceuticals may be emitted to water either directly as a result of application in fish farming or indirectly via runoff from animal-feeding operations in livestock industry. Pharmaceuticals ridden water exerts tremendous negative impacts on terrestrial and water-dwelling lives. The occurrence of pharmaceutical compounds and their residues and metabolites in wastewater has been an area of growing concern and keen research interest. Esomeprazole, the S-isomer of omeprazole, is a pharmaceutical drug used for the treatment of certain stomach and esophagus problems, such as acid reflux and ulcers. Prominent among its side effects are diarrhea, nausea, constipation, kidney ailment, and autoimmune diseases like, cutaneous lupus erythematosus and systemic lupus erythematosus. Prolonged, interminable use likely lead to a few severe health risks such as vitamin B-12 deficiency, fundic gland polyps, osteoporosis, etc2−, etc.) for thorough destruction of toxic organic substances. Heterogeneous photocatalysis is a type of AOP. It takes on the surface of a semiconductor catalyst. In heterogeneous photocatalysis, the semiconductor particles are excited by photons of energy equal to or greater than the band gap leading to the formation of electron–hole pairs5. Thereafter, superoxide anion radicals (·O2−) are formed by the reaction of electrons with oxygen and hydroxyl radicals (·OH) are formed when holes react with water. The energetic radicals thus generated then make indiscriminate onslaughts on the molecules of the organic contaminant breaking them down to less persistent, innocuous organic compounds and simple end products such as CO2, H2O, etc. Superior detoxification ability, inexpensive operation and modest operational conditions of experiment together impart semiconductor photocatalysis a high ground in tackling water contamination. Enormous surface area, chemical stability and size-dependent properties make nano-scaled semiconductor materials apt for application in water decontamination6. Among several semiconductor nanomaterials, TiO2 has attracted attention as efficient, photo-chemically stable and ecofriendly photocatalyst vis-à-vis decomposition of organic pollutants8. ZnO, WO3, CeO2, In2O3, Nb2O5, Ta2O5, MOSx, WSx, CdS, ZnS, etc. have also been recently used for photocatalysis11. On one hand, narrow semiconductor materials are beset with severe recombination of photo-induced charge carriers, while on the other hand, semiconductors possessing large band gap primarily absorb in the ultra violet region and are therefore incapable of utilizing a large portion of the solar light rendering them exclusively UV active. This has driven development of strategies that rely upon integration of wide and narrow band gap semiconductors for fine tuning band edges and enabling absorption of photons in the visible spectrum while also preventing the rapid recombination of electron and holes. Such integrated photocatalysts have displayed superlative performance in the photodecomposition of organic water contaminants. MoS2/TiO212, Ag3PO4/TiO2/MoS213, Bi2O3/Bi2S3/MoS214, etc. have recently been designed for application in photocatalytic decomposition of emerging organic water pollutants. Additionally to facilitate easy and inexpensive recovery of photocatalysts from reactors, Fe3O4 has been used as the superparamagnetic component in coupled photocatalysts17. Fe3O4 has also been known to step up photodecomposition via charge separation through trapping of light-generated electrons by Fe3+ ions19. Besides, recently, Fe as a component in electrocatalysts has been reported to play a key role in triggering oxygen evolution reaction (OER)20. The authors of the current work have thus designed an integrated system of photocatalyst that responds in the visible spectrum, is photo-chemically stable, bears the capacity to disintegrate recalcitrant organic pollutants and is magnetically recoverable. Indium oxide (In2O3) is an important n-type semiconductor. It has three phases, namely, hexagonal, cubic and hexagonal corundum. Of late, In2O3 has seen wide applications in solar cells, sensor modules, transparent electrode materials for both electrochromic cells and for liquid crystal display devices, flat-panel displays, light-emitting diodes, thin-film transistors, etc22. Chief advantages of In2O3 for use in photocatalysis include high photosensitivity, environmental stability and it being an efficient sensitizer to extend the absorption spectra of metal oxide-based semiconductor photocatalysts from the UV spectrum into the visible spectrum. Its drawback is a rapid recombination of photo-induced electrons and holes22.Among a host of advanced processes for detoxification of wastewater, advanced oxidation processes (AOPs) have seen a surge in application among researchers engaged in investigations geared towards water decontamination. AOPs usually refer to techniques of generating reactive oxygen species , a kind of two-dimensional layered transition metal dichalcogenide, has unique advantages in photocatalysis in view of its unique physical, chemical and electrical properties and tunable band structure24. MoS2 matrix prevents agglomeration of nanoparticles. Also MoS2 has of late been employed as a cocatalyst in photocatalysis induced decontamination of water25. Facile procedure of synthesis was adopted in the current work for construction of In2O3/MoS2/Fe3O4 hybrid nano-scaled photocatalyst. The ternary nano-scaled photocatalyst was found to exhibit excellent photocatalytic activities disintegrating esomeprazole, the target pollutant, within 50 min. The photodecomposition of esomeprazole by this nano-photocatalyst was found to follow pseudo-first order kinetics. In2O3/MoS2/Fe3O4 attained 92.92 ± 2.01% photodecomposition at a velocity constant of 0.06208 min−1 with TOC and COD reduction up to ~ 77.06% and ~ 66.71%. The fabrication of novel ternary nano-photocatalyst with excellent capacity to disintegrate an emerging water contaminant, esomeprazole, with superlative velocity constants marks the novelty of this work.Coupling In3)3.xH2O), sodium hydroxide (NaOH), ferrous sulphate heptahydrate (FeSO4 0.7H2O), ferric chloride (FeCl3), molybdenum(IV) sulfide (MoS2), esomeprazole magnesium, deionized water etc.The following items of AR grade were acquired from Sigma Aldrich and used without further purification: Indium (III) nitrate hydrate  (224.8 mmol) was slowly added to it. The reaction mixture was left for stirring for 18 h. Next, 20 mmol of Indium (III) nitrate hydrate (In(NO3)3.xH2O) was added to this reaction mixture followed by stirring. An aqueous solution of sodium hydroxide (NaOH) (30 mmol) was then slowly poured into it. The final reaction mixture was stirred and then taken in a Teflon autoclave maintained at 110 °C for 18 h. The precipitate thus obtained was centrifuged and washed thoroughly with ethanol. After collection, the sample was dried and thereafter annealed at 300 °C for 3 h. The brown sample thus obtained was treated as In2O3/Fe3O4.Deionized water was taken in a beaker and to it were added 20 mmol of FeSO2 was exfoliated by ultrasonication in distilled water following which In2O3/Fe3O4 was added to it. This mixture was also ultrasonicated for 5 min and then dried to obtain the final nanohybrid of In2O3/MoS2/Fe3O4.Next, MoS2O3, Fe3O4, exfoliated MoS2 and the binary nanohybrid were likewise prepared following the procedure illustrated above but only in presence of the respective starting materials. Indium (III) nitrate hydrate (In(NO3)3.xH2O) was used for fabricating In2O3 while Fe3O4 was prepared using the aforementioned iron salts in aforesaid proportion.Pristine samples of In2O3/MoS2/Fe3O4, In2O3/Fe3O4, In2O3, MoS2, and Fe3O4 were analyzed for X-ray diffraction studies on Phillips XPERT powder X-ray diffractometer with Cu-Kα radiation. TEM, HRTEM and SAED were executed on JEOL JEM 2100 instrument for determination of size, morphology and diffraction rings of In2O3/MoS2/Fe3O4. X-ray photoelectron spectroscopy (XPS) of the final nanocomposite was carried out with PHI 5000 Versa Prob II spectrometer to investigate the electronic environment and valence states of elements in the two ternary nanocomposites. Photoluminiscence data were acquired with Hitachi F4600 equipment. Magnetic behaviour of Fe3O4 and In2O3/MoS2/Fe3O4 was ascertained employing Vibrating Sample Magnetometer. HRLCMS graphs were obtained using 1290 Infinity UHPLC System, Agilent Technologies, USA. TOC results were collected following analyses carried out using Elementar, Liqui TOC. GENESYS 10S UV–visible spectrophotometer was used for obtaining the absorbance spectra of various samples.In−2) from an LED bulb affixed atop (Philips 23 W white LED) was shone over the reaction cell within a homemade chamber. A luxmeter was employed for the measurement of lux and radiation intensity. In a typical photocatalytic experiment, specified amount of the photocatalyst (0.7 gL−1 of In2O3/MoS2/Fe3O4) is suspended in 50 mL of aqueous solution of esomeprazole prepared using distilled water with at an initial pH of 5. Other requisite substances were incorporated before the dispersal of the photocatalyst in order to simulate aqueous matrices. Water matrices replaced distilled water in other investigative studies about the effect of environmental water samples on photodecomposition. Each test was carried out in