Gastric cancer (GC) remains an important cancer worldwide. Further understanding of the molecular mechanisms of gastric carcinogenesis will enhance the diagnosis and treatment of GC.The expression of DLEU2 and ETS2 was analyzed in several GC cell lines using GEPIA online analyze, qRT-PCR and immunohistochemistry. The biological behavior of GC cells was detected by CCK8, clone formation, transwell, wound healing, western blot, and flow cytometry assay. More in-depth mechanisms were studied.DLEU2 was significantly up-regulated in GC tissues and cell lines. The expression of DLEU2 was significantly associated with pathological grading and TNM stage of GC patients. Furthermore, knockdown of DLEU2 inhibited the proliferation, migration, and invasion of AGS and MKN-45 cells, while overexpression of DLEU2 promoted the proliferation, migration, and invasion of HGC-27 cells. MiR-30a-5p could directly bind to the 3’ UTR region of ETS2. Moreover, DLEU2 bound to miR-30a-5p through the same binding site, which facilitated the expression of ETS2. Knockdown of DLEU2 reduced the protein level of intracellular ETS2 and inhibited AKT phosphorylation, while overexpression of DLEU2 induced the expression of ETS2 and the phosphorylation of AKT. ETS2 was highly expressed in GC tissues. The expression of ETS2 was significantly associated with age, pathological grading, and TNM stage. ETS2 overexpression promoted cell proliferation and migration of AGS and MKN-45 cells. Furthermore, ETS2 overexpression rescued cell proliferation and migration inhibition induced by DLEU2 down-regulation and miR-30a-5p up-regulation in AGS and MKN-45 cells.DLEU2 is a potential molecular target for GC treatment. Gastric cancer (GC) remains an important cancer worldwide. The 2018 global cancer data released by the American Cancer Society shows that there are more than 1,000,000 new cases and an estimated 783,000 deaths  [Human whole-genome sequencing results show that about 1.2% of the mammalian genome is used to encode proteins , and mosDLEU2 gene encodes an lncRNA (1.0–1.8 kb)—DLEU2, which is polyadenylated and cleaved [H. pylori [The host gene of lncRNA DLEU2 is located on chromosome 13, 4,995,852–50,125,541, which is a common deletion mutation in leukemia and solid tumors . DLEU2 g cleaved . Its seq cleaved . Further. pylori .Unlike lncRNAs, microRNAs (miRNAs) are generally 18–25 nucleotides in length. MiR-30a-5p is considered to be involved in the development and drug resistance of GC and is closely related to the recurrence-free and overall survival rate of GC patients . ETS2 is75 pairs of GC tissues and matched adjacent normal tissues were collected from patients who were diagnosed and underwent surgical resection at the Jinan Central Hospital. None of the patients had other serious diseases except for GC and did not receive chemotherapy or radiotherapy before operation. All collected samples were immediately snap-frozen in liquid nitrogen and stored until needed. The Histological grade was staged according to the seventh TNM staging of the International Union against Cancer/American Joint Committee on Cancer system. All research complied with the principles of the Declaration of Helsinki and was approved by the Medical Ethics Committee of the Jinan Central Hospital. The enrolled patients gave written informed consent for publication.2.Human GC cell lines  and normal gastric mucosal epithelial cell line (GES-1) were purchased from the American Type Culture Collection  and cultured in Dulbecco’s modified Eagle medium  supplemented with 10% FBS  and 1% antibiotic/antimycotic in a humidified incubator at 37 °C containing 5% COsiRNAs targeting lncRNA DLEU2 (si-DLEU2) were designed and synthesized . The sequence of si-DLEU2 was used as follows: 5'-CUCAUUGAAUACUAUCAAAAAGGAA-3'. miR-30a-5p mimics were purchased from RiboBio . The cDNA of DLEU2 and ETS2 was synthesized by GENEWIZ and cloned into the pcDNA3.1 expression vector, respectively . si-DLEU2, miR-30a-5p mimics, and pcDNA3.1 vectors were transfected using Lipofectamine 2000 reagent , according to the manufacturer’s instructions.