In such films, the amounts of S and Se control the bandgap. Therefore, it is crucial to control the concentration of S/Se to improve efficiency. In this study, Cu2MnxZn1−xSnS4 (CMZTS) films were fabricated using the sol-gel method and treated in a Se environment. The films were post-annealed in a Se atmosphere at various temperature ranges from 300 °C to 550 °C at intervals of 200 °C for 15 min to obtain Cu2MnxZn1−xSn4 (CMZTSSe). The elemental properties, surface morphology, and electro-optical properties of the CMZTSSe films were investigated in detail. The bandgap of the CMZTSSe films was adjustable in the scope of 1.11–1.22 eV. The structural propeties and phase purity of the CMZTSSe films were analyzed by X-ray diffraction and Raman analysis. High-quality CMZTSSe films with large grains could be acquired by suitably changing the selenization temperature. Under the optimized selenization conditions, the efficiency of the fabricated CMZTSSe device reached 3.08%.Cu CZTSSe absorbers are made up of non-toxic and earth-abundant elements; they exhibit a high absorption coefficient of 104 cm−1 and a bandgap energy (1.0–1.5 eV) that can be obtained by adjusting the ratio of S/Se [oc) and low fill factor (FF), which largely originate from the absorber layer’s quality [Cu of S/Se . Althouggy 1.0–1. eV that  quality ,7. There quality , using t quality ,12,13, a quality ,15. Only2MnxZn1−xSn4 (CMZTSSe) with various Mn amounts using the sol-gel method in our previous work [To ameliorate the crystallinity of CZTSSe thin films, we successfully fabricated Cuous work . Howeverous work . The banous work ,18. In tous work . Adjustious work . They foous work . The eleous work . The res2 environment. The results showed that dense and smooth CMZTSSe films could be acquired after the selenization-annealing process. The effects of the selenization temperature on the crystallinity and properties of the CMZTSSe films, as well as the photoelectric performance of the corresponding solar cell, were systematically studied.Hence, in this study, high-quality CMZTSSe absorber films were fabricated by synthesizing CMZTS precursor films using the sol-gel method, accompanied by a post-annealing treatment. Annealing was performed at temperatures from 500 °C to 560 °C under a Se/N4H6CuO4·H2O), zinc chloride (ZnCl2), manganese (II) chloride (MnCl2), tin chloride dihydrate (SnCl2·2H2O), dimethyl sulfoxide (DMSO), and thiourea (CH4N2S) were purchased from Aladdin Inc. C4H6CuO4·H2O (1.1979 g), ZnCl2 (0.5394 g), MnCl2 (0.0553 g), SnCl2·2H2O (0.8462 g), and CH4N2S (2.2836 g) were dissolved in 10 mL DMSO and mixed for 2 h to obtain a transparent yellow solution. Mo-coated soda lime glass (SLG) was used as substrate; the precursor solution was spin-coated at a speed of 3000 rpm for 29 s to prepare the CMZTS films, and then it was annealed on 300 °C hot plate for 3 min under a N2 environment. The processes of spin-coating and drying were duplicated 10 times. The CMZTS precursor film was then selenized in a graphite box under a N2 flow. The selenization procedure involved the following steps: increase the temperature to 200 °C in 80 s, heat to a certain selenization temperature  in 350 s, hold the temperature for 15 min, and enable natural cooling. The process is illustrated in Copper (II) acetate monohydrate . Subsequently, using RF sputtering, an intrinsic ZnO layer with 50 nm thickness was deposited, and an Al-doped ZnO layer with 300 nm thickness was prepared. Finally, a Ag collection grid with 300 nm thickness was prepared on the top of the device by thermal evaporation.X-ray diffraction (XRD) was implemented using an X-ray diffractometer , wherein Cu Ka (λ = 0.15406 nm) was the radiation source. Raman spectra were recorded using a Renishaw system  with a 514 nm excitation wavelength. Scanning electron microscopy (SEM) was performed using a Hitachi S-4800 , which was provided with energy-dispersive X-ray spectroscopy (EDS) under 15 kV at various magnifications. The composition of the CMZTSSe was characterized by X-ray photoelectron spectroscopy (XPS)  with monochromated Al Kα radiation. An ultraviolet–visible–near-infrared (UV–vis–NIR) spectrophotometer  was used to characterize the optical properties of the CMZTSSe. A Hall-effect measurement system  was used to test the electrical performance. The current–voltage measurements under AM 1.5 G simulated sunlight illumination  was performed to characterize the electrical properties of the solar cell. An external quantum efficiency (EQE) measurement system was applied to measure the spectral response of the device .2ZnSnS4 (CZTS) with the kesterite phase, according to the International Center for Diffraction Data standards of JCPDS-26-0575 [-26-0575 ,23. No d2SnS(Se)3, making it very hard to identify the secondary phases using XRD [−1, which match the Raman characteristic vibrations of the A2, A1, and B modes of the CZTSSe phase [−1. When the selenization temperature reaches 540 °C, the peaks shift toward 172.2, 192.9, and 231.4 cm−1. As the selenization temperature further rises to 560 °C, the peaks shift to lower wavenumbers of 170.3, 190.6, and 229.4 cm−1. Therefore, as the selenization temperature increased from 500 °C to 560 °C, all the Raman peaks moved to lower wavenumbers. The inset of The XRD peaks of the CZTS(Se) films with a kesterite structure are extremely similar to those of cubic ZnS(Se) and ternary Cusing XRD . Raman sSe phase . The Ram+ lies in the CMZTSSe film [3/2 and Mn 2p1/2 with an energy gap of 8.6 eV, coinciding with the binding energy value of Mn2+ [5/2 and Sn 3d3/2, and the energy gap is 8.5 eV, which implies that the valence state of Sn is +4 Since the S 2p core level and Se 3p core level nearly overlap, in 3/2, S 2p3/2, S 2p1/2, and Se 3p1/2. The S 2p3/2 (160.2 eV) and S 2p1/2 (161.1 eV) peaks are in the representative reference standard value scope (160–164 eV), implying that S lies in S2− [3/2 and Se 3d1/2. The peak-spacing value is the same as that of standard Se2− [1+, Zn2+, Mn2+, Sn4+, S2−, and Se2−, respectively, in the CMZTSSe films.The chemical valence state of the constituent elements in the CMZTSSe films annealed at 540 °C was determined by XPS. SSe film . Figure SSe film ,30. In F of Mn2+ . Figure s in S2− . In Figuard Se2− . The XPS2, Zn, and Zn2, with the increasing selenization temperature. Thus, 540 °C can be considered the optimal selenization temperature.An appropriate selenization temperature is significant in order to gain large crystal particles and few voids in the CMZTSSe films. The SEM images of the CMZTSSe films annealed at 500, 520, 540, and 560 °C are presented in 2 for the CMZTSSe films annealed at distinct selenization temperatures  with the photon energy (hν). For the direct bandgap semiconductor, the correlation between the absorption coefficient (α) and Eg was determined by the formula:To survey the influence of the selenization temperature on the optical bandgap (Eg), the absorption spectra of the samples were acquired by a UV–vis–NIR spectrophotometer. and tail . Accordiand tail . Thus, t15 to 6.97 × 1016 cm−3, but then decreases to 7.53 × 1015 cm−3 at 560 °C. The mobility decreases from 4.35 cm−2V−1s−1 at 500 °C to 1.93 cm−2V−1s−1 at 540 °C, but increases to 3.90 cm−2V−1s−1 at 560 °C. Meanwhile, the resistivity initially decreases from 4.89×102 Ω·cm at 500 °C to 3.74 × 101 Ω·cm at 540 °C, then increases to 2.19 × 102 Ω·cm at 560 °C. This signifies that the CMZTSSe film has the best electrical properties at 540 °C. At this temperature, the carrier concentration reached its maximum value, and the resistivity attained its minimum value owing to the enhancement in the crystallinity of the film, and the decrease in the recombination of the carrier at grain boundaries, which remarkably increases the number of the carrier concentration [The electrical properties of the CMZTSSe films annealed at various temperatures  were measured using a Hall-effect apparatus, as displayed in ntration . Neverthntration .oc, Jsc, and the FF with the PCE are similar, as shown in oc, Jsc, and FF increase significantly, and then decrease from 540 °C to 560 °C. These variations in the device performance parameters may result from the change in the crystallinity of the absorber layer and the alteration in the optical bandgap induced by annealing at different selenization temperatures. 2, and an FF of 39.30% when the selenization temperature was 540 °C.In conclusion, pure-phase CMZTSSe films were acquired by the simple sol-gel method and post-annealed CMZTS precursor films. The effect of the selenization temperature on the device performance was surveyed by analyzing the change in the crystallization quality and the photoelectric properties of the CMZTSSe films. The results indicated that the Eg of CZTSSe could be changed in the range from 1.22 eV to 1.11 eV with the increase in the selenization temperature from 500 to 560 °C. All the CMZTSSe films displayed p-type electrical conductivity, and by changing the selenization temperature, films with optimal conduction properties could be achieved. When the selenization temperature was 540 °C, the CMZTSSe films acquired the preferred crystal quality as well as electrical and optical properties and acted as the absorber layer to fabricate a solar cell. Finally, the best PCE of 3.08% was achieved with a Voc of 334 mV, a Jsc of 23.46 mA/cm


Tsukamurella pumonis TP-B0596 to induce secondary metabolism by actinomycetes and have found several natural products. We also observed that MACB attached to the mycelium of Streptomyces lividans forming coaggregates during combined-culture. This stimulated interest in the interactions among actinomycetes and MACB, and we found that soil isolated cultures contained a mixture of actinomycetes and MACB. Our previously observed interactions were the result of selective screening and combination of bacteria in the lab, which warranted investigation of the existence of these interactions in the natural soil environment. Therefore, in this paper, we report the interaction between a co-isolated natural pair of actinomycetes and MACB in terms of morphology and metabolic changes. A natural pair of actinomycetes and MACB co-aggregated in liquid culture and showed metabolic changes. Interestingly, co-aggregated actinomycetes and MACB were re-isolated from soil with no obvious morphological colony differences from the colony of a single strain. The results demonstrate that there is a stochastic chance of picking colonies containing co-aggregated actinomycetes and MACB, which suggests that the pair can exist in co-aggregate form in the soil environment and interact with each other.Co-culture is an efficient strategy for natural product discovery. We have used mycolic acid-containing bacteria (MACB)  Each strain contains more than 30 putative biosynthetic gene clusters for secondary metabolites3. Although they are considered to have potential for producing more than 30 secondary metabolites, the number of detectable products in normal lab culture conditions are very limited4. Previous reports have shown that responses to environmental factors affect secondary metabolism of actinomycetes4. As many of bacterial interaction contains uninterpreted mechanism involving alteration of environmental factors, and thereby excite the response systems intricately, application of bacterial interaction will be a promising strategy for the activation of the cryptic biosynthetic genes for secondary metabolites, which were demonstrated by a number of reports.Actinomycetes are Gram-positive soil bacteria that produce pharmaceutically important natural products as