quintet, putting the suspension in a dark environment under agitation for 30 min to attain adsorption–desorption equilibrium followed by exposure to LED illumination for 50 min for photodecomposition. The photodecomposition was recorded by noting the absorbance at the λmax of esomeprazole at 301 nm after every 10 min.The esomeprazole photodecomposition experiments were carried out in reaction cells that had working volume of 100 mL. LED light irradiation  at 2θ angles of 30.63°, 35.52° and 51.06° respectively11 besides displaying peaks that could be indexed to (422), (431), (611), (620) and (541) planes at 2θ angles of 21.66°, 41.97°, 43.84°, 45.91°, 56.07°, 57.68° and 59.27° respectively. The presence of a prominent (023) peak at 34.23° along with accompanying peaks of (020), (100) and (151) planes at 18.96°, 32.08° and 60.09° respectively confirmed Fe3O4 phase (JCPDS 89–6466) in the nanohybrid. Furthermore, MoS2 phase (JCPDS 73–1508) could be ascertained by peaks indexed to (002), (100), (102), (103) and (006) planes at 14.45°, 32.86°, 35.21°, 39.72° and 44.34° respectively. The average crystallite size obtained using Scherrer formula in the ternary nanohybrid, In2O3/MoS2/Fe3O4, was 13.47 nm. In the powder XRD pattern of In2O3/Fe3O4  and Fe3O4 (JCPDS 89–6466) phases could be identified. Peaks associated with (222), (400) and (440) planes of the cubic In2O3 phase at 2θ angles of 30.62°, 35.50° and 51.04° respectively along with those corresponding (211), (332), (422), (431), (611), (620) and (541) planes at 2θ angles of 21.62°, 41.90°, 43.79°, 45.81°, 56.02°, 57.61° and 59.19° respectively showed up11. Also were present peaks indexed to (020), (100), (023), and (151) planes at 18.96°, 32.08°, 34.21° and 60.09° respectively of Fe3O4 phase (JCPDS 89–6466) in the XRD plot of In2O3/Fe3O4. The average crystallite size obtained using Scherrer formula in the binary nanohybrid, In2O3/Fe3O4, was 13.78 nm. The XRD plots of the ternary and binary nanocomposites have been reproduced with the corresponding JCPDS cards of the pristine samples in Figure 2O3 sample  at 2θ angles of 30.56°, 35.42° and 51.01° respectively19 as well as other peaks (211), (332), (422), (431), (611), (620) and (541) planes at 2θ angles of 21.57°, 41.86°, 43.78°, 45.79°, 55.97°, 57.54° and 59.14° respectively. The average crystallite size obtained using Scherrer formula of the nano-scaled In2O3 was 14.41 nm. The XRD plot of pristine MoS2  at 14.39°, 32.80°, 39.65°, 44.15°, 49.88° and 58.58° respectively26. The powder XRD pattern of Fe3O4  plane of cubic-phased In2O3 particle11 while that of 0.471 nm could be linked to (020) plane of Fe3O4. Although MoS2 nanosheet could not be clearly detected in the higher 2 nm resolution, a slightly lower 5 nm resolution image of the same region revealed flakes of MoS2 having a crystalline phase showing facet (002) that could be recognized from an interplanar spacing of 0.61 nm  and (023) planes of Fe3O4 and the (002) plane of MoS2 nanosheet were identified and marked in the SAED pattern  spectroscopy was carried out to access further insights into the optical properties and to make a comparison of the photoluminiscence behaviour of the as-fabricated nanohybrid samples with the pristine moieties. The emission spectrum of In2O3 Fig. b is obtaoS2 Fig. c recordered Fig. d. A shar3O4 and In2O3/MoS2/Fe3O4 towards externally applied magnetic fields for proper evaluation of their magnetic properties, the initial magnetization versus field measurements of the samples were performed by magnetometer operating at ambient temperatures, with the field sweeping from − 15,000 to 15,000 Oe following which the magnetization hysteresis curves were obtained  of 10.768 G . There was initially an intensification of photodecomposition behaviour as pH was increased but after a pH of 5, there was a slight reduction in photodecomposition efficiency 51.Under alkaline conditions, there could also be the formation of a less reactive ferryl ion species  is a O2−· scavenger and is widely known to quench the radical leading to a slowdown of the photocatalytic kinetics driven by O2−· radicals. The addition of TEMPOL had a marked negative effect on the photocatalytic decomposition dynamics of esomeprazole decomposition over In2O3/MoS2/Fe3O4. With insertion of 1 mmol of TEMPOL, the esomeprazole photodecomposition efficiency dropped to ~ 20.77%. Likewise, tert-butanol (t-BuOH) is a ·OH scavenger and has been reported to inhibit photocatalysis instigated by ·OH radicals. Indeed, photodecomposition was appreciably but moderately inhibited upon incorporation of 1 mmol of t-BuOH. The insertion of t-BuOH brought on a drastic slowdown in the photodecomposition dynamics of esomeprazole decomposition over In2O3/MoS2/Fe3O4. Efficiency dropped to ~ 51.21% over In2O3/MoS2/Fe3O4. The hole scavenger, triethanolamine TEOA and the electron scavenger, K2S2O8 didn’t have any significant disruptive effects on the photodecomposition dynamics of esomeprazole decomposition evidently suggesting the minor roles played by holes and electrons. Therefore, it emerged from the study that ROS such as O2−· and ·OH were chiefly involved in triggering photodecomposition of aqueous esomeprazole over the photocatalyst. Figure 2O3/MoS2/Fe3O4.The influences of incorporating radical scavengers were also determined. The objective behind this study was to properly asses the roles played by the reactive species such as ·OH, O2O3/MoS2/Fe3O4 is illustrated in Fig. 2O3 (~ − 0.6 eV versus SHE) being more negative than that of MoS2 (~ − 0.5 eV versus SHE), there was a migration of photogenerated electrons from the CB of In2O3 to that of MoS2 following which there occurred interaction of these electrons with adsorbed O2 species to form O2−·47. With a band gap of 1.91 eV, the VB of MoS2 (~ + 1.39 eV versus SHE) would be placed higher than that of In2O3 (∼ + 2.2 eV versus SHE)53. This would drive holes from the VB of In2O3 to that of MoS2. The interaction of holes with H2O would enable production of ·OH. The involvement of FeIII/FeII redox couple would cause additional generation of ·OH radicals. Such interactions to produce reactive species have been previously reported over Fe3O4 containing photocatalytic systems56. In our experiment, we admit, it is difficult to prove the formation of such an intermediate hypervalent Fe-IV species during photodegradation mechanism. However, certain works have cited the possible generation of such species59. These reactive species caused decomposition of esomeprazole as illustrated below: In view of the aforementioned experimental results, the mechanism of photocatalysis by In3O4 to that of MoS2 eventually leading to the generation of the aforementioned reactive species. Furthermore, HRLCMS conducted at a halfway stage of light assisted disintegration of esomeprazole over In2O3/MoS2/Fe3O4, made it possible to outline a plausible pathway of esomeprazole decomposition 3. The data thus generated is presented in Table 3+ ions at the active sites of the photocatalysts.Several metal cations are usually present in wastewater and therefore their impacts on the decomposition of organic contaminant were studied. Na−1 of each of these acids was incorporated in the photocatalysis reaction vessel and their effects on photocatalytic activity was recorded. All of them accelerated photodecomposition and also the photodecomposition yield. In presence of oxalic acid with two -COOH groups in which one -OH of a -COOH influenced by ortho-COOH functions like α-OH group the highest photodecomposition yield and the maximum magnitude of velocity constant were registered. Citric acid having three -COOH groups and an α-OH group came next followed by tartaric acid (two -COOH groups and two α-OH groups) and then lactic acid (one -COOH group and one α-OH group). While investigating the effect of pH on photodegradation, although a slight initial increase up till pH 5 followed by a slight decrease in the yield was noted, there wasn’t any reduction in the optimal contact time of the reaction. However, in presence of organic acids, there was a marked acceleration of photodecomposition that led to a considerable reduction of optimal contact time to 40 min. Hence, the effect of pH in this phenomenon may be ignored. The number of -COOH and α-OH groups, however, could be correlated to this acceleration of photodecomposition64. Although precise elaboration of parameters affecting this phenomenon will require further work along this direction. The decomposition profile is shown in Fig. The decomposition of living organism generates organic acids that are found to accumulate in aquatic environment and therefore the influences of a few organic acids like oxalic acid, citric acid, tartaric acid and lactic acid on photodecomposition of esomeprazole was investigated. 