−ΔΔCt using GAPDH as an endogenous control.Total RNA was extracted from tissue samples or cells with TRIzol reagent  and purified according to the manufacturer’s instructions. RNA concentration was measured spectrophotometrically at an optical density of 260 nm. The Reverse Transcription Kit  was used to generate cDNA. Real-time PCR was performed using SYBR® Green . PCR primers were used as follows: DLEU2-Forward, 5'-GGCGGCGGGTACTTATCTC-3'; DLEU2-Reverse, 5'- CCAGGGAAGGATGTAGCTGTG-3'; GAPDH-Forward, 5'- GGTGTGAACCATGAGAAGTATGA-3'; GAPDH-Reverse, 5'- GAGTCCTTCCACGATACCAAAG-3'. Relative fold expression was calculated by 22000 cells were cultured in a 96-well plate for 0, 24, 48, and 72 h. Then, the medium was replaced with 100 μl fresh DMEM containing 10 μl CCK8 solution . Incubate at 37 °C for 2 h. Absorbance at 450 nm was detected by a microplate reader .2000 cells were plated into 6-well plates and cultured for 10 days. Then, cells were fixed with methanol and stained with 0.1% crystal violet solution. Colonies were counted and images were obtained. Colonies with at least 50 cells were considered significant.5 cells in 200 μl of FBS-free DMEM were seeded into the upper chamber of transwells precoated with Matrigel (BD Bioscience), and 600 μl of DMEM containing 10% FBS was added to the lower chamber. After 24 h of incubation, non-migrating or non-invasive cells remaining on the upper surface were removed with a cotton swab. Then the membranes were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 15 min. Three random fields were counted in each chamber using an inverted microscope (Olympus).1 × 10A sterile plastic was used to create a wound in a single cell layer. After washing with PBS, the cells were cultured in FBS-free medium for 24 h. Five random fields of each wound were measured for quantification and images were obtained.Extraction of proteins from tissue samples or cells using RIPA buffer and protein concentration was measured by the BCA method . 15 μg proteins were separated by SDS–PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% skim milk and immunoblotted with primary antibodies, followed by incubation with matched secondary antibodies. Then blots were visualized using an enhanced chemiluminescence kit  and detected using the GelCapture version software . GAPDH was employed as an endogenous control.6 cells were harvested after transfection for 24 h and resuspended in Annexin V binding buffer. 5 μl FITC-Annexin V and 5 μl PI were added to stain using the Apoptosis Detection Kit . Then, 400 μl PBS was added to the cells, which were analyzed using a FACScan flow cytometry system . Cell apoptosis was analyzed using FlowJo V7 software .1 × 10Luciferase reporter assay was performed using psiCHECK2 vector (Promega). To construct the psiCHECK2-ETS2 recombinant vector, the complete 3’UTR of human ETS2 mRNA containing the putative or mutative miR-30a-5p binding sites was amplified and cloned into the psiCHECK2 vector. For the lncRNAs, wild or mutative full-length sequences of lncRNA DLEU2 were amplified and cloned into the psiCHECK2 vector. AGS cells were co-transfected with one of the psiCHECK2 recombinant vectors and miR-30-5p mimics, or miR-NC by Lipofectamine 2000 according to the manufacturer’s guidelines. The relative luciferase activity was valued using the Dual-Luciferase Reporter Assay System (Promega) and Infinate M200 PRO microplate reader .Tissue samples were fixed in formalin for 24 h at 4 °C and embedded in paraffin. The paraffin block was cut into 4 μm sections. Tissue sections were deparaffinized in Van-Clear  and concentration gradient ethanol, then microwaved in 0.01 M citrate buffer for 10 min. After blocking with 5% goat serum for 1 h at room temperature, the sections were incubated with the primary antibody and then incubated with enzyme-labeled goat anti-mouse/rabbit IgG polymer . The immune response was visualized by the enhanced DAB chromogenic kit , and hematoxylin was used for counterstaining. The immunostaining score was evaluated blindly by two independent investigators as the product of positive staining cell ratio (R) and staining intensity score (S). R was divided into four levels: 0 , 1 , 2 , 3 . S was also divided into four levels: 0 (negative), 1 (weak), 2 (middle), 3 (strong). A total of 0–3 was considered to be low expression, while 4–9 was considered to be high expression. Finally, images were collected by a vertical microscope system .P < 0.05.Statistical analyses were performed with SPSS software version 22.0 . The experimental results were expressed as mean ± standard deviation. The differences between groups were calculated using the Student's t-test or one-way ANOVA. The correlation among lncRNAs, miRNAs, and mRNAs was analyzed by Pearson's correlation analysis. A statistically significant threshold was defined as P = 0.0087) and TNM stage (P = 0.0382) of GC patients (Table GEPIA collected the RNA expression information of 408 stomach adenocarcinoma (STAD) and 211 normal tissues. The analysis results of GEPIA showed that DLEU2 was highly expressed in