secondary metabolites5. Actinomycetes respond to environmental factors including biogenic factors of other microorganisms4. We previously reported that a group of mycolic acid-containing bacteria  affected the secondary metabolism of actinomycetes in lab culture6. MACB are actinomycetes containing specific long chain fatty acids (mycolic acids) on the cell wall8. Using MACB (such as T. pulmonis TP-B0596) as a partner, Streptomyces lividans TK23 was activated to produce undecylprodigiosin (RED) and actinorhodin (ACT), which are not produced under general lab culture conditions6. Using T. pulmonis TP-B0596 as a combined-culture partner, a number of natural products have been isolated17, and this strategy was also found to be efficient to enhance production during heterologous expression20. This co-culture method using the combination of actinomycetes and MACB is named combined-culture  imaging showed that they formed co-aggregates21. Streptomyces species generally grow by forming a pellet in liquid culture22, and significant attachment of MACB cells on Streptomyces mycelium was observed21. Co-aggregation and the production of RED and ACT was not observed with addition of killed MACB 21. This suggested that co-aggregates formation through continuous physical contact might be important for the activation of secondary metabolism by actinomycetes.The activation of RED and ACT did not occur through provision of culture extracts or killed bacteria23, and these aggregates can also occur between bacteria from different environments24. Several theories have been reported to explain the interactions in interspecies aggregates for oral or sludge environments23. However, there is limited understanding of the mechanism and function of Streptomyces/MACB aggregates in the general soil bacterial community25, and it will be important to elucidate the ecological relevance or benefits of those bacterial interactions in the future.Bacteria can form interspecies aggregates in the environment21. However, these results suggested that the characteristic interactions such as forming interspecies co-aggregates and affecting each other including secondary metabolism, might be naturally occurring events in the soil environment. In this study, we report the morphology and metabolic changes of a co-isolated pair of actinomycetes and MACB from environmental soil of Hegura Island. The natural co-isolated pair formed interspecies aggregates and their interaction under combined-culture lab conditions affected the production of secondary metabolites. We performed additional experiments to re-isolate the co-aggregated cell pellets and demonstrated that mixed aggregates of actinomycetes and MACB were co-isolated with no obvious morphological change of the growing colony.The previous observation of the interactions between actinomycetes and MACB was the result of deliberate combinations of bacteria selected by screening in the lab for activation of secondary metabolismStreptomyces species , non-Streptomyces actinomycetes , MACB , and other bacteria .  in Ishikawa prefecture, Japan, is known as a stopover point for migratory wild birds in the middle of the Sea of Japan. We expected that those migratory wild birds might bring bacteria associated to their surface and stool and give rich bacterial diversity to this isolated island. We collected environmental samples from 71 points on Hegura Island in June 2008. General methods were applied for the isolation of actinomycetes  and T. pulmonis TP-B0596 (Tp) formed co-aggregates, where cells of Tp were attached to pellets of Sl. Therefore, it was predicted that the naturally co-isolated pairs of actinomycetes and MACB would have an affinity for each other and thus form similar mixed aggregates. To test this prediction, we grew the naturally co-isolated pair of actinomycetes and MACB in combined-culture and observed the morphological interaction by SEM imaging. The pairs of actinomycetes and MACB  were cultured in liquid medium, and the cultured cells were fixed for SEM imaging. From now on, we will use the letters A for actinomycetes, M for MACB, and B for other bacteria as abbreviation for strain identification.2O dilution and ISP4 agar plate  and S. variegatus HEK138A (filamentous hyphae) generated co-aggregates, where the cell clump of M. septicum HEK138M coexisted with the cell pellet of S. variegatus HEK138A mycelium , cocci-shaped actinomycetes–cocci-shaped actinomycetes (HEK654), Streptomyces–cocci-shaped actinomycetes–other bacteria (HEK652 and HEK282), and Streptomyces–Streptomyces (HEK332 and HEK423)  whereas P. marinus HEK654A2 cells were smaller (0.65 µm). Cells with both different sizes were observed in the cell pellet of the co-culture, indicating that they formed co-aggregates . We found significant differences in metabolites extracted from the culture after combined-culture of the natural pair of actinomycetes and MACB when compared with monoculture Figs.  and 5. Feus Fig.  and tranStreptomyces species and MACB, we performed a co-isolation experiment to test whether co-aggregated bacteria could be co-isolated in what looks like a single colony (see “S. variegatus HEK138A (Sv) and M. septicum HEK138M (Ms) combined-culture was compared with Sv and Bacillus subtilis (Bs) co-culture as negative control. The identity of isolates was confirmed by selective colony PCR. Sv + Ms showed significant co-isolation rate  of colonies were formed by two strains. This demonstrated that co-aggregating strains could be co-isolated under these conditions.T. pulmonis such as: streptoaminals and 5-alkyl-1,2,3,4-tetrahydroquinolines from Streptomyces nigrescens HEK61614, dracolactams from Micromonospora wenchangensis HEK79712, and streptogramins from Streptomyces albogriseolus HEK74042. Therefore we asked whether the original combination of actinomycetes and MACB affects their metabolism. As expected, we observed significant differences in the HPLC chromatograms of extracts from combined-culture when compared with monoculture of the naturally isolated pair. The extracted metabolites of HEK138A and HEK138M showed specific growth inhibition activity indicating potential antibacterial activity. Antibiotic production by the actinomycete (HEK138A) would be beneficial for the MACB (HEK138M), providing protection (by antibiotic) to secure the natural habitat without killing each other. MACB  have a degradative role in the environment, which might provide easily available nutrient sources for the actinomycete (HEK138A). There are no reports to date of the isolation of natural products from Isoptericola species (HEK904A), therefore identification of induced metabolites in combined-culture of HEK904A and M would be interesting for future analysis. Taken together, the approach of co-culturing naturally isolated bacterial co-aggregates could be an option for screening new bioactive secondary metabolites.We tested the effect of combined-culture on metabolic changes in the co-isolated pairs. In our previous work using the culture collection obtained from Hegura Island, we isolated natural products from combined-culture with 2O and divided in half for phenol or water treatments. Phenol solution (150 mg/l) was added to the one half of the suspension to kill phenol-sensitive bacteria. Subsequently the phenol-treated suspension was spread with appropriate dilution on ISP4 agar or humic acid agar with antifungal antibiotics. Ten milliliters of sterilized ddH2O was added to 1 g of environmental sample and suspended with agitation (on a shaker at 300 rpm) for 10 min. The suspended solution was further diluted by addition of 9 ml of sterilized ddH2O. In the next step, 9 ml of sterilized ddH2O or phenol suspension (1 g/140 ml) was added to the 1 ml of diluted suspension and mixed for 10 min (on a shaker at 300 rpm). The bacteria were grown on ISP4 medium or humic acid medium at 30 °C for 7–10 days. ISP4 agar medium contained soluble starch (1.0%), (NH4)2SO4 (0.2%), CaCO3 (0.2%), K2HPO4 (0.1%), MgSO4⋅7H2O (0.1%), NaCl (0.1%), and trace salt solution (0.1% v/v) containing FeSO4⋅7H2O (0.1%), MnCl2⋅4H2O (0.1%), ZnSO4⋅7H2O (0.1%), and agar (2.0%) in distilled water (pH 7.2). Humic acid agar medium contained humic acid (0.1%), Na2HPO4 (0.05%), KCl (0.171%), MgSO4⋅7H2O (0.005%), FeSO4⋅7H2O (0.001%), CaCO3 (0.001%), B-vitamins (0.0005% of each except for 0.0025% biotin), in distilled water (pH 7.2). To prevent fungal growth, amphotericin B  and cycloheximide  were dissolved in dimethyl sulfoxide (DMSO) and added to the agar medium after autoclaving.We collected 71 environmental samples from Hegura Island in June 2008 . Universal primers (16S-F:5ʹ-GAGAAGCTTAGAGTTTGATCCTGGCTCAG-3ʹ and 16S-R5ʹ-GAGAAGCTTAC-GGCTACCTTGTTACGACT-3ʹ) and GoTaq Green Master Mix (Promega Co.) were used for PCR by following the manufacturers protocol44. Sanger sequencing was performed at Eurofins Genomics Co. using the same primer sets. The partial 16S rRNA gene sequence data obtained was compared by nblast to identify the most similar strains in the National Center for Biotechnology Information (NCBI) database. We named the bacterial species in this study based on the closest identified relatives of the 16S rRNA gene sequences (if above 98%). Sequences of the 16S rRNA genes are registered in accession number of LC656369, LC656370, LC656371, LC656372, LC656373, LC656374, LC656375, LC656376, LC656377, LC656378, LC656379, LC656380, LC656381, LC656382, LC656383, LC656384, LC656385, LC656386, LC656387, LC656388, LC656389, LC656390, LC656391, LC656392.Genomic DNA of the isolated bacteria was extracted and purified using the cetyltrimethylammonium bromide methodEscherichia coli K12 was used as outgroup, and manually deleted from the figure.Partial 16S rRNA gene sequences were obtained from 182 culture stocks and used to construct a phylogenetic tree. 182 culture stocks were selected at random. ClustalW was used for the sequence alignment and the maximum likelihood method was used to generate the phylogenetic tree using MEGA X software. The phylogenetic analysis was tested using bootstrap values based on 1000 replications, and recommended parameters: the General Time Reversible model and Gamma Distributed with Invariant Site (discrete Gamma categories = 5). The 16S rRNA gene sequence from 43  or V-22 medium  as seed culture. ISP2 medium contained glucose (0.4%), malt extract , and yeast extract  in distilled water (pH 7.2) and V-22 medium contained soluble starch (1.0%), glucose (0.5%), NZ-case (0.3%), yeast extract (0.2%), Bacto tryptone (0.5%), K2HPO4 (0.1%), Mg⋅7H2O (0.05%), and CaCO3 (0.3%) in distilled water (pH 7.0). The test tubes were shaken on a rotary shaker (180 rpm) at 30 °C for 2 days. Broth containing the actinomycete (1 ml) and the MACB (1 ml) were transferred simultaneously into 100 ml flasks containing 20 ml of YGGS medium (described below) for morphological observation and cultured on a rotary shaker (180 rpm) at 30 °C for 5 days. For the metabolite analysis, 1 ml of actinomycetes broth and 1 ml of MACB broth were transferred simultaneously into 500 ml K−1 flasks containing 100 ml of A-3 M production medium and cultured on a rotary shaker (200 rpm) at 30 °C for 7 days. A-3 M medium contained glucose (0.5%), soluble starch (2.0%), glycerol (2.0% w/v), pharmamedia , and yeast extract  in distilled water (pH 7.2). YGGS was A-3 M medium without pharmamedia.Bacteria were grown on ISP2 agar plates for 3–5 days. Each isolated actinomycete and MACB were cultivated separately in test tubes containing 10 ml of ISP2 mediumB. subtilis and S. lividans TK23 as control. We did not observe the aggregated pellet of