0.5 mmolL2O3/MoS2/Fe3O4 was also investigated. With remarkable solvent properties acetone is frequently used in industrial applications and everyday life. Humic acids and SDS are also frequently found in wastewater.The influence of organic moieties such as, acetone, humic acid sodium salt (HAS) and sodium dodecyl sulfate (SDS) on esomeprazole photodegradation over InAs a photosensitizer, acetone has the repute to enhance photodegradation besides also exerting a decelerating effect. Acetone-photosensitization may proceed through the route delineated below:·OH radicals that led to enhanced esomeprazole decomposition. The retarding drag exerted by acetone could be due to the decomposition of acetone that competed with esomeprazole decomposition. Table In very low doses of acetone (0.001 M), the suppressing effect showed up. However, as the acetone concentration increased, photosensitization took over producing 2O3/MoS2/Fe3O4. In both the cases, HAS retarded the rate of decomposition. Besides, lowering the light intensity through water, HAS quenches hydroxyl radicals and also deactivates photocatalyst surface, thereby impeding the process of photocatalytic decomposition of water pollutants. Figures  were used and so, the slump in photocatalytic activity upon incorporation of SDS could be chiefly ascribed to competitive adsorption and suppression by sulfate ions. Figures , the suppressing effect due to its predominant competitive decomposition was evident. But with rise in acetone concentration increased, photosensitization took over producing ·OH radicals that reinforced esomeprazole decomposition. Organic acids like oxalic acid, citric acid, tartaric acid and lactic acid markedly increased photodecomposition yield and pseudo-first order velocity constant. Both HAS and SDS induced marked fall in the photocatalytic decomposition efficiency. Environmental water samples negatively affected photocatalytic performance of the ternary photocatalyst chiefly due to the combined effect exerted by inorganic and organic moieties. The ternary photocatalyst of In2O3/MoS2/Fe3O4 could be further used in esomeprazole photo-decomposition up to five consecutive cycles of the photocatalysis experiment sans any significant downturn in their performances.The present study is geared towards making an assessment of the photocatalytic activities of two novel ternary nanohybrids thus obtained vis-à-vis decomposition of aqueous esomeprazole and the interactive influences exerted by operating parameters, co-existing substances and environmental water samples. The target pollutant underwent ~ 92.92 ± 2.01% decomposition over InSupplementary Information


EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA–guide RNA complex residing at the 5′ of the crRNA. The activity of Cas13a was maximal for guide RNAs 24–30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70–76%), mCherry fluorescence (72%), and EML4-ALK at the protein (>80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine.RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the  RNAi technology has gained wide acceptance as a tool for knocking down specific cellular transcripts; hence it has been valuable for elucidation of molecular pathways, factors involved in diseases, and potential treatments ,2,3,4. HDNA manipulation tools such as CRISPR-Cas9 enable researchers to dissect the functions of particular genetic elements ,11, and EML4 and ALK genes are located 12 Mb apart on chromosome 2 (2p21 and 2p23), but are oriented in different directions. The genes can be fused through paracentric inversion, resulting in development of lung adenocarcinoma [EML4-ALK v1 [The arcinoma . At leasarcinoma . All vararcinoma ,20. Uponsome 2 2p and 2p234-ALK v1 ,23,24. TALK RNA in EML4-ALK fusion transcripts in H3122 lung cancer cells, with the goal of decreasing cell viability and providing proof-of-principle of a novel therapeutic approach.In this study, we examined CRISPR-Cas13a and crRNA–guide RNA from the standpoint of stability, effective knockdown efficiency, orientation of crRNA–guide RNA complex, and interference specificity on guide RNA design using firefly luciferase as a model target. Next, we used the programmable knockdown capacity of the Cas13a machinery to target 18S rRNA, rather than GAPDH [mRNA turnover is a key determinant of the abundance of cellular transcripts and, in turn, the level of protein or functional RNA. We first checked the stability of Cas13a mRNA and crRNA–guide RNA to characterize their decay rates and expression pattern. Both plasmid DNAs were transfected separately into HEK293T cells, and expression started to decrease 48 h after transfection . To calcan GAPDH , but inhRenilla luciferase, RLuc, was used as an internal control). A lower concentration (59 fmol/mL) of Cas13a was not sufficient to decrease luciferase luminescence (We next investigated the ability of the programmable endonuclease Cas13a to knock down firefly luciferase (FLuc) mRNA in HEK293T cells. First, we optimized the quantities of Cas13a and guide RNA for knockdown of FLuc  see A. As shoALK in the EML4-ALK fusion oncogene in the H3122 cell line model for human lung adenocarcinoma.We speculated that the endonuclease activity of Cas13a would vary among target transcripts. With this in mind, we targeted mCherry mRNA in HEK293T cells, and found that mCherry fluorescence was diminished up to 72% . In addiALK expression pattern for prognosis of lung adenocarcinoma patients was determined based on information in the PrognoScan microarray database, using a threshold COX p-value of 0.05. As shown in ALK was inversely proportional to patient survival. Moreover, ALK not only fuses with EML4 but also forms genetic fusions with other mRNA partners such as STRN, TPM1, PPP4R3B, ACTG2, STK39, KCNQ5, KCNQ5, MALAT1, GTF2IRD1, GALNT14, and SCEL, which, according to data retrieved from the TCGA PanCancer Atlas using cBioPortal , contribute to the development of eight other cancer types  [EML4 and ALK between H3122 and a nonfusion cell line, HEK293T . Our optimization experiments revealed that 177 fmol/mL Cas13a vector  and a 1:3 molar ratio of vector-to-guide RNA were sufficient to achieve 70–76% knockdown of FLuc mRNA  to be 5′ of the guide RNA (spacer) B. This iTo understand Cas13a’s target RNA binding and cleavage activity, we explored the length constraints for guide RNAs in human HEK293T cells. We found that a 24–30 bp guide RNA was sufficient for maximal FLuc mRNA knockdown (>75%)—cleavage activity was reduced when the guide was shorter (12 bp) or longer (40 or 50 bp) A. A simiEML4-ALK-positive cells [EML4-ALK oncofusion transcript in this study.Cas13a activity varies among target transcripts ,16,17. Ive cells ,31,32. Hve cells ,16. TherEML4-ALK transcript not only knocked down ALK expression but also changed cell physiology, as reflected by reductions in cell viability at three different time points  using siRNA in H3122 cells and H3122 CR (crizotinib resistance) cells . Initiale points . This leEML4-ALK oncogene. More rational guide design aimed at inhibiting expression of the EML4-ALK transcript has potential as an RNA-based therapy.Despite the remarkable advantages of Cas13a, it has some drawbacks. Cas13a still retains some off-target effects  and protRenilla luciferase pRL-TK Vector (GenBank Accession Number: AF025846) were purchased from Promega . pRSET-B mCherry was a gift from Dr. Hidekazu Tsutsui, JAIST, Japan. mCherry DNA was amplified from pRSET-B mCherry by PCR and inserted into pCS2+ vector at the BamHI and EcoRI sites. Plasmid LwCas13a-msfGFP was purchased from Addgene (plasmid #91902) [XhoI restriction site, a Kozak sequence, and an NLS were inserted at the 5′ site, and an NLS, 2 × Myc tag, TAA stop codon, and XbaI restriction site were inserted at the 3′ site adjacent to the Cas13a-GS-msfGFP insert. Oligo sequences used for mCherry, Cas13a, and pcDNA3 amplifications are listed in Firefly luciferase pGL3-Promoter Vector (GenBank Accession Number: U47298) and  #91902) . The CasDpnI: 25 µL PCR product, 9.5 µL Milli-Q water, and 0.5 µL DpnI . PCR products were electrophoresed on 1% agarose gels and purified using the Gel PCR purification kit . The assembly reaction contained 1 µL vector PCR product, 3 µL insert PCR product (1:3), and 4 µL HiFi assembly master mix (NEB). The final vector sequence was confirmed by Sanger sequencing , 0.75 µL (10 pmol) of each oligo, and 1 µL (5 ng/µL) template plasmid. PCR conditions were as follows: preheating at 95 °C for 2 min, 17 cycles of denaturation at 98 °C for 10 s, annealing at 60 °C for 5 s; extension at 68 °C for 1 min, and final extension at 72 °C for 7 min. Next, the vector PCR product was treated with cing see . The vechttp://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) to avoid self-complementarity and complex secondary structure. Following design, the cDNA sequences of crRNA (direct repeat) and guide RNA (spacer) with 5′ BamHI and 3′ EcoRI overhangs  on both strands were ordered as oligos from Eurofins Genomics, Tokyo, Japan. The oligos were annealed in a thermocycler. Reactions were performed in a total volume of 30 µL, consisting of 15 µL Milli-Q water, 4 µL (10 pmol) of each oligo, and 7 µL of 2 × ligation buffer (Promega). Annealing conditions were as follows: 95 °C for 5 min, decreased 7 °C every 5 min to 32 °C, 25 °C for 10 min, 4 °C for ∞ min. The annealed fragments were then ligated with BamHI- and EcoRI-digested pCS2+, and cloned into E. coli DH5 alpha competent cells. Construct was validated by restriction digestion and Sanger sequencing. Sequences of guide RNAs and crRNAs are provided in The guide RNAs were designed as the reverse complement of the target cDNA site to occupy the Cas13a machinery. The sequences were selected as follows: PFS on target A, T, or C; GC content 40–60%, off-target effects minimized by the BLAST tool in NCBI, and RNA secondary structure checked using the RNAfold web server  