STAD tissues compared with normal tissues , the expression of DLEU2 was down-regulated by approximately 60% compared with the negative control (NC) Fig. D. FurtheKnockdown of DLEU2 significantly inhibited the proliferation of AGS and MKN-45 cells, as evaluated by CCK-8 assays  predicted that DLEU2 could act as a ceRNA to target ETS2 via miR-30a-5p. We verified this in AGS cells . In addition, the association between ETS2 expression and clinical characteristics of patients was analyzed. As shown in Table P = 0.041), pathological grading (P < 0.001) and TNM stage (P = 0.023). No significant correlation with other characteristics was observed, such as gender and tumor diameter.We then verified the expression of ETS2 in GC tissues. As shown in Fig. Finally, to test whether DLEU2 exerted its role by regulating the miR-30a-5p/ETS2 axis, cells were transfected with pcDNA3.1-ETS2 overexpression plasmid (ETS2), pcDNA3.1-ETS2 plasmid and miR-30a-5p mimics (ETS2 + miR-30a-5p), pcDNA3.1-ETS2 plasmid, and si-DLEU2 (ETS2 + si-DLEU2), respectively. The results showed that the overexpression of ETS2 promoted the proliferation  is a critical step in tumor progression, which increases cell infiltration and promotes the occurrence of distant metastases . It is cETS2 gene is located on chromosome 21q22.1-q22.3 with a span of 17.6 kb, which has no TATA box or CAAT box in its promoter and has a major CpG island at its 5’ untranslated region [In the present study, we found that miR-30a-5p could directly bind to the 3’ UTR region of ETS2, thereby inhibiting the translation and protein stability of ETS2. Moreover, DLEU2 bound to miR-30a-5p through the same binding site, which facilitated the expression of ETS2. Knockdown of DLEU2 reduced the protein level of intracellular ETS2. The d region . The ETSd region . As a trd region , 35. In d region . In humad region , 35. Mord region . The aboIn addition to affecting ETS2, DLEU2 is used as ceRNA to regulate the expression of SOX9, PDK3, E2F7, and other target proteins , 24. ETSIn conclusion, our data revealed that DLEU2 was highly expressed in GC tissues and its expression was statistically correlcated with the clinicopathological characteristics of GC patients. DLEU2 promoted the proliferation, migration, and invasion of GC cell as well as induced the EMT process and inhibited apoptosis. We found that DLEU2 acted as a ceRNA targeting ETS2 via miR-30a-5p. Furthermore, the AKT signaling pathway was required. Taken together, we suggested that DLEU2 is a potential molecular target for GC treatment.


In vitro and in vivo experiments showed that dual drugs treated groups have better tumor inhibition than solo drug under near infrared laser irradiation, indicating the effectivity of combined photodynamic-chemotherapy.With hollow mesoporous silica (hMSN) and injectable macroporous hydrogel (Gel) used as the internal and external drug-loading material respectively, a sequential drug delivery system DOX-CA4P@Gel was constructed, in which combretastatin A4 phosphate (CA4P) and doxorubicin (DOX) were both loaded. The anti-angiogenic drug, CA4P was initially released due to the degradation of Gel, followed by the anti-cell proliferative drug, DOX, released from hMSN in tumor microenvironment. Results showed that CA4P was mainly released at the early stage. At 48 h, CA4P release reached 71.08%, while DOX was only 24.39%. At 144 h, CA4P was 78.20%, while DOX release significantly increased to 61.60%, showing an obvious sequential release behavior. Photodynamic properties of porphyrin endow hydrogel (The online version contains supplementary material available at 10.1186/s12951-021-01066-1. Cancer is still one of the significant threats to human health. Current cancer therapies, such as chemotherapy , photothHydrogel is composed of a large amount of water and cross-linked polymers. The abundant water content (70–99%) in hydrogels resembles biological tissues, and they usually have good biocompatibility and loading hydrophilic drugs capacity . Smart hTo further enhance the therapeutic effect of drugs, photodynamic therapy (PDT) has been used in combination with chemotherapy, magnetic therapy or immunity therapy because of its significant advantages such as causing less trauma, low toxicity, and palliative treatment . Belali Herein, we designed and prepared a novel injectable macroporous hydrogel for therapy of 4T1 breast cancer, which could load dual distinct tumor drugs (CA4P and DOX) offering a sequentially delivery profile. Furthermore, porphyrin derivatives in hydrogel trigger the formation of reactive oxygen species (ROS), achieving synergistic anticancer effects in photodynamic-chemotherapy combination therapy. The therapeutic efficacy from this combinational photodynamic/sequential chemotherapy was evaluated in vitro and in vivo.2-TPP), dextran and doxorubicin hydrochloride (DOX·HCl) were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 3-Aminopropyltrimethoxysilane (APS) was purchased from Braunwell Chemical Technology Co., Ltd. CA4P was acquired from Solarbio Science & Technology Co., Ltd. Cetyltrimethylammonium chloride (CTAC) was purchased from Sigma-Aldrich Co., Ltd. Dulbecco’s modified Eagle’s medium (DMEM), memorial institute medium (RPMI)-1640, 3--2,5-diphenyltetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI), 1,3-diphenylisobenzofuran (DPBF), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), live/dead cell viability/toxicity kit, and Annexin V-FITC/PI staining kit were all purchased from Vicmed . Caspase 3 polyclonal antibody (caspase 3 PAb) was purchased from Proteintech Co. (USA). PV-6001 rabbit polymer detection system was purchased from Zhongshan Jinqiao Co., Ltd. Trypsin and dimethyl sulfoxide (DMSO) were purchased from Nanjing Key Gen Biotechnology Inc. Dextran (W=70 kD), chitosan with medium viscosity (200–400 mPa·s), and sodium periodate were purchased from Macklin Biochemical Technology Co., Ltd. Other reagents were all purchased from Sinopharm Group Chemical Reagent Co., Ltd.Tetraethyl silicate (TEOS), triethanolamine (TEA), 5,10,15,20-tetraphenylporphyrin (TPP), -tetra(4-aminophenyl)porphyrin (NH2). After washing with water, dSiO2 was added into the mixed solution of CTAC and TEA, followed by drop-wise adding TEOS and the reaction was carried out at 80 °C for 1 h to synthesize dSiO2@SiO2.Na2CO3 powder was then added to it to allow etching dSiO2@SiO2 at 50 °C for 30 min. After washing with NaCl:methanol (1%), hMSN was achieved. Finally, hMSN was modified with amino groups on the surface via hydrolyzing APS in absolute ethanol.Hollow mesoporous silica nanoparticle (hMSN) was prepared according to our previous work . Briefly2-TPP (dissolved in 1 mL DMF) to form OD/TPP, the unreacted OD and TPP was removed using dialysis bags (MWCO 12000–14000) against distilled water for 3 days (the water was renewed at least 5 times per day). 30 mg chitosan (dissolved in 1% acetic acid) was applied to adjust the gelation level of OD/TPP (with the mass ratio of TPP:OD:chitosan of 10:1:30) to form ‘hydrogel’. hMSN was loaded in hydrogel by simply mixing during the gelation of OD/TPP, the hydrogel loaded hMSN was referred as ‘Gel’. The excessive and unreacted reagents were removed by washing with distilled water based on the ‘no flow’ status of hydrogel.Hydrogel OD/TPP was synthesized through a Schiff base reaction between the aldehyde groups from oxidized dextran (OD) and amino groups of porphyrins with the molar ratio of 1:10. Firstly, the adjacent diol groups were oxidized to aldehyde groups by sodium periodate to prepare aldehydated oxidized dextran . The oxi2-TPP with a mass ratio of 1:5 was dissolved in DMF to acquire system ‘A’, and CA4P was mixed with oxidized dextran at a ratio of 1:1 (in PBS) to prepare system ‘B’. DOX-CA4P@Gel was synthesized through a Schiff base reaction between A and B. The unreacted reagents were removed by washing with distilled water.DOX and CA4P was loaded into Gel in different methods to synthesize the composite system DOX-CA4P@Gel. Briefly, DOX·HCl aqueous solution was adjusted to pH 8.0, followed by mixing with hMSN at a mass ratio of 1:1. The mixture was stirred for 24 h at room temperature and the excess DOX was removed by dialyzing with molecular weight cut-off of 2 kDa to acquire drug-loaded microspheres DOX@hMSN [The gelation time and the degradation behavior of the hydrogel under different acidic conditions were detected by an MCR 302 rheometer. Transmission electron microscopy (TEM) images were taken on a G2T12 transmission electron microscope . The morphology of the hydrogel was recorded on a Q25 scanning electron microscope . The hydrodynamic size and zeta potentials were measured by dynamic light scattering (DLS) . The chemical compositions were determined by X-ray photoelectron spectroscopy (XPS) (PHI Quantera SXM). All XPS spectra were calibrated by the C 1 s peak at 284.6 eV. Infrared absorption spectrum (IR) was recorded on a FTIR 8400 spectrophotometer. UV-Vis and fluorescence spectra were recorded on Hitachi U-3010 and F-4600 spectrophotometer, respectively.Drug release behavior was evaluated at 37 °C by respectively incubating DOX-CA4P@Gel in PBS solution with