S. lividans TK23 with B. subtilis by method using centrifugation. (data not shown) Then, 1% OsO4 in 100 mM sodium phosphate buffer (pH 7.4) was added and incubated for 1 h on ice. After discarding the solution, 1 ml of 0.1 M sodium phosphate buffer (pH 7.4) was added to rinse the sample. Subsequently, the sample was dehydrated by stepwise addition of 50%, 70%, 90%, and 100% ethanol (1 ml each), and finally substituted by t-butyl alcohol, and stored at 4 °C until use. The sample was dried under vacuum before Pt/Pd sputter coating  and observation by SEM . Stereomicroscope images of bacterial colonies formed on agar plates were obtained by Nikon SMZ25 equipped with a SHR Plan Apo 1.6 × lens (Nikon) and DS-Ri2 digital camera (Nikon).Samples for SEM were prepared as follows. One milliliter of liquid culture was collected into a 1.5 ml tube. After centrifugation  and removal of the supernatant, 1 ml of 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) was added to the cell pellets and incubated for 1 h at 4 °C. After discarding the solution, 1 ml of 0.1 M sodium phosphate buffer (pH 7.4) was added to rinse the sample. Prior to establishing the current method using a centrifuge, we tested isolates aggregation by using combination of S. lividans (Sl), T. pulmonis (Tp), HEK138A, HEK138M, and B. subtilis (Bs) were inoculated into 10 ml of ISP2 medium and precultured for 2 days at 180 rpm and 30 °C. The following ratios of aliquot from preculture were used to inoculate 20 ml of YGGS medium in a 100 ml Erlenmeyer flask for co-culture . After culture for 5–6 days , 1 ml of culture was used for further analysis. First, the whole culture broth was passed through a stainless-steel sieve  to trap the cell pellets. Then 500 ml of PBS buffer was used to thoroughly wash the bacterial pellets that were trapped in the sieve. Subsequently, the bacterial pellets were recovered from the sieve and the washing step was repeated. After the washing step, the bacterial pellets were transferred to the center of an ISP4 agar plate and further incubated for 7 days. The growing colony images were obtained by stereomicroscope and the primers  were used to amplify specific regions of the 16S rRNA gene by PCR using GoTaq Green master mix (Promega). Agarose gel (1.5% w/v) was used for DNA electrophoresis.2O was added to wash the cell pellets. This washing step was repeated twice. Then 1 g of autoclaved soil (obtained from Yayoi campus) was added to the collected cell pellets. After addition of soil, the process for bacterial isolation was repeated as described above. .HEK138A or HEK138M were inoculated in 10 ml ISP2 medium and precultured for 2 days . The following ratio of aliquot from preculture was used to inoculate 20 ml of YGGS medium in a 100 ml Erlenmeyer flask for combined-culture (HEK138A : HEK138M = 1 ml : 300 µl). After culture for 5 days , the whole culture broth was transferred to a conical tube (50 ml) and left to stand for 2 min to allow pellet sedimentation. The upper layer was discarded and 20 ml of ddHS. lividans, Tp: T. pulmonis) Sl-Fw: 5ʹ-GAACCACTTCGGTGGGGA-3ʹ, Sl-Rv: 5ʹ-CCAACACCCCGAAGGGCT-3ʹ, Tp-Fw: 5ʹ-TTCCCCTGCATGGGGGTT-3ʹ, Tp-Rv 5ʹ-AGAAGGCACAAGACAAACCGA-3ʹ. For Sl and Bs co-culture, the following primers were used to specifically amplify the partial 16S rRNA gene region. (Bs: B. subtilis) Sl-Fw: 5ʹ-CAGGCATCTGCGAGGTTC-3ʹ, Sl-Rv: 5ʹ-CACGGACAACGTGGAATGTTG-3ʹ, BsFw: 5ʹ-CAAGTGCCGTTCAAATAGG-3ʹ, Bs-Rv: 5ʹ-AGGGGCGGAAACCCCCTA-3ʹ. For Sv and Ms combined-culture, the following primers were used to specifically amplify the partial 16S rRNA gene region.  Sv-Fw: 5ʹ-GGGCAACATTCCACGTTG-3ʹ, Sv-Rv: 5ʹ-CATCACCCCGAAGGGCAT-3ʹ, Ms-Fw: 5ʹ-CGTTGTTCGTGAAAACTCACA-3ʹ, Ms-Rv: 5ʹ-GAACCAATATCTCTACTGGCG-3ʹ.For Sl and Tp combined-culture, the following primers were used to specifically amplify the partial 16S rRNA gene regions. (Sl: in vacuo. Dried samples were dissolved in 1.2 ml of DMSO (to concentrate the solution tenfold), placed on a 96 well plate (Agilent Technologies), and then covered with a silicone closing mat (Agilent Tech.). Metabolites were analyzed by PDA-HPLC . Four microliters of sample was applied for each data acquisition. The HPLC system was equipped with a Cosmosil 5C18-AR-II column . Acetonitrile and H2O (MilliQ) containing 0.1% formic acid was used as mobile phase. Acetonitrile at 5% was the mobile phase for the first 5 min, and then increased to 95% by linear gradient from 5 to 25 min, and then maintained at 95% until 30 min. The column temperature were kept at 40 °C and the flow was 1.0 ml/min.Fifteen milliliters of culture broth was collected and an equal volume of butanol was added to extract the metabolites. After centrifugation, 12 ml of the butanol layer was collected and dried Bacillus subtilis ATCC6633, Staphylococcus aureus 209PJC-1, Micrococcus luteus ATCC9341, and Escherichia coli NIHJ-2 were inoculated in 10 ml Mueller Hinton (MH) medium (BD Biosciences) and precultured for 1 day . Preculture (0.6 ml) was added to 6 ml of soft agar (1.6% nutrient broth (BD Biosciences), 0.5% agar). The solution was applied as an overlay to an agar plate  containing 15 ml of MH agar. Twenty microliters of samples dissolved in DMSO (extracts from 0.2 ml culture) were transferred to paper discs  and placed on an agar plate. After incubation at 30 °C for 1 day, the zones of growth inhibition were measured. The experiment was performed twice for the biological replicate. Gentamicin (5 µg) was used as positive control and DMSO (20 µL) was used as negative control.Supplementary Information.


Medulloblastoma (MB) is the most common malignant brain tumor in children. Approximately one-third of MB patients remain incurable. Understanding the molecular mechanism of MB tumorigenesis is, therefore, critical for developing specific and effective treatment strategies. Our previous work demonstrated that astrocytes constitute the tumor microenvironment (TME) of MB and play an indispensable role in MB progression. However, the underlying mechanisms by which astrocytes are regulated and activated to promote MB remain elusive.Math1-Cre/Ptch1loxp/loxp mice, which spontaneously develop MB, primary MB cells and astrocytes were isolated and then subjected to administration and coculture in vitro. Immunohistochemistry was utilized to determine the presence of C3a in MB sections. MB cell proliferation was evaluated by immunofluorescent staining. GFAP and cytokine expression levels in C3a-stimulated astrocytes were assessed by immunofluorescent staining, western blotting, q-PCR and ELISA. C3a receptor and TNF-α receptor expression was determined by PCR and immunofluorescent staining. p38 MAPK pathway activation was detected by western blotting. Transplanted MB mice were treated with a C3a receptor antagonist or TNF-α receptor antagonist to investigate their role in MB progression in vivo.By taking advantage of We found that complement C3a, a fragment released from intact complement C3 following complement activation, was enriched in both human and murine MB tumor tissue, and its receptor was highly expressed on tumor-associated astrocytes (TAAs). We demonstrated that C3a activated astrocytes and promoted MB cell proliferation via the p38 MAPK pathway. Moreover, we discovered that C3a upregulated the production of proinflammatory cytokines, such as IL-6 and TNF-α in astrocytes. Application of the conditioned medium of C3a-stimulated astrocytes promoted MB cell proliferation, which was abolished by preincubation with a TNF-α receptor antagonist, indicating a TNF-α-dependent event. Indeed, we further demonstrated that administration of a selective C3a receptor or TNF-α receptor antagonist to mice subcutaneously transplanted with MB suppressed tumor progression in vivo.C3a was released during MB development. C3a triggered astrocyte activation and TNF-α production via the p38 pathway, which promoted MB cell proliferation. Our findings revealed the novel role of C3a-mediated TNF-α production by astrocytes in MB progression. These findings imply that targeting C3a and TNF-α may represent a potential novel therapeutic approach for human MB.The online version contains supplementary material available at 10.1186/s12974-022-02516-9. Medulloblastoma (MB), the most common malignant brain tumor in children, accounts for approximately 30% of brain tumors in children and has high mortality and poor prognosis , 2. MB iAstrocytes are the most abundant glial cells in mammalian brains and express specific markers, such as S100β  and gliaComplement C3a is the product of complement system activation. The complement system, the critical component of the innate immunity, constitutes the first line of body defense. The complement system is composed of more than 30 proteins and can be activated through 3 different pathways: the classical, alternative, and lectin pathways. All 3 pathways merge at complement 3 (C3). Accompanied by complement activation, the C3 protein is cleaved to release the peptide C3a , 32. C3aC57BL/6 mice and CB17/SCID mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Math1-Cre/Ptch1loxp/loxp mice and eGFP mice were bred in our laboratory. Math1-Cre/Ptch1loxp/loxp mice were generated by Dr. Yang as described previously [Ptch1 is conditionally deleted in Math1-positive cells, which are MB original cells, granule neuron precursors (GNPs). These mice develop MB spontaneously starting from 6 to 8 weeks. Homozygous Math1-Cre/Ptch1loxp/loxp mice were screened from heterozygous breeding pairs for the MB tumor model, and Math1-Cre/Ptch1wt/wt littermates were used as controls. Math1-Cre/Ptch1loxp/loxp mice were cross-mated with eGFP mice to obtain eGFP-positive MB cells. All mice were housed in a specific pathogen-free facility at School of Pharmaceutical Sciences, Soochow University.Wild-type (WT) eviously . In thesMath1-Cre/Ptch1loxp/loxp or Math1-Cre/Ptch1loxp/loxp /eGFP mice were sacrificed, and their heads were exposed. Tumor tissues were removed from the cerebella and digested in papain buffer consisting of 10 U/mL papain (Worthington), 250 U/mL DNase (Sigma) and 200 µg/mL l-cysteine (Sigma) for 30 min at 37 °C to acquire a single-cell suspension. These cells were used to generate a subcutaneous transplantation MB model. To obtain purified MB cells, the single-cell suspension was then separated through 35–65% Percoll medium (Sigma). MB cells were located in the 65% concentration layer. Tumor cells were suspended in NB-B27 culture medium  and seeded on poly-d-lysine-coated coverslips placed in 24-well plates.As described previously , for priTo isolate tumor-associated astrocytes (TAAs), a single-cell suspension of MB tissue was obtained as above. The cells were stained with APC-conjugated anti-ACSA-2 antibody , and then TAAs were sorted and collected by FACS  according to APC positive staining. For primary astrocyte culture, the cerebella of WT mice at postnatal day 2 (P2) were dissected and digested as described above to acquire a single-cell suspension, and cells were cultured in DMEM/F12 medium (HyClone) containing 10% fetal bovine serum  and 1% penicillin/streptomycin. When the cells grew to 80% confluence, they were digested with 0.25% trypsin–EDTA (Beyotime) for passage. Cells were generally passed to two to three generations for experiments.Recombinant mouse C3a, TNF-α and IL-6 were purchased from Novoprotein. In addition, recombinant mouse C3a from R&D Systems was used in some of the experiments as indicated. C3a was used for primary astrocyte stimulation at the indicated concentrations; TNF-α and IL-6 were administered to MB cells at lower (200 ng/mL) and higher (500 ng/mL) concentrations, and