as described in our previous protocol . In some2. Unless otherwise noted, cells were plated in 48-well plates at 6 × 104 cells/well; cell counts were determined using a hemocytometer. The next day, cells were transfected with the indicated vectors using PEI reagent. H3122 and H2228 cells were kindly provided by Dr. Jeffrey A Engelman  [HEK293T cells  were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  at 37 °C in a humidified incubator under an atmosphere containing 5% COMA, USA) ,36. Both3 HEK293T cells were seeded per well; transfections were performed on the following day. Total RNA was extracted after 12, 24, 48, 72, and 96 h, and subjected to cDNA synthesis and RT-PCR. For half-life measurements, 6 × 103 HEK293T cells were seeded per well. The next day, 375 ng each of Cas13a and crRNA–guide RNA vector were transfected separately. Culture media was replaced the following day. On the third day, for time point 0 (t0), cells were washed with D-PBS , lysed with 100 µL TRIzol reagent (Invitrogen), and then kept in a −80 °C freezer. For the remaining time points , cells were simultaneously treated with 5 µg/mL ActD . Total RNA was isolated from the cells and subjected to cDNA synthesis and qRT-PCR. The CT value was normalized against human 18S rRNA and the corresponding value at t0. Half-life was estimated from t0-normalized data.To monitor the expression of Cas13a mRNA and crRNA–guide RNAs, 2.5 × 10The half-life of a transcript was calculated as follows: half-life = ln(2)/kdecay . This eqg for 15 min at 4 °C, and about 50 µL supernatant was transferred to a new tube. Next, 62.5 µL of 100% isopropanol was added to the supernatant, mixed by inversion, and incubated at room temperature for 10 min. The samples were centrifuged at 12,000× g for 10 min at 4 °C, and the supernatant was discarded. The pellet was washed with 200 µL of 80% ethanol, and the supernatant was discarded. The pellet was then air-dried at room temperature for 5–10 min and dissolved in Milli-Q water.Cells were washed quickly with D-PBS and lysed by addition of 100 µL/well TRIzol reagent (Invitrogen). The lysed samples were homogenized by vortexing for 2–3 min. Next, 25 µL chloroform was added to the solution and mixed by hand for 15 s. The sample was centrifuged at 12,000× cDNA synthesis was performed in a total volume of 20 µL. Reaction mixtures contained 100 ng RNA, 0.5 µL of each oligo (dT) primer and random primer, 1 µL of 10 mM dNTPs, and water to a final volume of 14 µL. After mixing, the sample was incubated at 65 °C for 5 min and cooled on ice for 5 min. Next, 4 µL of 5 × buffer, 1 µL of ReverTra Ace (TOYOBO), and 1 µL of 0.1 M DTT were added to the solution, followed by mixing. Incubation was performed in a thermocycler  as follows: 50 °C for 30 min and 55 °C for 30 min for synthesis, 70 °C for 15 min for inactivation, and 4 °C hold.2O, 1 μL cDNA template, 4 μL of 5 × Green GoTaq Flexi buffer (Promega), 2 μL of 25 mM MgCl2, 2 μL of 2 mM dNTPs, 0.1 μL Taq polymerase (Promega), and 0.8 μL (10 pmol) of each primer. Sequences of all primers are provided in GAPDH was used as a loading control. Two-microliter aliquots of each PCR product was loaded on 6% polyacrylamide gels. The ImageJ software was used to quantify band intensities [PCR reactions were performed in a total volume of 20 μL on a thermocycler (GeneAmp PCR System 9700). Reaction mixtures contained 9.3 μL of Hensities .T values were normalized against GAPDH or 18S rRNA expression using the 2−ΔΔCT method [SYBR Green–based qPCR was performed in a total volume of 10 μL on an MX 3000P STRATAGENE Multiplex Quantitative PCR System . The reactions consisted of 3.5 μL water, 5 μL TB Green, 0.2 μL of 50 × ROX reference dye II , 0.5 μL cDNA template, and 0.8 μL (10 pmol) of each primer; sequences of all primers are provided in T method .4 cells/well of a 48-well, and cotransfected with firefly luciferase (as a target) and Renilla luciferase  at a 1:10 ratio with Cas13a and guide RNA vectors. Forty-eight hours after transfection, the media were removed, and the cells were washed with D-PBS. The cells were partially lysed in a 50 μL/well of 1 × passive lysis buffer (PLB)  and incubated for 15 min at room temperature with shaking. Next, the plate was kept on ice, and luminescence was measured in each well. First, a blank was measured: 10 µL of 1 × PLB was mixed into 30 μL luciferase substrate I (LARII) in a measuring tube, firefly relative light unit (RLU) was observed on a Gene Light 55 , 30 µL Stop and Glo reagent was added to the same tube, and Renilla RLU was observed. Second, the sample was measured: 2 μL cell lysate and 8 μL of 1 × PLB were mixed into 30 μL LARII, and then the RLU values were observed as described above for the blank. The blank value was subtracted from all sample values. The condition of the machine was set to dual-luciferase assay mode: first delay time, 3 s; first count time, 6 s; second delay time, 3 s; second count time, 6 s; repeat time, 1. Finally, the data were normalized as described for the genetic reporter assay [Bioluminescence of luciferase was measured according to the Dual-Luciferase Reporter (DLR) Assay System (Promega) protocol with minor modifications. Briefly, HEK239T cells were seeded at 6 × 10er assay .Cell viability was measured by MTT -2,5-Diphenyltetrazolium Bromide) (Invitrogen). Absorbance was quantified with a ThermoFisher Spectrophotometer 1000  at a wavelength of 540 nm. Crizotinib  (0.25 µM and 2 µM for H3122 and H2228 cells respectively) was used as a positive control.Cells were lysed in buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid sodium salt, 0.1% SDS, 25 mM Tris-HCL (pH 7.4), and protease inhibitor cocktail set III . Proteins were separated by 9% and 11% SDS-PAGE for blotting with ALK and β-actin antibodies, respectively, and then transferred onto a PVDF membrane . Proteins were detected by immunoblotting using ECL Select detection reagent . Anti-ALK antibody  was used at a dilution of 1:1000. Anti-β–actin antibody (Proteintech) was used at a dilution of 1:2000.ALK expression and patient prognosis in lung adenocarcinoma was retrieved from the PrognoScan cancer microarray database [t-test was used to evaluate the significance of comparisons between two groups. A p-value of 0.05 was considered statistically significant.The relationship between database . The gendatabase ,43. StatEML4-ALK resulted in alteration of cell physiology, reflected by a decrease in viability. Based on our successful robust knockdown of marker genes such as firefly luciferase, mCherry in HEK293T cells, and EML4-ALK in H3122 and H2228 lung cancer cells, we speculated that this approach could be used to knock down other transgenes or genes of interest  that affect the biological functions of cells.In this study, we determined the half-life of CRISPR Cas13a mRNA and crRNA in human cells. Subsequently, we successfully knocked down firefly luciferase in HEK293T cells (>70%) after optimizing the levels of Cas13a mRNA and guide RNA. In addition, we assessed the effects of the orientation of the crRNA–guide RNA complex, length, and mismatches on Cas13a activity. Knockdown of the oncofusion gene


These geothermal features may provide a suitable niche for the growth of chemolithotrophic thermoacidophiles, including the lanthanide-dependent methanotrophs of the phylum Verrucomicrobia. In this study, we started enrichment cultures inoculated with soil of the Favara Grande at 50 and 60°C with CH4 as energy source and medium containing sufficient lanthanides at pH 3 and 5. From these cultures, a verrucomicrobial methanotroph could be isolated via serial dilution and floating filters techniques. The genome of strain AP8 was sequenced and based on phylogenetic analysis we propose to name this new species Methylacidimicrobium thermophilum AP8. The transcriptome data at μmax  of the new strain showed a high expression of the pmoCAB2 operon encoding the membrane-bound methane monooxygenase and of the gene xoxF1, encoding the lanthanide-dependent methanol dehydrogenase. A second pmoCAB operon and xoxF2 gene were not expressed. The physiology of strain AP8 was further investigated and revealed an optimal growth in a pH range of 3–5 at 50°C, representing the first thermophilic strain of the genus Methylacidimicrobium. Moreover, strain AP8 had a KS(app) for methane of 8 ± 1 μM. Beside methane, a type 1b [NiFe] hydrogenase enabled hydrogen oxidation at oxygen concentrations up to 1%. Taken together, our results expand the knowledge on the characteristics and adaptations of verrucomicrobial methanotrophs in hydrothermal environments and add a new thermophilic strain to the genus Methylacidimicrobium.The Favara Grande is a geothermal area located on Pantelleria Island, Italy. The area is characterized high temperatures in the top layer of the soil (60°C), low pH (3–5) and hydrothermal gas emissions mainly composed of carbon dioxide (CO By oxidizing CH4, they act as the carbon sink in various environments and limit the emission of this greenhouse gas to the atmosphere  using the Calvin-Benson-Bassham (CBB) cycle (4 can be used as energy source and it is oxidized to methanol (CH3OH) using a copper-dependent enzyme called particulate methane monooxygenase (pMMO). The CH3OH in turn is oxidized to formaldehyde (CH2O) and formate (HCOOH), and finally to CO2  cycle . CH4 cany to CO2 .mxaFI. The