pH of 5.0, 6.4, and 7.2. CA4P release was measured by high performance liquid chromatograph (HPLC), where chromatographic separation was carried out on an Agilent Zorbax Eclipse Plus C18 column with particles size of 5.0 μm (250 × 4.6 mm) using methanol (0.01% acetic acid)-water (60:40) as the mobile phase at 1 mL/min .1O2 [1O2 capture agent, the 1O2 generation efficiency of TPP in Gel can be calculated from the slopes of DPBF absorbance variation (410 nm) in the presence of photosensizers. The laser irradiation was 808 nm (0.5 W/cm2), with TPP in DMSO as the control, the 1O2 yield (φ∆) was calculated as follows [The optical property of DPBF was sensitively affected by 1O2 . With DP follows :tTPP and tGel is the time for the decrease in absorption of DPBF in the presence of TPP and Gel, respectively.2. Cells were used when they reached ~80% confluence.4T1 breast cancer cells and L929 fibroblasts cells were maintained in RPMI 1640 and DMEM medium supplemented with 10% fetal bovine serum and proper antibiotics at 37 °C with the supply of 5% CO6 of 4T1 cells suspended in 50 µL of PBS into female Balb/c mice . Mice were used for therapy study when the tumor diameter reached 5–8 mm, monitored every other day by a caliper measurement.All animal procedures were performed in accordance with the National Academy of Sciences’ Guidelines on the Care and Use of Laboratory Animals , and wer2, and 5 min). All experiments were performed at least three times. Five groups including CA4P@Gel, DOX@Gel, DOX-CA4P@Gel, DOX and CA4P were compared in this work.MTT assays were carried to evaluate the biocompatibility (without laser irradiation) and the therapeutic efficacy (with laser irradiation) of DOX-CA4P@Gel. DOX-CA4P@Gel at the concentration of 0.01–0.20 mg/mL were respectively incubated with 4T1 and L929 cells for 24, 48, and 72 h. MTT measurement was subsequently carried out to evaluate the cellular toxicity of DOX-CA4P@Gel [1O2 and cytotoxic effect of DOX-CA4P@Gel in 4T1 cells. 4T1 cells were cultured at a 24-well plate with concentration of 5 × 104 cells/mL. After incubation with hydrogel extracted solution (0.5 mg/mL based on TPP) for 24 h with laser irradiation , the cells were stained with DCFH-DA and DAPI [The fluorescence microscopy was used to observe the production efficiency of and DAPI . Cells wand DAPI .5/well. CA4P@Gel, DOX@Gel, DOX-CA4P@Gel extracts at different concentrations were incubated with 4T1 cells for 24 h with or without laser irradiation . Flow cytometry assay was carried out after Annexin V-FITC 5 µL and PI 5 µL staining for 10 min.MACSQuant 10 flow cytometry  was also used to study the drug release behavior of CA4P and DOX from DOX-CA4P@Gel. 4T1 cells were dispersed with a final concentration of 1 × 102 power intensity was used as the near-irradiation source. During therapy, the body weight and tumor volume (V = L × W2/2) were monitored every other day, where L (mm) and W (mm) are the long and short axes of tumor size, respectively. The tumor inhibition rate (TIR) was calculated using the equation of TIR = (Vc−Vt)/Vc×100%, where Vt and Vc represent the tumor volumes in the treatment group and control group, respectively. After treatment, major organs including heart, liver, spleen, lung, and kidney were harvested for the hematoxylin and eosin (H&E) analysis. The 4T1 tumors harvested on day 0, 3, 7, and 14 were also frozen for histological analysis.The mice bearing 4T1 tumor nodules were randomly divided into 10 groups (n = 8) including PBS (Control), free DOX/CA4P (mixture of 0.4 mg DOX and 1 mg CA4P), Gel, CA4P@Gel, DOX@Gel, DOX-CA4P@Gel, Gel+NIR (near infrared laser), CA4P@Gel+NIR, DOX@Gel+NIR, and DOX-CA4P@Gel+NIR. 100 µL of each sample were intravenously injected into tumor. Except for PBS and free DOX/CA4P, the concentrations of hydrogel were consistent (44 mg/mL) in eight groups. During preparation of CA4P@Gel and DOX@Gel, proper amount of DOX and CA4P were added to ensure the consistent concentration in DOX-CA4P@Gel. An 808 nm laser with 0.5 W/cmMajor organs were fixed in 4% formalin, transferred routinely into paraffin, sectioned into 6 μm thick slices, stained with H&E and examined by a microscopy with a magnitude of 200×. Tumors were cut into frozen slices of 6 μm thickness. After being fixed with cold acetone for 10 min, the tumor slides were stained with caspase 3 PAb  for 1 h, followed by detecting by PV-6001 rabbit kit. The caspase 3 expression was by a confocal microscope at a magnitude of 200×.2-TPP, in which the OD-to-NH2-TPP ratio was 1:10, and chitosan (W% = 10%) was to adjust the elasticity. The