the data shown in the results are representative of the 500 ng/mL treatment groups. C3aR antagonist (SB290157), p38 inhibitor (SB203580) and TNF-α receptor antagonist (R-7050) were all purchased from ApexBio. SB290157 and SB203580 were used at 2 μM for astrocyte administration, and R-7050 was used at 500 nM for MB cell administration in vitro.Primary astrocytes and purified MB cells were mixed at a 1:10 ratio and cocultured in MB medium NB-B27 for 48 h. In certain experiments, astrocytes were pretreated with C3a or both C3a and p38 inhibitor for 24 h, and then the cells were harvested and washed with PBS for 3 times before being cocultured with MB cells. To collect astrocyte-conditioned medium, astrocytes were cultured in DMEM/F12 medium with C3a or vehicle as a control for 24 h, and the supernatant was removed before the addition of fresh medium. Astrocytes were cultured for another 24 h for conditioned medium collection. MB cells were then cultured in astrocyte-conditioned medium mixed with NB-B27 at a 1:1 ratio for 48 h.CB17/SCID mice were subcutaneously injected with 2 × 106 MB cells per mouse in the hind flanks. Tumor volume was measured and calculated as (W2 × L)/2 . When the tumor volume reached 200 mm3, the mice were randomly grouped and received treatments. For both in vivo treatment experiments, the C3aR antagonist SB290157 and the TNF-αR antagonist R-7050 were dissolved in 3% DMSO in MCT . MB tumor-bearing mice were injected intraperitoneally with SB290157 (30 mg/kg body weight), R-7050 (30 mg/kg body weight) or vehicle control once a day for 8 days at designated sites. Tumor volume was measured daily. Tumors were harvested at the end of treatment and underwent sectioning and RNA or protein sample preparation.Four-to-six-week-old male Human MB paraffin sections were provided by Dr. Liu from Sanbo Brain Hospital Capital University and obtained from Children's Hospital of Soochow University, respectively. The use of the sections was reviewed and approved by the Ethics Committee of Soochow University. For immunohistochemistry, MB-bearing and WT normal mice were perfused, and cerebella and subcutaneous allografts were dissected and fixed in 4% paraformaldehyde  for 24 h at 4 °C, dehydrated in 30% sucrose and embedded in paraffin, followed by sectioning at 6 μm thickness. MB paraffin sections were sent to Wuhan Servicebio Technology for immunohistochemistry staining of C3 and C3a. The antibodies used for detection were anti-human/mouse C3 polyclonal antibody , anti-human C3a monoclonal antibody, clone 2991 , anti-mouse C3a monoclonal antibody, and clone I87-1162 . Hematoxylin–eosin (H&E) staining was performed to compare the histological properties between WT normal cerebellum, primary MB and subcutaneous tumors following the standard protocol.For immunofluorescent staining, tissues were harvested as above, frozen in Tissue Tek-OCT (Thermo Fisher Scientific), and then sectioned at 10–12 μm thickness. Sections were blocked with PBS containing 0.2% Triton X-100 (Biosharp) and 1% bovine serum albumin  for 1 h at room temperature. Then, the sections were incubated with primary antibody overnight at 4 °C. The next day, sections were exposed to secondary antibody conjugated with the appropriate fluorescein for 2 h at room temperature. DAPI (Beyotime) staining was performed for 10 min. Immunofluorescent staining for cells followed the same protocol. Before staining, cells were fixed in 4% PFA for 15 min and permeabilized with PBS containing 0.2% Triton X-100 for 10 min at room temperature. Finally, slides were mounted with Fluoromount-G (Southern Biotechnology) and visualized by a Nikon microscope. All antibodies were diluted in PBS containing 0.2% Triton X-100 and 1% BSA. The primary antibodies and dilution ratios were as follows: anti-C3aR , anti-Iba-1 , anti-Ki67 , anti-cleaved caspase-3 , and anti-GFAP . The secondary antibodies and dilution ratios were as follows: goat anti-rabbit-Alexa Fluor 594 , goat anti-mouse-Alexa Fluor 488 , and goat anti-rabbit-Alexa Fluor 488 .Cell lysate or MB tissue homogenate was prepared in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktail (Roche), and the total protein concentration was evaluated using a BCA Protein Assay Kit (Beyotime). Then, equal amounts of protein samples were loaded and separated by 10% SDS–PAGE (Beyotime) and transferred onto PVDF membranes (Millipore). The membranes were blocked with TBS containing 0.05% Tween-20 and 5% defatted milk powder and incubated overnight at 4 °C with primary antibodies against GFAP , GAPDH , total p38 , phosphorylated p38 , β-Tubulin , phosphorylated Erk1/2  and total Erk . The following day, the membranes were incubated with the HRP (horseradish peroxidase)-conjugated secondary antibodies anti-mouse IgG  and anti-rabbit IgG  at room temperature for 2 h. Bands were visualized using high-signal ECL substrate (Beyotime) and exposed on a ChemiScope 3300 mini (Clinx).2SO4 10 min later. The optical density (OD) in each well was read with a microplate reader at 450 nm. The C3a level was calculated according to the standard curve. TNF-α was determined utilizing a mouse TNF-α ELISA Kit (Fcmacs) in accordance with the manufacturer’s instructions.Tissue homogenate and cultured cell conditioned medium were centrifuged, and the supernatant was collected to perform ELISA. The total protein concentrations of the samples were detected with a BCA Protein Assay Kit (Beyotime) and adjusted to be equal in each group. To detect C3a, a 96-well plate was precoated with purified anti-mouse C3a monoclonal antibody  overnight at 4 °C. The plate was washed with PBST (PBS containing 0.05% Tween-20) and blocked with 1% BSA in PBST. Samples and standards  were added to the wells and incubated for 1 h at room temperature. Biotinylated anti-mouse C3a antibody  was later used to bind to C3a antigen for 1 h at room temperature. Then, streptavidin–HRP  was added and incubated for 30 min. Finally, the substrate TMB (Beyotime) was added for color development, and the reaction was stopped by 1 M H−ΔΔCt method. The primers  used in the experiments included: GAPDH ; C3 (forward: 5-CGCAACGAACAGGTGGAGATCA-3); reverse: 5-CTGGAAGTAGCGATTCTTGGCG-3); C3AR1 ; GFAP  IL-6 ; TNF-α ; and TNFRSF1A .Total RNA was extracted from cell lysate or tissue homogenate using TRIzol reagent (Sigma-Aldrich) in RNase-free conditions, and the purity of RNA was detected with a Nanodrop 2000 spectrophotometer (Thermo). cDNA was synthesized using oligo (dT) and Superscript II reverse transcriptase (Invitrogen). Conventional PCR was performed following a standard protocol, and PCR products were visualized in a 1.5% agarose gel. Quantitative PCRs were performed in triplicate using SYBR qPCR Master Mix (Vazyme Biotech) and the ABI 7500 TaqMan Real-Time PCR Detection System. The differences in mRNA expression were calculated by the 2https://hgserver1.amc.nl/cgi-bin/r2/main.cgi).Relevant patient data were acquired via the R2 database , while P < 0.01 was considered to be a very significant difference. Data are expressed as the mean ± SEM.Experimental data were analyzed using GraphPad Prism software. Student’s Math1-Cre/Ptch1loxp/loxp mice) was utilized for investigation. In these transgenic mice, the Hh ligand antagonistic receptor Patched 1 (Ptch1) is conditionally knocked out in Math1-positive cells. Math1 is the cell marker of granule neuron precursors (GNPs), which have been approved to be the Hh-type MB origin [Math1-Cre/Ptch1loxp/loxp mice develop MB spontaneously starting from 6 to 8 weeks with a 100% incidence rate. MB tumor tissues derived from Math1-Cre/Ptch1loxp/loxp mice and normal cerebella of WT littermate controls were collected and sectioned for C3 and C3a protein detection by IHC. Similar to human patient samples, both intact C3 and C3a were distributed intensively in tumor tissue, whereas they were barely detected in the normal cerebella control  were counted. As shown in Fig. C3a–C3aR is universally accepted to fulfill bioactivities mediated by the MAPK pathway , 43. To ion Fig. : Fig. S4ion Fig. : Fig. S5Next, we explored the mechanism by which C3a-activated astrocytes regulate MB tumor cell proliferation. Our previous work demonstrated that TAAs could produce Hh ligands to maintain Hh signaling in MB cells via the noncanonical Hh pathway ; therefoFurthermore, we investigated whether TNF-α production induced by C3a could contribute to MB growth in vivo. We first compared TNF-α mRNA expression in normal primary astrocytes and TAAs, it was found that TAAs expressed more TNF-α Fig. a. We theCurrent therapies for MB are far from optimal. Patients always suffer from side effects caused by surgery combined with radio- and chemotherapy. Targeted drugs have been developed in the past decade; however, some drugs still show defects. For example, Hh signaling pathway inhibitors targeting SMO, vismodegib and sonidegib, have been reported to impact skeletal growth in pediatric patients, since Hh signaling is also required for bone development , 46, 47.GFAP-cre and C3aRloxp/loxp mice, and then crossmate these mice with MB mice. Unfortunately, our MB model is based on Math1-cre/Ptch1 loxp/loxp, which makes it infeasible to further manipulate mouse breeding.Here, we described that C3, the central factor of the complement system, was activated and released C3a in MB, and consistent with other publications, C3a activated astrocytes in vitro: C3a upregulated the expression of the astrocyte activation marker GFAP and some proinflammatory cytokines; C3a stimulated p38 MAPK pathway signal transduction in astrocytes; and C3a enhanced the tumor-promoting ability of astrocytes. Furthermore, C3aR antagonist treatment successfully suppressed astrocyte activation and MB progression in vivo, indicating that C3a contributes to MB development by activating astrocytes. We cannot exclude the involvement of other cells in the MB TME, such as microglia/macrophages, given that microglia/macrophages in the brain express C3aR , 53, andThe function of C3a in medulloblastoma has seldom been reported. Maurer et al. found that knockdown of C3aR in Daoy cells, a human MB cell line, by siRNA reduced Daoy cell proliferation in vitro . InconsiThen, in the current study, we further investigated the mechanisms of astrocyte activation by C3a and their promotion of MB cell proliferation. We found that C3a triggered p38 MAPK pathway activation and TNF-α production in astrocytes; blockage of the p38 pathway in astrocytes or the TNF-αR pathway in MB cells significantly decreased MB cell proliferation in the coculture system, indicating that astrocytes activated by C3a promote MB cell proliferation by secreting TNF-α. TNF-α and other proinflammatory cytokines have been proven to be expressed based on C3a–C3aR signal transduction, and TNF-α is reported to have controversial roles in cancers; several studies have shown that it has protumor functions , 45. HerHh-type MBs are driven by aberrant activation of Hh signaling, and our previous work demonstrated that astrocytes provide Hh ligands to maintain Hh signaling in MB cells. However, in the current study, we did not find that Hh ligand mRNA was remarkably upregulated in astrocytes after C3a stimulation. Meanwhile, neither C3aR nor TNF-αR antagonist treatment in vivo impacted Hh signaling pathway target gene expression in MB tumors (data not shown). These data suggest that C3a-triggered astrocyte TNF-α secretion dose not promote MB tumor growth by targetting Hh signaling. It seems that there is no crosstalk between the C3aR or TNF-αR and Hh signaling pathways in our model. Further efforts are needed to address