genomes of verrucomicrobial methanotrophs lack mxaFI and accessory genes, but do possess a homologous gene called xoxF, that encodes an Ln-MDH with lanthanides instead of calcium in the active site  and heavy lanthanides . In contrast to their name, REEs are fairly abundant in the Earth’s crust, especially in acidic geothermal soils from which all verrucomicrobial methanotrophs have been isolated to date  methanol dehydrogenase (Ca-MDH) encoded by the genes ive site . Lanthan to date . For a l to date . XoxF frproteins , suggestproteins . Consequproteins . Furtherproteins .Methylorubrum extorquens PA1, a TonB-dependent receptor and an ABC transporter are required for growth on methanol in the presence of REEs  for growth. The thermophilic M. fumariolicum SolV genome encodes two [NiFe] hydrogenases, belonging to the groups 1d (O2 sensitive) and 1 h/5 , acidic pH (3–5), and gas emissions mainly consisting of CO2, CH4, and H2 . The top layer of a soil core was used as inoculum in air-tight serum bottles containing 10 ml of Pantelleria medium (see below) adjusted to pH 3 and 5. The headspace of the bottles was composed of 87% NM. thermophilum AP8 were obtained from enrichment cultures originating from the soil top layer of FAV1, using dilution-to-extinction and floating filter techniques, as described previously 2SO4, 1 mM NaH2PO4.2H2O, 2 mM MgSO2.7H2O, 2 mM CaCl2.2H2O, 5 μM nitrilotriacetic acid (NTA), 0.5 μM CeCl3 or Nd2O3, 0.5 μM Na2SeO3, and a trace element solution containing 10 μM FeSO4, 10 μM MnCl2, 15 μM CuSO4, 5 μM NiCl2, 0.5 μM ZnSO4, 0.5 μM Na2MoO4, and 0.5 μM CoCl2. The trace elements solutions, CaCl2.2H2O, NTA, CeCl3/Nd2O3, and Na2SeO3, were autoclaved separately and added afterward, to avoid precipitation. The pH was adjusted with 1 M H2SO4 or 0.1 M NaOH using a 691 pH meter (Metrohm). Bottles were incubated at 50°C with 100 or 250 rpm shaking. To reach maximum growth rate, the headspace contained 49–64% N2, 10–20% CH4, 5–10% CO2, and 21% O2.Pure cultures of eviously . Brieflyd before . Single M. thermophilum AP8 batch culture at OD600 0.3 and operated at 50°C with stirring at 1000 rpm. To achieve maximum growth rate, CH4/CO2 (95:5) was sparged through a sterile filter with a flow rate of 169 ml/min. The dO2 was regulated at 5% O2 and growth of the culture was recorded by measuring the OD600 with Spectronic 200 spectrophotometer (Thermo Science) in 1 ml microcuvettes. After achieving maximum growth rate, the stirring was decreased to 500 rpm and CH4/CO2 was limited to a flow rate of 11.6 ml/min. The chemostat was supplied with Pantelleria medium [with 20 mM (NH4)2SO4] at a dilution rate of 0.005 h−1 (50 ml.h−1). The pH was kept stable with controlled supply of 0.5 M NaOH.A bioreactor  containing 10 L of Pantelleria medium was inoculated with 300 ml of M. thermophilum AP8 were incubated in Pantelleria medium from pH 1.5 to 5.5. For pH 4 and above, medium was buffered with 50 mM 2-(N-morpholino)ethanesulfonic acid (MES), as described previously , or in 96-well plate in the SpectraMax 190 plate reader . During and after experiments, pH was measured using the Hanna HI 5221 pH meter.Cultures of eviously . Bacteri600 in 96-well plate in the SpectraMax 190 plate reader (Molecular Devices). All experiments were conducted in triplicate.For temperature optimum experiments, cultures were incubated in Pantelleria medium at pH 4 in shaking incubators at the following temperatures: 30, 37, 45, 50, 55, and 60°C. Bacterial growth was determined by an increase in density, measured as ODM. thermophilum AP8 cells (OD600 0.2–0.5) were transferred to a capped 120 ml serum bottle. Methane was injected into the bottles to reach a concentration of 0–80 μM CH4. To ensure active methane oxidation, the bottles were incubated at 50°C with 350 rpm shaking for 20 min prior to the start of measuring. Consumption of methane was measured over a 90 min period by injecting 100 μl of headspace gas with a glass syringe into a HP 5890 gas chromatograph (Agilent) equipped with a Porapak Q column and a flame ionization detector. The experiment was conducted in triplicate. The obtained methane consumption rates were normalized against the cell density measured as OD600. Origin Software (OriginLab Corporation) was used to fit a Michaelis–Menten curve and calculate the Ks and Vmax values according to the equation V = Vmax ∙ [S}/(Ks+[S]), in which V was measured as methane consumption rate and [S] represents the methane concentration.Exponentially growing Methylacidimicrobium thermophilum AP8 cells grown under CH4 limitation were inoculated into sterile capped 120 ml serum bottles. Bottles were made anaerobic by using a mixture of Ar/CO2  and supplied with 5% H2 and a varying concentration of O2 (0–5%). Starting OD600 was 0.1 in 20 ml of medium. The bottles were incubated at 50°C with 350 rpm shaking. The H2 consumption was measured by injecting 50 μl of headspace gas with a glass syringe into a HP 5890 gas chromatograph (Agilent) equipped with a Porapak Q column and a thermal conductivity detector. The respective H2 consumption rates were determined by converting the peak areas into percentage of H2 using a calibration curve. The growth on H2 was detected by measuring OD600 every 24 h with Spectronic 200 spectrophotometer (ThermoFisher) in 1 ml microcuvettes. The experiment was conducted in triplicate.M. thermophilum AP8 was isolated using the DNeasy Blood&Tissue kit according to manufacturer’s instructions . A combination of Illumina and Nanopore sequencing was used to obtain the complete genome.gDNA of Libraries for Illumina sequencing were prepared using the Nextera XT kit (Illumina), followed by enzymatic tagmentation, incorporation of indexed adapters, and amplification of the library. The amplified library was purified with AMPure XP beads, after which quality and size distribution were checked with the Agilent 2,100 Bioanalyser and High sensitivity DNA kit. After fluorimetric quantitation using Qubit dsDNA HS Assay Kit (ThermoScientific), the library was normalized and diluted to a final concentration of 4 nM. Pooled libraries were denatured and diluted using Denature and Dilute Libraries Guide (Illumina) and loaded into cartridge of the Illumina Miseq sequence machine. Paired-end sequencing of 2 × 300 bp was used.Nanopore MinION sequencing was performed by shearing the DNA using Covaris g-TUBE (Covaris). Sheared fragments were end-repaired using NEBNext FFPE DNA Repair Mix (New England Biolabs) and purified using AMPure XP beads. Barcodes were ligated using Blunt/TA Ligase Master Mix (NEB) and cleaned using AMPure XP beads. Adapters were ligated using NEBNext Quick Ligation Module (NEB). The library was purified with AMPure XP beads and quantified using Qubit dsDNA HS Assay Kit (ThermoScientific).A total of 6,769,626 sequenced Illumina reads were trimmed  using CLC Genomics Workbench 11.0 , resulting in 6,237,920 trimmed reads. After base calling and quality assessment with ONT Albacore Sequencing Pipeline Software 2.1.10 , 141,891 Nanopore reads passed the check . The Nanopore reads were assembled with Canu , resulting in one circular contig of 2,298,237 bp. The assembly was optimized with Racon  and protein  sequences of strain AP8 were used in Blast searches against Genbank databases. Representative homologous sequences were downloaded and aligned by the ClustalW or MUSCLE tool available in MEGA7 . MEGA7 w600 3.5, was centrifuged for 10 min at 4,400 rpm, supernatant removed, and the pellets frozen at −80°C. RNA was extracted from thawed cells using RiboPure Bacteria Kit according to manufacturer instructions (ThermoFisher). The total RNA concentration was measured with Qubit Fluorometer RNA HS assay kit (ThermoFisher). The total RNA was purified with MICROBexpress Kit (ThermoFisher) to remove non-coding RNA and the quality of the rRNA-depleted RNA was checked using the Agilent 2,100 Bioanalyzer (Agilent). A cDNA library was prepared from the rRNA-depleted samples using Ion Total RNA-seq Kit v2 according to the manufacturer protocol (Thermofisher). Briefly, RNA was first fragmented, and the fragments were purified with magnetic beads. The fragments were hybridized and ligated with random primers for cDNA production by reverse transcription and the cDNA was purified with magnetic beads. Then, cDNA was amplified with PCR into a barcoded library and the quality of the PCR products checked with Agilent 2,100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent). The concentration of cDNA was measured using a Qubit Fluorometer dsDNA HS Assay (ThermoFisher). Samples were diluted and loaded into a cartridge of the Illumina Miseq sequencing machine. The sequence reads were analyzed using CLC Genomic Workbench software . Reads shorter than 100 bp were trimmed and non-coding rRNA sequences were excluded to obtain the expression values of mRNA sequences presented as reads per kilobase per million reads  of bacterial culture, grown at maximum growth rate and ODds RPKM; .g, 4 min). The pellet was resuspended in 15 μl supernatant and subsequently, 0.6 μl samples were high-pressure frozen in a HPM-100 (Leica Microsystems) using gold-plated platelets (2 mm inner diameter and 100 μm sample thickness). Care was taken to keep the cells at their optimal temperature of 50°C until the very moment of freezing. Samples were freeze-substituted in an AFS2 (Leica Microsystems) using