degree of aldehyde was closely related to the oxidation time by sodium periodate. Considering the reaction time and the degree of aldehyde  was optimal for getting fast formation and slowly degradation. As shown in Fig. −1 for C=O) and porphyrin derivative (~1510 cm−1 for N–H), and presence of characteristic IR peak from Gel (~1650 cm−1 for C=N) [As shown in Fig. for C=N) , 19 conf2 significantly increased, indicating a modification effect of amino groups. No obvious difference was present between DOX-CA4P@Gel (38.4 ± 3.8 mV) and CA4P@Gel (33.5 ± 2.4 mV). Figure As shown in Fig. tpH 5.0=5.0 min, tpH 6.4=5.2 min, tpH 7.2=6.4 min). DOX-CA4P@Gel was dispersed in PBS with different pH values, and the degradation of hydrogels was evaluated by measuring the content of decomposed TPP in solution. Results in Fig. DOX-CA4P@Gel was dispersed in phosphate buffer solution (PBS) solution with different pH values, and the elastic modulus of hydrogel was measured using a rheometer. As shown in Fig. CA4P and DOX exhibited pH sensitive and the release profiles were also observed, where faster release rates were shown at acidic condition (pH 5.0) compared with that in neutral condition Fig. h, i. CA41O2) in solution. The results of the irradiation of the samples in the presence of DPBF (absorption monitored at 410 nm) with time are shown in Fig. φ∆(TPP) = 0.52) as the control [1O2 yield of hydrogel  was 0.91, which may be due to the high viscosity of hydrogel limiting the rotation of porphyrin molecules and avoiding the optical quenching induced by the collision between porphyrin molecules. DCFH-DA was an intracellular singlet oxygen capture agent, and its green fluorescence intensity of cells was directly proportional to the production of 1O2. The concentration of After incubation of hydrogel with cells, 1O2 detection was conducted. As shown in Fig. In order to prove that the photosensitivity of porphyrin was not changed significantly after the incorporation into the hydrogel, DPBF was used as the reagent to quantitatively detect the production of singlet oxygen . This result shown in Fig. p < 0.05), suggesting that hydrogel has an obvious photodynamic killing effect on 4T1 cells. In order to further intuitively demonstrate the PDT effect of hydrogel on cancer cells, the live/dead cells staining with calcein AM and PI was carried out. The fluorescence imaging results in Fig. The extracts of blank hydrogel with different concentrations  were incubated with L929 and 4T1 cells for 24, 48 and 72 h, respectively. As shown in Fig. 2, 5 min). PBS was used as the control. As showed in Additional file Gel, DOX@Gel, CA4P@Gel, and DOX-CA4P@Gel 0.1 mg/ml) were respectively incubated with 4T1 cells for 48 h, and the cell viabilities were measured with or without laser irradiation  without laser assistance, results showed that after 24 h of co-incubation with 4T1 cells, only CA4P@Gel (79.5%) and DOX-CA4P@Gel (66.6%) was significantly lower than that of control group (95.3%), while DOX@Gel group was not significantly different from the control group, which proved that early release of CAP4P from DOX-CA4P@Gel was the major reason for cell toxicity before 24 h. Comparing the Gel groups , it was found that the cell viability with laser assistance was significantly lower than that of the without laser assistance group, especially DOX-CA4P@Gel+NIR was only 50%.p < 0.01), indicating that sequential delivery therapy combined with photodynamic therapy had a significant impact on tumor growth. It should be noted that after 18 days of treatment, tumor growth was accelerated in the DOX/CA4P group, with tumor volume greater than that of CA4P-DOX@Gel, which may be due to the fact that CA4P stimulated the formation of tumor vascular collateral after a single dose, thus accelerating tumor growth. In the DOX-CA4P@Gel system, CA4P plays its role first, followed by DOX and PDT. Sequential drug release combined with photodynamic therapy can effectively inhibit tumor growth to a certain extent. The therapeutic effect of each group was also evaluated by measuring tumor weight, and the results were shown in Fig. p < 0.01).The 4T1 tumor-bearing mice were respectively treated by a single intratumor injection administration of PBS, Free DOX/CA4P (mixture of 0.4 mg DOX and 1 mg CA4P), Gel, CA4P@Gel, DOX@Gel, DOX-CA4P@Gel, Gel+NIR, CA4P@Gel+NIR, DOX@Gel+NIR, DOX-CA4P@Gel+NIR, where NIR was illuminated for 5 min every other day. The body weight, the volume and weight of the tumor were recorded every day, and the tumor inhibition rate was calculated with PBS as the control group. As