these questions.Collectively, our studies demonstrate that complement is activated in MB development, releasing C3a, which then induces TNF-α production in astrocytes via the p38 pathway, and TNF-α in turn promotes MB progression Fig. . For theAdditional file 1: Fig. S1. C3a is present in the tumor tissue of MB patients and C3aR is present on both astrocytes and microglia. a, IHC staining of C3a on human MB sections derived from 4 patients, respectively (P#2–P#5). b, Representative immunostaining of C3aR (in red) and GFAP (in green) or Iba-1(in green) on human MB sections. Fig. S2. C3aR antagonist SB290157 inhibits GFAP expression by C3a-administrated astrocytes. Primary astrocytes were stimulated with C3a (100 nM) with or without SB290157 (2 μΜ) for 48 h in vitro. Immunostaining of GFAP (green) was performed (a), and the percentage of GFAP + cells was quantified (b). Fig. S3. C3a does not activate the Erk pathway in astrocytes. Primary astrocytes were stimulated with 100 nM C3a for 2, 5, 10 and 20 min. Then, the cells were harvested and lysed for total Erk and phosphorylated Erk (p-Erk) detection by western blotting. β-Tubulin served as a protein sample loading control. Fig. S4. Phosphorylation of p38 in C3a-administrated astrocytes is inhibited by C3aR antagonist SB290157. a, Primary astrocytes were stimulated with C3a (100 nM) with or without addition of SB290157 (2 μΜ) for 4 h in vitro. Then, the cells were harvested and lysed for total p38 and phosphorylated p38 (p–p38) detection by western blotting. Fig. S5. GFAP and TNF-α expression and the phosphorylation of p38 in C3a-administrated astrocytes is inhibited by C3aR antagonist SB290157. Primary astrocytes were stimulated with C3a  in the presence or absence of SB290157 (2 μΜ) for 48 h (a and b), 4 h (c) and 12 h (d), respectively, in vitro. a, Immunostaining of GFAP (in green) was performed, and b, the percentage of GFAP + cells was quantified. c, the cells were harvested and lysed for total p38 and phosphorylated p38 (p–p38) detection by western blotting. d, the cells were harvested to perform qPCR for evaluating TNF-α mRNA levels. ***p < 0.001 vs. C3a (−) & SB290157 (−) group, ##p < 0.01 and ###p < 0.001 vs. C3a ( +) & SB2901570 (−) group. Fig. S6. C3aR antagonist treatment inhibits the p38 MAPK pathway activation in tumor tissue of subcutaneous MB mice. Tumor tissue of C3aR antagonist (SB290157)- and vehicle (MCT)-treated subcutaneous MB mice was harvested at the termination of treatment, and tumor tissue was homogenized to evaluate phosphorylated p38 (p–p38) and total p38 levels by western blotting. β-Tubulin served as a protein sample loading control. Fig. S7. TNF-α receptor is expressed on MB cells. Primary MB cells (pMB), TAAs and BV2 cells were harvested and lysed for RT–PCR assays to detect TNF-α receptor (TNFSF1A) expression. GAPDH served as an internal control.


Porous organic cages have shape persistent cavities which provide a suitable platform for encapsulation of guest molecules with size suitably fitting to the cavity. The interactions of the guest molecule with the porous organic cage significantly alter the properties of the guest molecule. Herein, we report the effect of encapsulation on the kinetics of various organic transformations including 2 + 4 cycloaddition, 1,5-sigmatropic, 6π-electrocyclization, ring expansion, cheletropic, dyotropic, trimerization and tautomerization reactions. Non-bonding interactions are generated between the CC2 cage and encapsulated species. However, the number and nature/strength of interactions are different for reactant and TS with the CC2 cage and this difference detects the reaction to be accelerated or slowed down. A significant drop in the barrier of reactions is observed for reactions involving strong interactions of the transition state within the cage. However, for some reactions such as the Claisen rearrangement, reactants are stabilized more than the transition state and therefore an increase in activation barrier is observed. Furthermore, non-covalent analyses of all transition states (inside the cage) confirm the interaction between the CC2 cage and substrate. The current study will promote further exploration of the potential of other porous structures for similar applications. Porous organic cages have shape persistent cavities which provide a suitable platform for encapsulation of guest molecules with size suitably fitting to the cavity. Besides MOFs, zeolites are also porous materials with many similar applications. In 2009, Karapınar, N. reported some natural zeolites as a good choice for the removal6 of phosphorus and ammonium through aqueous solution. Later on Mingyu Li et al.7 published work on the removal of ammonia from edible or drinking water through modified zeolites. Catalysis,8 food production, agriculture,9 adsorption10 and separation11,12 are the most reported applications of zeolites.A rapid increase in the applications of porous materials in various applications has been witnessed recently because of their unique structural features such as porosity, persistent shapes and large surface area. Metal organic frameworks (MOFs) are typical examples of porous materials which find application in the field of catalysis,13 are one of the commodious and latest class of porous compounds with single and multiple cavities. Shape persistency and covalently bonded framework14 of porous organic cages make suitable substrate for guest molecules. POCs bear more than one opening door through which analytes can enter to the cavity. Sensing ability of certain porous compounds depends upon their structural skeleton. Porous organic cages (POCs) are one of highly potent affinity15 compounds which are used as a sensing object in quartz crystal microbalances (QCMs). For the adsorption of sulfur hexafluoride (SF6),16 porous organic cages are reported as superior platform than metal organic framework and fullerene. Similarly, Chen et al. successfully separated the rare gases and chiral compounds17 with the help of porous organic cages, which is also a challenging task ever. In 2015, CC10 was reported for separation of enantiomers.18Over the last few decades, several porous materials have been synthesized including covalent organic frameworks. Porous organic cages (POCs)19 such as strontium and cesium ions. These confinements have greater effects in separation, adsorptions catalysis and kinetic controls.20 Selectivity has been shown by the cage system in some common industrial gases such as H2/N2,21 CO2/CH4,22 SF6/N2,23 CO2/N2.24,25 Large sized porous organic cages have been investigated for the separation of large organic molecules such as organic molecules and noble gases. Recently, In 2019, another chiral porous organic cage CC3-R was introduced with potentiometric sensing properties26 of chiral alcohol amines (S-2-amino-1-butanol and R-2-amino-1-butanol).The porous organic cages are also reported for the adsorption of radioactive species27 coated with molecular cage have shown very good selectivity for chiral resolution and separation of a number of structural isomers, such as aromatic hydrocarbons, n-alcohols and n-alkanes. Specific adsorption properties can be incorporated into porous compounds, such as, water tolerance. Stable non-covalent organic frameworks which are based on electron rich pyrazoles have shown very good adsorption properties for ozone-depleting substances (fluorocarbons and CFCs) and hydrocarbons.28 Some porous organic cages with high electron delocalization in their π-conjugated system along with rigid structure are fluorescent and these compounds have been used for chiral29 fluorescent sensing. In 2018 Zhenyu Lu and his co-workers have designed a new modified chiral porous organic cage with sensing abilities. The CD@RCC3 POC for 349 fluorescent sensing possesses a stable and strong fluorescent property. CD@RCC3 has been reported as fluorescence chemosensor30 for the sensing of phenyl alaninol, phenyl ethanol enantiomers and nitrophenol isomers.POCs have been reported multiple times for their application in gas chromatography columns. The capillary columns31 Isolation of nanoparticles also reduced the aggregation between nanoparticles. The obtained Rh/CC3-R-homo  exhibits very significant catalytic performance towards different liquid phase catalytic reactions (e.g. methanolysis of ammonia borane), while Rh/CC3-R-hetero32 did not show such efficiency. The recyclability and durability of Rh/CC3-R-homo is also excellent. Porous organic cage with well-dispersed embedded nanoparticles (NPs)33 have efficient catalytic application in cyanation, of aryl halide under additive free and heterogeneous conditions.34 Porphyrin-based POCs has been reported by Kim and Chang for electrochemical reduction of CO2.35,36 The encapsulation of metal nanoclusters with high catalytic activity inside POCs is elucidated by Xu et al.37 POCs (CC3-R cage) are reported to exhibit remarkably improved catalytic activities towards the selective hydrolysis of ammonia borane. Dong et al. further reported that CC3-R cage exhibits higher selectivity and conversion under normal conditions for the tandem hydrogenation of quinoline and nitroarenes in water.38–40 Recently, Wang et al. reported triphenylphosphine-based quasi-POCs as remarkable organic cage system to catalyze hydroformylation reactions with more than 97% aldehyde selectivity.41 Furthermore, the performance of Pd doped RCC3 cage toward semi-hydrogenation of alkyne and hydrogenation of 4-nitrophenol compounds was demonstrated both experimentally and theoretically by Kou et al.42 Covalent organic cages (COCs) are also reported with high catalytic efficiency for hydrogen evolution reaction (HER).43Covalently bonded crystal structure of porous organic cages makes them feasible for catalysis applications. Cage immobilized catalyst have the potential to solubilize the heterogeneous catalytic particles. The metal nanoparticles confinement in the cavity of cage also raised the possibilities of size selective catalysis by the control of guest accessing through the window of cage. CCR-3 cage has been used to support rhodium (Rh) nanoparticles with reasonable size.44 In 2002 Halls et al. theoretically investigated activation energy and enthalpies for Menshutkin SN2 reaction45 inside CNTs and achieved a significant reduction in the reaction barrier. Decomposition of chloromethanol and dichloromethanol46 was carried out inside the carbon nanotube of different diameters, where the CNT with smaller diameter resulted in lowest activation barrier. Thermodynamic and kinetic study of Diels Alder reactant inside CNTs47 was investigated theoretically; whereas minimum energy of activation was obtained in case of SWCNT. Recently Inês Alves and his co-worker48 reported the DFT studies on SN2 reaction inside the different single walled carbon nanotubes (SWCNTs) for their kinetic barrier. Among the selected SWCNT, BN doped SWCNT lowered the energy barrier for SN2 reaction. Up till now, successful alteration in activation barriers for organic transformations is achieved only with carbon nanotubes. It has been shown previously by Alves et al. that BN doping in SWCNT creates electrophilic and nucleophilic sites which significantly affect the energy barriers by interaction with the substrates either in the reactants or transition states. Moreover, these nanotubes have quite wide cavities where the reagents may not feel the proper influence of encapsulations. Another drawback associated with doped SWCNT is the selective placement of dopant because of associated challenges, and to have reproducible results. We, instead