anhydrous acetone  containing 0.2% Uranyl Acetate . The substitution started at −90°C for 48 h, followed by a +2°C/h slope to −70°C, where the sample remained for 12 h. Afterwards, the temperature was raised with 2°C/h to −50°C, where the sample remained for 12 h. Next, the sample was washed twice with anhydrous acetone at −50°C and stepwise infiltrated with Lowicryl HM20  in anhydrous acetone at −50°C. After the four changes of 100% HM20, the samples were polymerized by UV irradiation at −50°C for 96 h and +2°C/h to 0°C followed by 24 h at 0°C. Ultrathin sections (ca. 50 nm) were cut using an ultramicrotome  and applied to 100 mesh copper grids  containing a carbon-coated formvar film. Micrographs were recorded in a JEOL Jem-1400 Flash transmission electron microscope operating at 120 kV.Biomass (4 ml) was harvested from a bioreactor and concentrated by centrifugation  together with a single separate pmoD gene  and xoxF2  encodes for a homologous of the TonB dependent transporter of Methylobacterium extorquens PA1 .The electrons generated during methane oxidation can be carried to the respiratory chain by a cytochrome C2. Furthermore, a methanethiol oxidase (MTHMO_v1_1990) was detected in the genome; this enzyme converts methanethiol to H2O2, formaldehyde, and H2S. Toxic formaldehyde can be metabolized by XoxF or by the tetrahydrofolate-dependent formaldehyde oxidation pathway, where the gene folD (MTHMO_v1_0721), encoding a methylenetetrahydrofolate dehydrogenase/cyclohydrolase generates formate, that can be converted to CO2 by formate dehydrogenase (The presence of a carbonic anhydrase (MTHMO_v1_0262) and of enzymes for the Calvin cycle , indicatrogenase . Genes frogenase . Glycoge4, H2 seemed to be a possible substrate for M. thermophilum AP8, as a type 1b [NiFe] hydrogenase (MTHMO_v1_1378-1380) was detected in the genome . NH4+ and NO3− can also be transported inside the cells by NH4+  and NO3− transporters . Finally, cyanate can be converted to NH4+ by a cyanate hydratase . So N2 gas, NH4+, NO3−, and cyanate could serve as nitrogen source and ammonium can be incorporated into biomass via the glutamate dehydrogenase  and/or glutamine synthetase/glutamate synthase  pathways. Nitrate (NO3−) would be converted via assimilatory nitrate reductase  into nitrite and further on to NH4+via nitrite reductase .Similar to other verrucomicrobial methanotrophs , M. ther2O) by nitric oxide reductase  as also described by  by a copper-containing nitrite reductase . NO would then be converted to nitrous oxide . NH2OH needs to be rapidly metabolized due to its toxicity and potential inhibition of XoxF  of 0.051 ± 0.001 h−1. This corresponded to a doubling time of approximately 14 h.Growth was minimal at 30°C and maximal at 50°C . No growmax were used to measure the affinity constant for CH4 (Ks(app)). Cultures were incubated in bottles containing 0–80 μM CH4 and the consumption of methane was monitored over time  of 8.2 ± 0.9 μM CH4 . The CH9 μM CH4 .M. thermophilum AP8, the cell morphology was investigated via electron microscopy . The second operon, pmoCAB1 (MTHMO_v1_0893-0895), was hardly expressed (xoxF2 (MTHMO_v1_1756) expression was 85-fold lower (xoxG (MTHMO_v1_1702) had a higher expression value compared to xoxJ , exhibited the highest expression values among the other putative lanthanide transporters . To study their differential expression in xpressed . Similarld lower . The cytuens PA1 . Most ofsporters .2, which can be assimilated in the CBB cycle by a type I RuBisCO. Formate dehydrogenases , genes involved in the tetrahydrofolate pathway , as well as carbonic anhydrase  and the ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) encoding genes  were expressed  was constitutively expressed even if cyanate was not supplied in the medium. The genes responsible for NO  and NO2− detoxification  had expression levels between 200 and 65 RPKM. Furthermore, the NH4+ and NO3− transporters  did not have high levels of expression (<50 RPKM) and the assimilatory nitrate reductase/nitrite reductase (MTHMO_v1_1031) was basically not expressed  rather than via glutamine synthetase/glutamate synthase (GS-GOGAT) pathway  and their maturation factors had expression values up to 800 RPKM .The genome and transcriptome of rogenase . We studve 1% O2 . In a sece of O2 . DecreasM. thermophilum AP8 was not strictly O2 sensitive but it required very low concentrations of O2 (<1%) in order to actively oxidize H2.These results showed that the type 1b [NiFe] hydrogenase encoded by M. thermophilum AP8. Strain AP8 was discovered in a soil sample from the geothermal area Favara Grande on Pantelleria Island. This area is characterized by low pH and high temperatures  and a faster growth rate (0.05 h−1 vs. 0.01–0.04 h−1) compared to other Methylacidimicrobium species. In this regard, M. thermophilum AP8 represents the first thermophilic strain of the genus Methylacidimicrobium.The genus w as 0.5 . MethylaM. tartarophylax 4AC and M. cyclopophantes 3B, membrane stacks were not detected, which questions the location of the pMMO enzyme. The only exception is represented by M. fagopyrum 3C, whose membrane stacks are visible in the cytoplasm  vs. 9 μM (PmoCAB1)], suggesting that differential expression of pmoCAB might depend on the concentration of methane ) of 8 ± 1 μM, which is close to that reported for the thermophilic M. fumariolicum SolV  1–12 μM; Methylocapsa gorgona MG08 was shown to have KS(app) of 5 μM and still be able to grow on atmospheric methane concentrations  XoxF-type MDHs. XoxF from Methylomonas sp. LW13 and Bradyrhizobium sp. MAFF211645 functioned with lanthanides from La3+ to Nd3+ (atomic number 57–60), whereas in Methylotenera mobilis, activity was detected with REEs until atomic number 64  and a component of the ABC transport system (MTHMO_v1_2099) showed expression of 392 and 587 RPMK, respectively. These genes are highly conserved in verrucomicrobial methanotrophs and probably responsible for REEs uptake. MTHMO_v1_2099 was located next to the gene MTHMO_v1_2097, a putative copper binding protein (248 RPKM), and MTHMO_v1_2098 that encoded a transport permease protein -dependent formaldehyde oxidation were detected and their expression found to be >160 RPKM  was isolated from volcanic soil of the Favara Grande area on Pantelleria Island, Italy. Cells are rod-shaped, Gram-negative, with glycogen and phosphate particles present in many cells. Growth occurred between pH 1.5–5.5 and 30–55°C. Optimum was registered at pH from 3 to 5 and 50°C. Doubling time at maximum growth rate was about 14 h. KS(app) for methane was calculated at 8 ± 1 μM CH4. Hydrogen oxidation was observed below 10 μM O2.https://www.ncbi.nlm.nih.gov/genbank/, PRJEB37308 https://www.ncbi.nlm.nih.gov/genbank/, LR797830.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NP, AP, MJ, and HC designed the projects and experiments. NP, AP, CH, AG, WD’A, PQ, and HC sampled the geothermal soils. NP and CH performed the enrichment and isolation experiments. NP, PB, and AW conducted the physiology experiments. RM performed the electron microscopic analyses. GC and PB sequenced the genome and transcriptome, reconstructed the genome, and analyzed the gene expression. NP, PB, AP, and HC carried out the data analysis. NP, PB, and HC wrote the manuscript. All authors contributed to revision of the manuscript, and read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Autophagy starts with the initiation and nucleation of isolation membranes, which further expand and seal to form autophagosomes. The regulation of isolation membrane closure remains poorly understood. CK1δ is a member of the casein kinase I family of serine/threonine specific kinases. Although CK1δ is reported to be involved in various cellular processes, its role in autophagy is unknown. Here, we show that CK1δ regulates the progression of autophagy from the formation of isolation membranes to autophagosome closure, and is essential for macroautophagy. CK1δ depletion results in impaired autophagy flux and the accumulation of unsealed isolation membranes. The association of LC3 with ATG9A, ATG14L, and ATG16L1 was found to be increased in CK1δ-depleted cells. The role of CK1δ in autophagosome completion appears to be conserved between yeasts and humans. Our data reveal a key role for CK1δ/Hrr25 in autophagosome completion. Autophagy is an evolutionarily conserved pathway in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes, and then transported into lysosomes or vacuoles for degradation . Autophade novo formation of a cup-shaped membrane, known as the isolation membrane or phagophore, which expands and seals to form the autophagosome (Autophagy is initiated with the hagosome . In mammhagosome . Autophahagosome .CK1δ (casein kinase I δ), a member of the CK1 family of serine/threonine specific kinases, is involved in the regulation of various cellular processes including circadian rhythms, Wnt signaling, cytoskeleton maintenance, the cell cycle, and DNA damage repair . Hrr25, In this study, we show that CK1δ is essential for macroautophagy in mammalian cells, and that CK1δ depletion or Hrr25 mutation results in blockade of the progression of isolation membranes to autophagosomes, thus revealing a key role of CK1δ/Hrr25 in autophagosome completion.2. For starvation, cells were washed with PBS three times and incubated with