can be seen from Fig. The expression of caspase 3 and DOX fluorescence intensity were used to characterize the location of CA4P and DOX. CA4P can specifically bind to vascular growth factor and prevent its interaction with the receptor, thereby causing apoptosis. The activity of caspase 3 reflects the apoptotic status of tumor cells. At different time points, the caspase 3 activity (apoptotic inducing factor) in the tumors of the DOX-CA4P@Gel group was detected by immunohistochemistry. Results in Fig. To evaluate the possible systemic toxicity, body weigh variation and H&E staining was carried out. Additional file With biocompatible dextran oxide, chitosan, porphyrin and hMSN as the starting materials, a dual drug carrying system DOX-CA4P@Gel was constructed, which could be injected in tumor and play therapeutic roles in situ. Good biocompatibility DOX-CA4P@Gel was verified by cell tests and H&E staining. Studies on the properties of the materials show that Gel can be degraded slowly under tumor microenvironment, and DOX and CA4P released sequentially for tumor therapy at different time. Animal model experiments showed that DOX-CA4P@Gel+NIR could effectively enhance therapy effect due to the sequential drugs release and photodynamic performance. The DOX-CA4P@Gel constructed in this work is simultaneously injectable, biodegradable, sequentially releasable and photodynamic, providing a feasible route to reduce drug resistance and improve cancer therapy efficiency.Additional file 1: Figure S1. Zeta potential of hMSN, DOX@hMSN-NH2, CA4P@Gel and DOX-CA4P@Gel. Figure S2. XPS survey spectra of hMSN, Gel and CA4P-DOX@Gel.  Figure S3. Visible spectra of DOX, TPP and CA4P.  Figure S4. Fluorescence images of 4T1 cells after incubation with hydrogel with irradiation assistance . Scale bar: 100 μm.  Figure S5. Cell viability of 4T1 cells post different treatment. ***p<0.001.  Figure S6. Body weight variation of mice post different treatment.  Figure S7. Histolodical analysis of the major organs of mice treated with various drug formulations at day 21 . Table S1. The degree of aldehyde affected by the oxidation time by sodium periodate. Table S2. Effect of chitosan on hydrogel formation and degradation.


Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, indicating that C1b stabilizes C2b. Moreover, the levels of several IFT and BBS proteins, HSP70, and enzymes that catalyze the final steps of the glycolytic pathway: enolase ENO1 and pyruvate kinase PYK1, are also reduced in the C1b-less mutants.Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate  The cytoskeletal scaffold of motile cilia, the axoneme, is typically composed of nine microtubule doublets arranged around the cilium’s circumference and two single microtubules positioned in its center (9 × 2 + 2). Both the outer doublets and the central microtubules serve as scaffolds for periodically attached multi-protein complexes. Outer and inner dynein arms, radial spokes, and the nexin-dynein regulatory complex are major complexes that are repeatedly docked onto the outer doublet microtubules4. In consequence, the CA is asymmetric, which could be important for the generation of asymmetric waveforms.The central microtubules, C1 and C2, are linked by so-called bridge and are a docking site for C1a-C1f and C2a-C2e complexes, called projections, and together form so-called central apparatus (CA). The CA projections differ in their size, architecture, protein composition, and likely functionChlamydomonas, loss of the entire CA causes flagella paralysis (mutants pf155 and pf196), while losses of single projections or their parts disturb the flagellar waveform, amplitude or beat frequency to varying extents, depending upon the affected projection or even its part. For instance, flagella of the hydin mutant lacking C2b and a part of C2c are mostly paralyzed7. Flagella of the pf6 mutant that lack C1a8 or those of the pf16 mutant that lack the entire C1a-c-e supercomplex10 twitch ineffectively at a low frequency and with slightly modified waveform. Smaller structural defects within the C1a-c-e supercomplex such as a loss of a part of the C1c (fap76 mutant), small parts of C1c and C1e (fap216), entire C1c-e (fap81), or minor defects of C1a (fap92) only reduce the beat frequency and lead to flagella asynchrony10. In fap46 and fap74 mutants, flagella lack the entire C1d projection and the sheath between C1d and C1b (recently described as C1f3) which causes a range of defects including reduced beat frequency, twitching, and even paralysis. Moreover, flagella that are able to beat, frequently