of creating electrophilic and nucleophilic sites through doping, were more interested in ordered predefined orientation of electrophilic and nucleophilic sites in porous organic cages where structure is compact and reproducible. Moreover, these cages possess nitrogen atoms which can act as hydrogen bond acceptor in the close proximity of reacting atoms. For this purpose, we have chosen CC2 cage because it contains not only the nitrogen heteroatom (for hydrogen bond type of interaction with the substrate or transition state), but it also has compact structure where the cavity inside is relatively small. This small cavity restricts freedom of reagents inside the cage and force the substrate or transition state to form some non-bonding interaction. Our objective was to study the influence of these interaction on the activation barriers for several organic transformations.Literature reveals several examples where confinement of reagents in certain cavities has remarkable effect on the activation barriers. Catalytic activities of self-assembled metallocage for Diels–Alder reaction were investigated through encapsulation of Diels–Alder reactants inside the cage.Therefore, in current study, we are interested in studying chemical reactions inside porous organic cage confinement which imparts significant effect on the reactivity. Porous organic CC2 cages exhibit high surface area, appropriate chemical composition, high rigidity, and persistency. The above-mentioned characteristics make CC2 cage an excellent cage to carry out organic transformations within its confined spaces. Therefore, we have selected this class of porous materials (CC2 cage) to investigate some simple chemical reaction for their kinetic transformations within the cage and without cage. We performed DFT study on nine different organic transformations including: 2 + 4 cycloaddition, 1,5-sigmatropic, 6π-electro-cyclization, ring expansion, cheletropic, dyotropic, trimerization and tautomerization reactions. Furthermore, we explored the positive as well as negative catalytic influence of CC2 cage on the studied organic chemical reactions. Since these organic transformations are quite diverse therefore, it is not expected that a similar type of effect may be seen in all of them. Therefore, some organic transformations are catalyzed whereas others are slowed down (increase of barrier).250 level of theory. [2 + 4] cycloaddition,51 6π-electrocyclization,52 dyotropic reaction,53 cheletropic,54 1,5-sigmatropic reaction,55 Claisen rearrangement,56 ring expansion and contraction reaction57 keto-enol tautomerization reaction58 and trimerization reaction59 have been selected to investigate their energies (activation energy and energy of reaction) without CC2 cage and inside the CC2 cage. All optimized structures were confirmed by means of frequency analysis. Transition states have been optimized through Berny algorithm. Whereas stationary points were characterized as minima (no imaginary frequency) or transition state (one imaginary frequency). Furthermore, imaginary frequencies of all transition states were evaluated to confirm that their associated eigenvectors correspond to the motion along the reaction coordinates. Obtained results (activation energy and geometric parameters) of reactions within the cage and without cage were compared. All calculated energies are in kcal mol−1 while bond lengths for all optimized structures are reported in angstrom (Å). Non-covalent interactions are studied through NCI analysis. Multiwfn and VMD software have been used for the analysis of non-covalent interactions inside the CC2 cage. Results of non-covalent interactions have been reported in the form of 3D isosurface images and 2D reduced density graphs.Gaussian 09 ref.  was used3etc. They are different from each other based on vicinal diamine. The cage of our interest is CC2 with tetrahedral geometry with four triangular windows which form window-arene stacks while methyl groups are attached at vicinal diamine. The diameter of pore limiting window for CC2 cage is 3.6 Å at 298 K under 1 bar pressure45 and BET surface area is 533 m2 g−1.Porous organic cages represent novel porous compounds with permanent and shape persistent cavities. Shape persistent [4 + 6] imine cages are generally represented by CC1, CC2, CC3 CC2 porous organic cage was optimized at M06-2X/6-31G(d) level of theory to get the minimum at PES . Accordi3.1Alkyne trimerization reaction was theoretically investigated at M06-2X/6-31G(d) level of theory without and within CC2 cage. After encapsulation of R1 inside the CC2 cage, non-covalent interactions have been generated between inner surface of CC2 cage and the encapsulated molecules. The generated non-covalent interactions include strong non-covalent interactions such as hydrogen bonding as well as weak non-covalent interactions such as van der wall forces. H atoms of acetylene molecules show multiple interactions with the atoms of CC2 cage. H atoms interact through H-bond as well as van der Waals interactions with the C atoms of CC2 cage for which interaction distances range from 2.4 Å to 3.9 Å. At the same time, H atoms of acetylene interacts with the N of CC2 cage through H-bond for which the interaction distances range from 2.3 Å to 2.7 Å. Interactions of the host affect the geometric parameters of TS1′ which appear different than the geometric parameters of TS1 (without cage). For example, C1–C6 bond length is 2.29 Å in TS1 which is reduced to 2.27 Å in TS1′. C2–C3 bond length is 2.29 Å in TS1 which is reduced to 2.18 Å in TS1′. Similarly, C5–C4 bond length in TS1 is 2.29 Å while, this bond length is reduced in TS1′ (2.26 Å). Encapsulation has caused the TS1′ to become early in nature as compared to TS. Their early nature of TS1′ leads to drop in activation barrier.Ea) for trimerization reaction without cage was 49.40 kcal mol−1 whereas it is reduced to 47.60 kcal mol−1 in the cage. The result showed an appreciable drop in the energy barrier of trimerization reaction within the cavity of the CC2 cage because of non-covalent interactions between CC2 and guest molecules. The total energy of reactants (ER) with and without host cage are −165.03 kcal mol−1 and −160.70 kcal mol−1 respectively.The activation barrier of the reaction has been calculated. The activation barrier  for sigmatropic reaction without cage was calculated as 17.09 kcal mol−1 where it is 14.26 kcal mol−1 inside CC2 cage. The result showed a significant drop in the energy barrier of cheletropic reaction within the cavity of the CC2 cage mainly due to favorable interactions of transition state (TS2′) with the guest molecules. The energy of reaction (ER) was calculated as −15.68 kcal mol−1 without host cage and −12.88 kcal mol−1 within the CC2 cage.The activation barrier  and weak non-covalent  interactions were observed. Br and H of di-bromo ethane interact with the C and N of CC2 cage through van der Waals forces. The interaction distances for the interaction of Br and H atoms of di-bromo and C and N atoms of CC2 are in the range of 3.4–3.9 Å and 3.5–3.9 Å, respectively. Besides van der Waals forces, H atoms of di-bromo ethane interact with C atoms of CC2 cage through H-bond in the range of 2.6–3.5 Å. Some important interactions have been reported in The geometric parameters of TS3′ (inside cage) are different than those of TS3 (without cage) at the reaction site. For example, the C2–Br3 bond length in TS3 is 2.25 Å which is reduced to 2.15 Å in TS3′, whereas C1–Br4 and C2–Br4 bond lengths are 2.25 Å each in TS3 which are elongated to 2.39 Å in TS3′.Ea) of the reaction is 56.06 kcal mol−1 without the cage and 52.88 kcal mol−1 inside the CC2 cage. The activation barrier of the dyotropic reaction is significantly dropped inside the CC2 cage. The calculated total energy of reaction (ER) is 0 kcal mol−1 without cage due to identical reactants and products. Whereas in case of reaction in CC2 the surrounding interactions are different, which results in differences in geometry of R3′ and TS3′. The energy of reactant in this case is −0.13 kcal mol−1.The observed activation energy  level of theory. Non-covalent interactions such as hydrogen bond and van der Waals forces have been observed when reactant was encapsulated inside the CC2 cage. H atoms of cycloheptatriene interacts with the C and N of CC2 through H-bonds for which interaction distance range from 2.7–3.5 Å and 2.6–3.4 Å, respectively. O atom of cycloheptatriene interacts with the H atoms of CC2 cage through van der Waals forces appearing in the range of 3.1–3.7 Å. The nature and numbers of non-covalent interactions in TS4′ are different than those in R4′ except the interaction between H10⋯C14. H10⋯C14 interaction distance is 2.71 Å in R4′ which is increased to 3.03 Å in TS4′. H8⋯C15, H12⋯C17 and H13⋯N17 interactions distances are 3.11 Å, 2.97 Å and 2.67 Å in R4′, respectively. Nonbonding interactions H12⋯C17, H8⋯N18 and H9⋯N19 for which interaction distances are 2.88 Å, 2.73 Å and 2.76 Å in TS4′, respectively . The intThe geometric parameters of both transition states (TS4 and TS4′) are not significantly different from each other . C3–C4 bEa) for cycloheptatriene–norcaradiene rearrangement without cage is 28.81 kcal mol−1 while it is 26.99 kcal mol−1 inside the cage. The result showed a reasonable drop in the energy barrier of cycloheptatriene–norcaradiene rearrangement within the cavity of the CC2 cage. The energy of reaction (ER) is calculated as 15.43 kcal mol−1 without host cage and 12.21 kcal mol−1 within the CC2 cage.The activation barrier  and van der Waal forces (3.3–4.00 Å). Both types of interactions have been observed during the study of complex (CC2 cage and encapsulated molecule), whereas the interactions lengths vary in each case. H atoms of 1,3 diene interacts with C and N atoms of CC2 cage through H-bonding and van der Waals forces whereas, the interaction lengths in each case inside the complex are 2.6–3.5 Å and 3.2–3.7 Å, respectively. The modes of interactions in R5′ are modified in case of TS5′ . The intThere is no significant difference observed in the bond lengths of TS5 and TS5′ except C1–H6 and C5–H6 which are 1.43 Å in TS5, C1–H6 bond length is reduced to 1.42 Å whereas C1–H6 bond length is elongated to 1.44 Å in TS5′ while remaining bond lengths remained unchanged.