Earle’s balanced salt solution  for 2–h at 37°C. Transfection of plasmids was carried out with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocols. Transfection of small interfering RNAs (siRNAs) was carried out with Lipofectamine RNAi MAX (Invitrogen). To knockdown CK1δ, double-stranded siRNAs were purchased from GenePharma. The following sequences were used: human CK1δ siRNA 5′-CGACCUCACAGGCCGACAATT-3′ and control siRNA 5′-UUCUCCGAACGUGUCACGUTT-3′.HeLa cells were cultured in DMEM (Hyclone) and 10% fetal bovine serum  supplemented with 1% penicillin-streptomycin (Gibco) at 37°C and 5% COYeast cells were grown at 25°C in yeast extract peptone dextrose media  or synthetic minimal media . To induce starvation, yeast cells were transferred to SD(–N) medium (0.17% yeast nitrogen base without amino acids and 2% dextrose) or treated with 400 ng/ml rapamycin.Total RNA was extracted from CK1δ-knockdown HeLa cells using the Cultured CellTotal RNA Extraction Kit (TIANGEN), and cDNA was reverse-transcribed using the FastQuant RT Kit(TIANGEN). Quantitative PCR was carried out on a Step One PlusTM RealTime PCR system using SuperReal PreMix Plus (TIANGEN). Data were normalized to the expression level of β-actin. Results are representative of at least three experiments. The following primers were used: F-CK1 Delta, 5′-CTCCGTGTTCCGTTTC-3′; R-CK1 Delta, 5′-TGCTACTCGCCATCCT-3′; F-GAPDH, 5′-GGCATCCTGGGCTACACTGA-3′; R-GAPDH, 5′-GTGGTC GTTGAGGGCAATG-3′.g for 30 min at 4°C. Supernatants were separated by SDS-PAGE and transferred onto a PVDF membrane, followed by incubation with primary and secondary antibodies  supplemented with 1 mM PMSF, and incubated for 30 min on 4°C. Cell lysates were centrifuged at 12000 tibodies ; then, tHeLa cells grown on cover slips were fixed with 4% paraformaldehyde for 20 min. Cells were permeabilized in 10 μg/mL digitonin (Sigma) for 15 min at 25°C. After blocking with 5% goat serum for 60 min at 25°C, cells were incubated with the indicated primary antibodies overnight at 4°C. After three washes with PBS, cells were stained with FITC or Rhodamine-labeled secondary antibodies for 1 h at room temperature. Nuclei were stained with 0.2% Hoechst 33258 for 30 min. Images were acquired using a FV3000 confocal microscope (Olympus). The colocalization was measured using the ImageJ software.g and at 4°C, for 10 min. The supernatant was then centrifuged again at 100,000 g for 30 min to acquire the pellet fraction. The pellet was resuspended in homogenization buffer and then treated with 100 μg/ml proteinase K (ProK) with or without 0.5% Triton X-100. After incubation for 20 min on ice, 10% Trichloroacetic acid (TCA) was added, and the samples were centrifuged at 15,000 g for 10 min. The pellet was washed with ice-cold acetone, resuspended in SDS-PAGE sample buffer, and boiled at 100°C for 10 min. Proteinase K digestion products were detected by immunoblotting.HeLa cells were starved in EBSS for 2 h, treated with 100 nM bafilomycin A1 for 6 h, and suspended in ice-cold homogenization buffer . Cells were then passed 10 times through a 27-gauge needle using a 1 ml injection syringe, and the post-nuclear supernatant (PNS) was recovered by centrifugation at 4500 To analyze phagophore formation, yeast cells were fixed with 3.7% formaldehyde at 25°C for 30 min, and then visualized by super-resolution structured illumination microscopy (SIM), at 25°C, on an Applied Precision DeltaVision OMX Super Resolution System using an Olympus UPlanSApo 100 × 1.4 NA oil objective. The data were acquired and processed using Delta Vision OMX Master Control software and SoftWoRx reconstruction and analysis software.t test. Data were analyzed for statistical significance after at least three repeated experiments. Threshold for statistical significance for each test was set at 95% confidence (p < 0.05). Error bars represent SEM. ∗p < 0.05, ∗∗p < 0.01. ∗∗∗p < 0.001.The significance of differences was evaluated by an unpaired two-tailed To determine whether CK1δ is essential for autophagy, we used siRNAs to deplete HeLa cells of CK1δ, and examined the autophagic flux by monitoring the level of p62, an autophagy substrate . As showhrr25-5 mutant cells; in a previous study, we observed numerous punctate GFP-Atg8 structures in the hrr25-5 mutant, despite the decreased autophagic activity in these cells  is conjugated to PE to form the lipidated LC3-II form, which associates with autophagic membrane structures and forms punctate structures. Analysis of LC3-II levels serves as a useful assay to determine which step of autophagy is affected . We examse cells .Collectively, these results indicate that CK1δ depletion impairs autophagic flux downstream of LC3 lipidation and leads to the accumulation of LC3-II-positive structures.hrr25-5 mutant cells. The tandem red fluorescent protein (RFP)-green fluorescent protein (GFP)-LC3 reporter was transfected into CK1δ-depleted cells and control cells as previously described. The GFP fluorescence signal is quenched in acidified compartments; accordingly, prior to fusion with the lysosome, the isolation membranes and immature autophagosomes decorated with RFP-GFP-LC3 are visible as yellow puncta, whereas red puncta represent acidified autolysosomes , a dominant-negative VPS4A closure . In yeas closure , 2019a,b closure ; therefoAll datasets generated for this study are included in the article/JW designed the research. YL, QX, XC, YC, HZ, and JS performed the experiments. JW, YL, BZ, and QX analyzed the data. JW and MT wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This translated to a functional outcome in both human and mouse primary macrophages, where SB290157 significantly dampened C5a-induced ERK signaling. We also confirmed that SB290157 acts as a potent agonist at human C3aR in transfected cells, but as an antagonist in primary human macrophages. Our results therefore provide even more caution against using SB290157 as a research tool to explore C3aR function. Given the reported immunomodulatory and anti-inflammatory activities of C5aR2 agonism, any function observed with SB290157 could be due to these off-target activities.Innate immune complement activation generates the C3 and C5 protein cleavage products C3a and C5a, defined classically as anaphylatoxins. C3a activates C3aR, while C5a activates two receptors (C5aR1 and C5aR2) to exert their immunomodulatory activities. The non-peptide compound, SB290157, was originally reported in 2001 as the first C3aR antagonist. In 2005, the first report on the non-selective nature of SB290157 was published, where the compound exerted clear agonistic, not antagonistic, activity in variety of cells. Other studies also documented the non-selective activities of this drug in vivo. These findings severely hamper data interpretation regarding C3aR when using this compound. Unfortunately, given the dearth of C3aR inhibitors, SB290157 still remains widely used to explore C3aR biology (>70 publications to date). Given these issues, in the present study we aimed to further explore SB290157's pharmacological selectivity by screening the drug against three human anaphylatoxin receptors, C3aR, C5aR1 and C5aR2, using cell models. We identified that SB290157 exerts partial agonist activity at C5aR2 by mediating  The C5aR2 selective ligand, P32, was synthesized in house as previously described . Recombi2) and subcultured at 80–90% confluency using 0.05% trypsin-EDTA in DPBS.The following cell lines were cultured as previously described . Briefly+ monocytes were isolated from blood using Lymphoprep density centrifugation  followed by CD14+ MACS magnetic bead separation . The isolated monocytes were differentiated for 7 days in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin and 15 ng/ml recombinant human macrophage colony stimulating factor  on 10 mm square dishes . Non-adherent cells were removed by washing with DPBS, and the adherent differentiated HMDMs were harvested by gentle scraping.Human monocyte-derived macrophages (HMDMs) were generated and cultured as previously described . BrieflyMouse bone marrow-derived macrophages (BMDMs) were obtained and cultured as previously described . BrieflySurefire Ultra p-ERK1/2 (Thr202/Tyr204) kit  following the manufacturer's protocol as previously described (Ligand-induced ERK1/2 phosphorylation was assessed using the AlphaLISA escribed . Brieflyβ-arrestin 2 recruitment to C5aR2 was measured using bioluminescence resonance energy transfer (BRET)-based assay as previously described (β-arrestin 2-Renilla luciferase 8 (Rluc8) and human C5aR2-Venus constructs using XTG9  for 24 h. Transfected cells were then seeded  onto white 96-well plates  in phenol-red free DMEM containing 5% FBS overnight. For BRET assay, cells were firstly incubated with the substrate EnduRen  for 2 h . All ligands were prepared in SFM containing 0.1% BSA. On a Tecan Spark 20 M microplate reader  (37°C), the BRET light emissions (460–485 and 520–545 nm) were continuously monitored for 25 reads with respective ligands added after the first 5 reads. The ligand-induced BRET ratio was calculated by subtracting the emission ratio of Venus (520–545 nm)/Rluc8 (460–485 nm) of the vehicle-treated wells from that of the ligand-treated wells.The C5a-mediated escribed . Brieflyt-test or two-way ANOVA as indicated. Differences were deemed significant when p < 0.05.All experiments were conducted in triplicates and repeated on 3 separate days (for cell lines), using cells from 3 or more independent donors (for HMDMs) or mice (for BMDMs) unless otherwise specified. Data was analysed using GraphPad software (Prism 8.4). Data from each individual repeat was normalised accordingly before being combined and expressed as mean ± standard error of the mean (S.E.M.) unless otherwise described. For all dose-response studies, logarithmic concentration-response curves were plotted using combined data and analysed to determine the respective potency values. Statistical analysis was performed using unpaired Student's 50 of 0.46 nM , similar to a prior report  of 16.1 µM (50 = 5.9 µM). The maximum level of β-arrestin 2 recruitment to C5aR2 induced by SB290157 however was higher relative to that of P32. We next compared the C5aR2 activity of SB290157 to that of human C5a , was ∼16-fold more potent than previously reported data (IC50 = 3.8 µM), determined using an intracellular calcium mobilisation assay in the same cells   , with a  1.6 nM) . Pre-tre 1.6 nM) . Pre-tre 1.6 nM) . Therefoi = 210 nM for SB290157, vs. 0.09 nM for human C3a)  . To accooff rate . This proff rate . Thus, ooff rate .β-arrestin-mediated, yet-to-be-identified mechanisms , exhibit biased agonism among different cell systems and signaling pathways  administration of any dose greater than 1 mg/kg could result in a sufficient circulatory concentration of SB290157 to target both C3aR and C5aR2 based on its calculated IC50 and EC50. Likewise, intraperitoneal (i.p.) administration of dose greater than 10 mg/kg could also lead to a circulatory concentration that ultimately targets both receptors. Hence, administering a dose greater than 10 mg/kg (i.p.) or 0.3 mg/kg (i.v.), would likely result in both agonistic activity at C5aR2 and antagonist/agonist activity at C3aR. High in vivo doses of SB290157 could therefore lead to numerous on- and off-target adverse effects such as neutropenia , in macrophages that naturally express complement-peptide receptors, SB290157 effectively and potently inhibited C3a-induced phospho-ERK1/2 signaling, without any agonistic effect. This confirms prior reports that the pharmacological C3aR agonist activity of SB290157 may be dependent on cellular receptor density . For insin vivo dose of no more than 1 mg/kg (i.p.) should be considered to minimise off-target activity at C5aR2.In conclusion, our results confirm that the widely used C3aR antagonist SB290157, exerts potent C3a agonist activity in transfected cells, and partial C5aR2 agonist activity at higher concentrations. As many studies continue to use this drug at concentration ranges that would exert activity at C5aR2, it is possible some of the reported phenotypes are due to activity at this receptor, in addition to any potential activity at C3aR. Thus, our data further caution against the use of this compound as the sole tool to delinate C3aR biology and function. Until further validated selective C3aR inhibitory tools become available eg. , we stroThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The University of Queensland Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.TW, JL, and XL conceived the project and designed the research; XL performed the research, analysed data and wrote the first paper draft. RC and JL generated key reagents for the study, and VK provided additional pharmacokinetic data analyses and insight. All authors edited the manuscript, and approved the final version.This work was supported by National Health and Medical Research Council of Australia (NHMRC) (Grant APP1118881 to TW and RC).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To achieve a highly balanced comparison of trabecular bypass stenting  with ab interno trabeculectomy  by exact matching.Fifty-three IS2 eyes were matched to 3446 T eyes. Patients were matched using exact matching by baseline intraocular pressure (IOP), the number of glaucoma medications, and glaucoma type, and using nearest neighbor matching by age. Individuals without a close match were excluded. All surgeries were combined with phacoemulsification.p < 0.05 at 1, 12, 18 months). Glaucoma medications decreased to 2.0 ± 1.5 in IS2 and to 1.5 ± 1.4 in T.A total of 78 eyes (39 in each group) could be matched as exact pairs with a baseline IOP of 18.3 ± 5.1 mmHg and glaucoma medications of 2.7 ± 1.2 in each. IOP in IS2 was reduced to 14.6 ± 4.2 mmHg at 3 months and in T to a minimum of 13.1 ± 3.2 mmHg at 1 month. In IS2, IOP began to rise again at 6 months, eventually exceeding baseline. At 24 months, IOP in IS2 was 18.8 ± 9.0 mmHg and in T 14.2 ± 3.5 mmHg. IS2 had a higher average IOP than T at all postoperative visits (T resulted in a larger and sustained IOP reduction compared with IS2 where a rebound occurred after 6 months to slightly above preoperative values. Following reports of fibrosis  and biofThe study was approved by the local ethics committee of the University of Würzburg (protocol #20191016 01). Because of its retrospective nature, informed consent was waived. The study was in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study included all patients who underwent either IS2 or T in our clinic and associated satellites between January 2008 and March 2018. The indication for surgery was a stable IOP with a desire to reduce glaucoma medications at the time of cataract surgery or an above-target IOP as determined by a glaucoma specialist, while on maximally tolerated topical treatment. To increase the chances of an exact match to the new IS2, of which fewer data exist, data from the Trabectome Study Group database , 14 was U test and chi-squared test. Using exact matching, both groups were matched using preoperative IOP, glaucoma medications, and type of glaucoma, and using Nearest Neighbor Matching for age [p values of less than 0.05 were considered statistically significant. Mean ± SD was used to express continuous variables. Statistical analyses were performed using R [Data were described as frequency, percentage, mean ± SD, median, and range. Continuous and categorical variables were compared with the Mann-Whitney  for age . Each un using R .IS2 and T were combined with phacoemulsification and lens implantation in all cases. IS2 implantation was done after cataract surgery, while T was done first and followed by cataract surgery. IS2 was implanted through a temporal clear corneal incision and with viscoelastic . SchlemmT was performed as described before . BrieflyIn both IS2 and T, postoperative treatment comprised a topical antibiotic for 1 week and a steroid tapered over 4 weeks. Glaucoma medications were stopped on the day of surgery and restarted as needed.p > 0.05). Table p = 0.04 compared with preoperative). IOP continued to slowly increase in IS2 from 6 months onward, eventually reaching the baseline average . At 24 months, IOP in IS2 was 18.8 ± 9.0 mmHg, while in T, it was 14.2 ± 3.5 mmHg. T had a lower average IOP than IS2 at all postoperative visits , increased to a slightly higher average starting at 6 months, and became similar preoperative counts by month 12 (p > 0.05). This medication increase did not prevent the IOP from rising. At 24 months, the medication count in IS2 was 2.0 ± 1.5 , and declined to 1.5 ± 1.4 at 24 months (p = 0.04, Fig. Glaucoma medications started at a matched value of 2.7 ± 1.2 in IS2 and T. In IS2, they decreased to 1.7 ± 1.4 by month three (Using a definition of success commonly applied to trabecular bypass stents, a final IOP of ≤ 21 mmHg or a 20% IOP reduction from baseline, 97% of T and 95% of IS2 achieved this goal (Fig. This study applied exact matching to detect essential differences between IS2 and T using real-world data. Studies that use simple statistics or study design can miss those , 22. ExaIS2 is a second-generation trabecular bypass stent  made of In the IS2 patients followed here, we observed a slow loss of efficacy after 6 months. By 24 months, the IOP became indistinguishable from preoperative values and appeared to trend towards exceeding it. In contrast, T had a 20% reduced IOP through the study. Medications in IS2 were increased around 6 months but not above the preoperative level. In T, medications declined throughout the study. Our experience with the IS2 resembled the IS1  except tIS2 and IS1 are safe add-on procedures that were approved in conjunction with cataract surgery , 28, andThe lowest IOP any TM bypass or ablation surgery could theoretically achieve is limited by the episcleral venous pressure of about 8 mmHg present in the episcleral venous veins . This liOur study had several limitations. It was specifically designed to detect differences between IS2 and T which cannot easily be discovered by other analysis methods. Because of this, the IOP and medication changes seen here should not readily be generalized to patients who undergo these procedures but have other baseline parameters. For instance, patients with a high preoperative IOP undergoing T typically have a much more substantial IOP reduction than those with a low preoperative IOP, as examined here , 30. AllIn summary, a highly balanced comparison of IS2 and T by exact matching showed that reduction of IOP and the number of glaucoma medications is lower and sustained in T. IOP in IS2 rebounds 6 to 24 months after surgery, eventually reaching preoperative levels.