−1 (without cage) and 36.82 kcal mol−1 for the reaction inside the CC2 cage. The exothermicity of 1–5 sigma tropic shift are calculated 0 kcal mol−1 without cage and −0.33 kcal mol−1 (inside the CC2 cage). The activation barriers for both reactions (without CC2 cage and inside CC2 cage) are almost similar which is attributed to similar strength of interactions in R5′ and TS5′.The observed activation energies of the reaction are 36.34 kcal mol3.6Optimization of the reactants and transition state of 6-π electro-cyclization reaction performed at M06-2X/6-31G(d) level of theory within and without CC2 cage. The values of interactions lengths showed the presence of non-covalent interactions between CC2 cage and encapsulated molecules. Interactions distances for R6 are between 2.7 Å to 4.00 Å which indicate the presence of hydrogen bond and van der Waals forces inside the complex. H atoms of 1,3,5-hexatriene shows multiple interactions with the C atoms and N atoms of CC2 cage through H-bond. The nature and number of interactions are different in R6′ and TS6′. H12⋯N16, H14⋯C15, H11⋯C17 and H7⋯C18 distances in R6′ are 2.70 Å, 2.88 Å and 2.91 Å, respectively . H14⋯C15The geometric parameters of TS6 and TS6′ are shown in Ea) of the reaction are 20.44 kcal mol−1 without the cage and 20.10 kcal mol−1 for the reaction inside the CC2 cage. The activation barriers for both reactions (without CC2 cage and inside CC2 cage) are almost similar. It is due to the same strength of interactions of the cage in R6′ and TS6′. The exothermicity of 6-π electro-cyclization reaction are calculated where the calculated value of ER of the selected reaction is −26.87 kcal mol−1 without cage and −28.49 kcal mol−1 for the reaction inside the CC2 cage.The observed activation energies  are indicative of non-covalent interactions such as hydrogen bond and van der Waals forces. H atoms of allyl vinyl ether interact with the C and N atoms of CC2 cage through H-bonding. O atom of allyl vinyl ether interacts with the H atoms of CC2 cage through van der Waals interactions in the range of 3.1 Å to 2.8 Å. Some important interactions distances are highlighted in −1 without the cage and 51.24 kcal mol−1 for the reaction inside the CC2 cage. The result shows increase in the energy barrier of Claisen rearrangement inside CC2 cage. The calculated ER is 17.64 kcal mol−1 for reaction without cage and 18.32 kcal mol−1 inside the CC2 cage.Unusual behavior is observed by the Claisen rearrangement due to structural features and modified interactions distances in R7′ and TS7′. The observed activation energies are 47.82 kcal mol3.8−1 and total energy of reaction is −13.61 in kcal mol−1. After encapsulation H, C and O atoms of reactants interacts through non-covalent interactions such as hydrogen bond and van der Waals forces with the atoms of CC2 cage such as N, C and H. whereas, the interaction lengths in each case are in between 2.6–3.5 Å and 3.1–3.7 Å, respectively. O atoms of the reactant interact with the C atoms of CC2 cage through van der Waals interaction range from 3.1 Å to 3.8 Å.To investigate the keto-enol tautomerism reaction, reaction was carried out individually without cage as well as inside the selected CC2 cage. Studied activation barrier for individual reaction without cage is 27.71 kcal molInteractions in R8′ are different than the interactions in TS8′ except some interactions. For example, H8⋯C16 and H10⋯C15 are the interactions which are common in both R8′ and TS8′. H8⋯C16 interaction distance is 3.01 Å in both R8′ and TS8′ while H10⋯C15 interaction distance is 2.83 Å in R8′ which is increased to 2.89 Å in TS8′. H11⋯C17, H12⋯C1815 interactions distances are 2.37 Å and 2.35 Å in R8′, respectively. H11⋯C14, H12⋯C13 interactions distances are 2.98 Å and 2.33 Å in TS8′, respectively. Interaction distances of R8′ and TS8′ are shown in The geometric parameters of TS8 are different than those of TS8′. C3–H11, O5–H13 and C1–C2 bond lengths are 1.48 Å, 1.25 Å and 1.50 of TS8 which are increased to 1.49 Å, 1.29 Å and 1.51 Å in TS8′. C2–C3 bond lengths remain same in both TS8 and TS8′ which is 1.40 Å each .−1 without the cage and 27.97 kcal mol−1 for the reaction inside the CC2 cage. In this case, both R8′ and TS8′ have almost same stabilities inside the cage, due to which the energy barrier of keto-enol tautomerism inside the cage is almost same as calculated for reaction without cage. Calculated values of ER of the keto-enol tautomerism are −13.61 kcal mol−1 without cage and −15.97 kcal mol−1 for the reaction inside the CC2 cage.The observed activation energies are 27.67 kcal mol3.9To investigate the kinetics of [2 + 4] cycloaddition reaction, the reactants and transition state were optimized at M06-2X/6-31G(d) level of theory. Reaction was studied individually in its gaseous state as well as inside the selected CC2 cage. Butadiene and ethene (R9) were encapsulated inside the optimized CC2 cage. Atoms of R9′  interact with the inner surface of CC2 cage which behaves as guest for the encapsulated molecules through non-covalent interactions such as hydrogen bond and van der Waals forces. H atoms of butadiene and ethane interact with the C and N atoms of CC2 cage through H-bond. Where interaction lengths are 2.6–3.0 Å and 2.6–3.5 Å, respectively.The nature and number of non-covalent interactions in R9′ and TS9′ are differing at certain points. H133⋯C24, H135⋯C76, H137⋯C57 and H137⋯C34 interaction distances in TS9 are 2.76 Å, 2.75 Å, 2.81 Å and 2.63 Å, respectively. The interaction bond distances of H133⋯C24, H135⋯C26, H137⋯C57 and H137⋯C34 are 2.81 Å, 3.43 Å, 2.81 and 3.18 Å, respectively inTS9′ see . OverallThe geometric parameters of TS9 are different than TS9′. For example, C1–C2 bond length is 1.38 Å in TS9 which is decreased to 1.37 Å in TS9′. C1–C6 bond length is 2.27 Å in TS9 which is elongated to 2.29 Å in TS9′. Whereas C2–C3 and C4–C5 bond lengths are 1.41 Å each in TS9 and TS9′.−1 without the cage and 19.47 kcal mol−1 for the reaction inside the CC2 cage. The higher barrier is due to the greater stability of the reactant inside the cage compared to transition state. Calculated value of ER of the 2 + 4 cycloaddition reaction is −47.86 kcal mol−1 without cage and −50.25 kcal mol−1 for the reaction inside the CC2 cage.The observed activation energies of the reaction are 17.62 kcal mol3.10i.e., blue patches show hydrogen bonding, red patches show repulsion forces and green patches shows non-covalent interactions. The green patches in the 2D-RDG graph ranging from 0.000 a.u. to 0.015 a.u. confirm the presence of van der Waals forces. Slight blue patches are also observed in all organic transformations except 1,5-sigmatropic shift reaction, 6-π electron cyclization reaction and keto-enol tautomerism reaction, which is responsible for hydrogen bonding. 2D-RDG graph also confirms the presence of hydrogen bonding in the above-mentioned reactions  were generated to characterize the type of interactions. The obtained 3D isosurface (NCI of reactions) generally there are two types of non-covalent interactions are found hydrogen-bonding and van der Waals forces. NCI plots reveal the presence of strong hydrogen-bonding and van der Waals forces in all organic transformations. In the 2D reduced density graph, three types of interactions are appeared ions see )The obtained 3D isosurface and RDG-2D graph for trimerization reaction is presented in 4−1 and 17.52 kcal mol−1 without cage, whereas these barriers are 47.70 kcal mol−1 and 15.09 kcal mol−1, respectively, in the cage. Moreover, reactions where the strength of non-covalent interactions between cage and guest molecules is almost same for reactants and transition states, no change in activation barrier is observed. The activation barrier for 6-π electrocyclization is 20.44 kcal mol−1 and 20.10 kcal mol−1 without and within CC2 cage, respectively. Similarly for keto-enol tautomerization the activation barrier is almost same with in the cage and outside of the cage. An unusual behavior has been observed in case of Claisen rearrangement R7′ is more stabilized inside the cage as compared to the TS7′ due to its structural geometry, and as a result, the activation barrier increased inside the cage for Claisen rearrangement.In summary we have studied various organic transformation in confined space of CC2 cage. Mullikan charges show nitrogen as the most electronegative specie whereas carbon is electronegative and hydrogen atoms, the most positive specie of optimized cage. Reactions were investigated theoretically within and without CC2 cage. Reactants after encapsulation interacts with the atoms of CC2 cage through non-covalent interactions such as hydrogen-bond and van der Waals forces. For some reactions such as trimerization, cheletropic reaction, ring expansion reaction, the non-covalent interactions of the transition states and CC2 cage are stronger as compared to the reactants which results in the significant drop of activation barrier. NCI analysis also confirms the presence of strong non-covalent interactions for these reactions. For trimerization reaction and cheletropic reaction the activation energies are 49.40 kcal molAyesha Mukhtar: methodology, conceptualization, investigation, writing-original draft. Sehrish Sarfaraz: software, validation, visualization. Khurshid Ayub: supervision, writing-review & editing.There are no conflicts to declare.RA-012-D2RA03399B-s001


Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)], produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels  KCNN1-3) with different but overlapping tissue expression. For example, all three isoforms are present in pancreatic islet cells, whereas levels of SK1 transcript are negligible in atrial and endothelial cells where SK2 and SK3 mRNA is detected at a significantly higher level  have been identified and characterized by their conductance, voltage- and calcium-sensitivity . The cloer level , 5. Wither level , 7. As aer level , 10, 11.er level , 15, 16.50 of 30–140 pM) and the two other SK isoforms o 9.5 nM , 23, 24. , 19, 20o 9.5 nM , 26.Functional SK channels are tetramers, and it has been suggested that they favor assembling as heteromeric channels , 28. TheBoth human and animal models show that SK subunit expression levels are higher in the atria than the ventricles, and subsequently SK current inhibitors prolong the atrial action potential significantly more than the ventricular action potential , 29, 30.A). In contrast, cells transfected with hSK3 subunits exhibited a distinctive expression pattern at the cell membrane (B). This difference in localization was confirmed by plotting the signal intensity along a line, across a section of cells. As illustrated in Fig. 1C, hSK3 subunits showed clear peaks at the cell edge. In contrast, expression of hSK2 subunits produced even distribution of subunits within the cell.Immunostaining for expressed hSK2 subunits revealed a diffuse pattern of staining which showed a strong degree of correlation between hSK2 and the ER marker DsRed2ER (DsRed) A. In conmembrane B. This dA). As seen when hSK2 subunits were expressed alone, signal intensity plots showed a strong correlation between the ER and hSK2 (Ci). In contrast to the clear membrane labeling observed for hSK3 subunits when expressed alone in HEK293 cells, hSK3 subunits labeling showed that the intracellular distribution during co-expression with hSK2 subunits. Co-expression with hSK2 subunits dictated that hSK3 subunit distribution was similar to that of hSK2 subunits, with labeling showing a reduction in membrane staining and more diffuse distribution (B and Cii).Immunolabeling of hSK2 subunits in cells co-expressing hSK2 and hSK3 protein showed a similar pattern of expression as observed during expression of hSK2 alone. No distinctive membrane expression was present, and a relatively consistent level of expression was exhibited across the cells A. As seeand hSK2 Ci. In coD; 67.4 ± 9.2 pA/pF versus 251.4 ± 27.0 pA/pF; n = 18 & 21; p < 0.0001). Co-expression of the two isoforms resulted in a current which had an amplitude similar to that seen with hSK2 subunit expression alone  and was significantly smaller than the current amplitude observed when homomeric hSK3 channels were expressed (p < 0.0001). These observations are consistent with the changes in cellular distribution shown in These data suggested that co-expression with hSK2 subunits reduced hSK3 protein reaching the cell membrane, with a higher proportion residing within the ER. Whole-cell patch clamp experiments were performed to investigate whether differences in intracellular distribution of SK channel subunits affected the magnitude of functional current. Whole-cell hSK2 currents were significantly smaller than those carried by hSK3 , with a current-voltage relationship exhibiting negative slope at positive voltages (A and B). Application of increasing concentrations of apamin led to hSK2 inhibition with an IC50 of 141 ± 21 pM . Expressed hSK3—mediated current was significantly less sensitive to apamin and was inhibited with an IC50 of 2.62 ± 0.36 nM . The Hill slope for inhibition by apamin in each channel was similar . Co-expression of hSK2 and hSK3 subunits produced an inwardly rectifying current which reversed at -78.6 ± 0.9 mV . Construction of a concentration-inhibition relationships showed current to be inhibited by apamin with an IC50 intermediate to those of the two homomeric channel currents , which was best fit by a monophasic Hill equation .Expression of homomeric hSK2 or hSK3 subunits produced currents that reversed at -75.9 ± 0.8 mV and -77.9 ± 1.8 mV, respectively . Co-expression of hSK2 and hSK3 subunits produced a current with an intermediate sensitivity to UCL1684 (2.37 ± 0.63 nM), the concentration–response relation for which was best fit by a monophasic curve and showed no difference in Hill slope compared with homomeric channels . These data indicate that co-expression of hSK2 and hSK3 channel subunits preferentially forms heteromeric channels that display an intermediate sensitivity to inhibitors.Expressed homomeric hSK2-mediated current was more sensitive to inhibition by UCL1684 than current mediated by hSK3 channels . These were reduced in amplitude by co-expression with WT-hSK2 subunits, and the amplitude was not significantly different from that observed with expression of WT channels  of one subunit and an outer pore histidine H336/H485) of an adjacent subunit , 28. UCL6/H485 ofA and 7A). Co-expression of WT-hSK2 and hSK3(H485N) subunits produced a current which was maximally inhibited by 100 nM UCL1684 to the same degree as observed with WT heteromeric channel current . Similarly, fractional inhibition by 100 nM apamin was not significantly reduced when hSK3(H485N) subunits were co-expressed with WT-hSK2 subunits compared with when WT-hSK3 subunits were used for co-transfection , demonstrating that no homomeric hSK3(H485N) channels were present during co-expression with hSK2 subunits. This co-expression of WT-hSK2 and hSK3(H485N) subunits produced a channel current which was inhibited by apamin with an IC50 of 588 ± 183 pM . This was best fit by a monophasic relation, indicating a single population of channels and the preferred formation of heteromeric hSK2:hSK3(H485N) channels.In agreement with previous work , 21, chaFigs. 650 of WT-hSK3 channel current  and a low sensitivity component with an IC50 of 25.5 ± 11.4 nM . Concentrations of up to 300 nM failed to fully inhibit current, suggesting that a population of homomeric hSK3(H485N) channels were also present  subunits produced concentration-inhibition responses to apamin which were best fit by a biphasic curve, with a high sensitivity component matching the ICM n = 7; , C and D present .E). This occurs as each molecule binds to the outer pore of one subunit and the S3-4 loop in an adjacent subunit. A low sensitivity component (25%) and an apamin-insensitive component (6%) are also predicted (E). The high sensitivity component of the biphasic curve in Fig. 7D constituted ∼65% of total inhibition during co-expression of WT-hSK3 and hSK3(H485N) subunits and therefore likely represents channels with at least two apamin-sensitive subunits. Low sensitivity inhibition accounted for ∼26% of current and represents channels formed with three hSK3(H485N) subunits. These data showed that co-expression of WT and mutant hSK3 subunits produced a mixed population of channels, with each channel containing none to four mutant subunits. In contrast, co-expression of WT-hSK2 and hSK3(H485N) subunits produced only one population of channels which exhibit a fixed subunit stoichiometry.Binomial distribution predicts that 69% of channels would be inhibited with a high sensitivity if the probability of incorporating WT-hSK3 or hSK3(H485N) subunits is equivalent E. This oredicted E. The hiThe three cloned SK channel subunits (SK1-3) are ∼90% homologous between the beginning of S1 and the distal end of the calmodulin-binding domain (CaMBD) in the C terminus, with the residues outside of this region showing little to no homology . Heteromsame isoform are co-expressed ; rSK2 & rSK2(YA246/7LV); hSK3 & hSK3-ex4), subunits form channels with stoichiometries which approximately follow the binomial distribution (D). The proportion of current inhibited with low (25 nM) and high (1.6 nM) sensitivities demonstrated that channels formed with a subunit organization which followed a binomial distribution. In contrast, co-expression of WT-hSK2 and hSK3(H485N) subunits produced a current which was inhibited by apamin with a high sensitivity and best fit by a monophasic curve (B and D). This is consistent with previously published data showing that co-expression of the apamin-insensitive rSK1 with rSK2 subunits produced a single population of channels which were inhibited by apamin with a high sensitivity (Heteromeric channel conformation was confirmed by co-expressing hSK3(H485N) with WT-hSK3 or WT-hSK2 channel subunits. As nonconserved C terminus sequences are key in channel assembly, when apamin-sensitive and apamin-insensitive subunits of the ribution , 19, 21.ribution D. The pric curve , B and Dsitivity .preferential, produce channels with a fixed stoichiometry, and also lead to an altered pattern of subcellular distribution. Co-expression of WT-hSK2 with hSK3(H485N) subunits showed a nonsignificant trend toward reducing co-operativity of inhibition (0.94 ± 0.14 versus 1.18 ± 0.19). The cooperative inhibition by apamin requires adjacent apamin-sensitive subunits to be present , no evidence of homomeric channels was present, suggesting mandatory formation of heteromeric channels. Taken together, these data show that hSK2 and hSK3 co-assemble to form channels of a fixed stoichiometry. The formation of heteromeric SK2:SK3 channels has been suggested to occur previously in both native tissue , 32 and  present , 28, andD). It is suggested that the hSK2 subunit appears to act as a chaperone and controls the surface expression of hSK3 subunits during co-expression. These data are consistent with previous studies showing that the rSK2 subunit acts as a molecular chaperone for rSK1 subunits and augments its subcellular localization  which is present on C terminus of the SK2 subunit but absent in the SK3 subunit  is present in both subunits but lies within the predicted coiled-coil region critical for channel assembly. This could therefore be masked during tetramerization or may contribute to the proportion of hSK3 channel homomers which do localize to the ER . The SK605879) in pcDNA3 and human SK3  in pcDNA3.1-c-DYK were used for all experiments. hSK2 was a generous gift from Dr Palle Christophersen  and hSK3 was purchased from GenScript. The SK3(H485N) mutation was made using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent). Successful mutagenesis was confirmed by Sanger sequencing of the complete open reading frame (Eurofins Genomics). Enhanced green fluorescent protein (eGFP) in pEGFP-C3 and DsRed (pDsRed2-ER) were purchased from Clontech.Human SK2 . Cells were replated into 35 mm tissue culture dishes and transfected using 3 μl Lipofectamine 2000 (Invitrogen) at least 24 h later and once they reached 80 to 90% confluency. For the study of homomeric currents, each dish was transfected using 1 μg ion channel, and 200 ng eGFP was included as a marker of successful transfection. Due to the difference in current size between expression of SK2 and SK3 subunits, only 100 ng SK3 was used for the study of heteromeric currents, and 400 ng empty vector (pcDNA3.1) was included in the transfection mixture to maintain a total of 1.2 μg DNA. The same quantities were used for cotransfection of WT-hSK2 and hSK3(H485N). These altered quantities of DNA did not affect current size (see HEK293 cells were maintained at 37 °C and 5% COsize see  but prod2 and 0.1 mM CaCl2 (PBS+) and fixed in 4% PFA for 20 min before being permeabilized with Triton X-100 . Cells were then blocked with 2.5% horse serum (Vector Laboratories) before a 1-hour incubation in primary antibody (Alomone APC-025 or -028) diluted 1:200 in blocking buffer. Following three washes in PBS+, secondary antibodies were then applied  for 1 hour, before cells were incubated in 1.2 μM DAPI (Sigma) for 5 min after excess secondary antibody was washed off with PBS+. Coverslips were mounted onto slides using ProLong Gold Antifade reagent (Invitrogen), which was allowed to set at overnight before being stored at 4 °C. All steps were performed at room temperature.Transfections were performed as above but DsRed was co-expressed with the subunits of interest in place of eGFP in order to label the endoplasmic reticulum. Additionally, cells were plated onto poly-L-lysine (Sigma Aldrich)–coated glass. Cells were washed in ice-cold PBS supplemented with 1 mM MgClStaining was imaged using a 63 × oil immersion lens with a Leica DM1600 confocal microscope. Fluorophores were excited at 488 (SK) or 568 nm (DsRed), and emission from a single confocal plane of 1 nm was detected. Images were processed and analyzed using FIJI . Signal 2 (1.2), CaCl2 (2.5) and titrated to pH 7.4 with NaOH. Glass patch pipettes  were pulled to a resistance of 2 to 3.5 MΩ and filled with intracellular solution containing (in mM): K-aspartate (97), KCl (20), Na2-ATP (1.5), Hepes-Na (10), EGTA (10), CaCl2, and MgCl2 and titrated to pH 7.2 with KOH. Homomeric currents were recorded in the presence of 500 nM free Ca2+ and 1 mM free Mg2+. Concentrations of Mg2+ and Ca2+ were calculated using Max Chelator V8 (2+ and 50 μM free Mg2+ stabilized currents without affecting current size (data not shown).Glass coverslips were placed into a recording chamber and superfused at room temperature with a solution of composition (in mM): NaCl (138), KCl (4), Hepes (10), D-glucose (10), MgCllator V8 . AlthougD and Channel current was elicited using voltage ramps running from 100 mV to +100 mV (1 s duration). Where current amplitudes are reported D and 5, Current was recorded using an Axopatch 200B amplifier (Axon instruments) and filtered at 1 kHz using a low pass Bessel filter  before sampling at 10 kHz using Pulse (HEKA). Electrode resistance was compensated for by at least 70%  when the whole-cell configuration was used. Liquid junction potentials were not corrected for.2O and DMSO, respectively, with aliquots stored at −20 °C. Aliquots were thawed on the day of use and diluted further in the external solution described above. Aliquots were sequentially added to the superfusate to obtain concentrations ranging from 10 pM to 300 nM. Concentration–inhibition relationships were fit and a standard Hill equation in the form of:Icont and I are the current magnitude in the absence and presence of a given concentration of drug  were diluted to 100 μM in ddHt tests and one-way ANOVAs were performed using Prism 9.2.0 (GraphPad) and Tukey’s post-hoc test was used to make multiple comparisons where appropriate. p values of less than 0.05 were taken as being statistically significant and are indicated by ∗. p values of less than 0.01, 0.001 and 0.0001 are signified by ∗∗, ∗∗∗, and ∗∗∗∗, respectively. This article contains supporting information.All experimental values are described in text as mean ± standard error of mean (SEM). Bar charts present all individual data points and the standard deviation (SD) to convey variation as previously . The t tData are available upon request to the corresponding author.This article contains The authors declare that they have no conflicts of interest with the contents of this article.