struggle to initiate the next effective stroke12. In contrast, a mutation in the Cpc1 subunit of C1b leads to a loss of the entire C1b projection and neighboring C1f, and reduces the beat frequency but does not affect the waveform14. Thus, each projection contributes to the overall ciliary motility in a unique way12. Because projections are interconnected, it is likely that a subunit loss in one projection could affect also the stability/functionality of other projections. The specific functions of individual projections remain obscure.In 15. Oda and colleagues16 showed that the expression of the C-terminally tagged radial spoke proteins partly rescues the motility defects of Chlamydomonas flagella lacking C1a (pf6 mutant) but has no effect on the movement of flagella lacking C1b projection (cpc1 mutant). Thus, likely the interaction between C1a and radial spokes is based on a mechanical collision. Such a transient physical contact between radial spoke head and a projection could involve electrostatic interactions between the negatively charged surface of the radial spoke head and CA projection17. Whether and how other projections interact with the radial spokes is less clear. In order to reveal such interactions, it is essential to identify all protein components of the CA projections and determine their individual roles in the context of ciliary motility.It has been proposed that the mechano-chemical signals originating at the CA are transmitted through the radial spokes to the inner dynein arms and regulate their activityChlamydomonas flagella isolated from wild-type and CA-less mutants revealed that the CA is composed of at least 25 proteins18. This number was significantly extended by recent comprehensive proteomic analyses20 and detailed genetic, biochemical, and microscopic studies of selected projections12.Early comparative analyses of the Chlamydomonas as a model. However, a significant number of the CA proteins are not present in other ciliated species20. Thus, it will be informative to learn more about the composition and functions of the CA subunits in other species.The vast majority of data concerning the CA was obtained using Chlamydomonas, C1b and C1f , missing in the cpc1 mutant are composed of CPC1/SPEF2, FAP42, FAP69, HSP70, and enolase14. All those proteins co-purify as a 16S complex21. Recent proteomic analyses suggest that FAP39, FAP174, FAP246, phosphoglycerate mutase, WD-domain containing CHLREDRAFT_170023, and an ankyrin domain-containing CHLREDRAFT_177061 could also build a part of either C1b or C1f or be loosely associated with these structures20. A Chlamydomonas FAP42, the adenylate/guanylate kinase-like protein with a predicted molecular mass of approximately 270 kDa, has obvious orthologs only in unicellular green algae (Chlorophyceae). This raises a question of the protein composition of C1b/C1f projections in cilia assembled by other species.In Tetrahymena and investigate their function in cilia beating regulation.Here we use genetic, biochemical, and microscopic approaches to identify the components of the C1b and C1f projections in Tetrahymena thermophila encodes two proteins with homology to Spef2/CPC1, here named Spef2A (TTHERM_01142770) and Spef2B (TTHERM_00633390). Both proteins were identified in the Tetrahymena ciliome22. However, the N-terminal calponin-homology (CH) domain was predicted only in Spef2A  or Spef2A-HA-BirA* expressing cells  showed that like Spef2-2V5, these proteins were enriched in cilia except for the distal tip  , a 102 kDa, armadillo motifs-containing protein Fig.  has orthio) Fig. B.Chlamydomonas FAP246, and human leucine-rich repeats and guanylate kinase domain-containing protein (LRGUK)  is a 53 kDa, leucine-rich repeats-containing protein, with homology to an N-terminal fragment of UK) Fig. C. In conion Fig. .Chlamydomonas FAP174 and human c-myc binding protein (MYCBP-1), and a ciliate-specific Tt170 (TTHERM_00205170) are small proteins  with predicted coiled-coils regions , orthologous to ns Figs. , S2D.24.Androglobin/Adgb (TTHERM_00290850) is a large protein (180 kDa) showing similarity to human androglobin and calpain-7 within the N-terminal region and to androglobin within the short C-terminal fragment Figs. , S2E. OrTetrahymena as fusions with a C-terminal 3HA tag under control of native promoters and interacting ciliary proteins were immunoprecipitated with resin-bound anti-HA antibodies 26 and SPEF2A-coDel, ADGB-coDel, and CFAP246-coDel mutants using the co-Deletion method based on the induction of scnRNAs27  and therefore we analyzed knockdown of CFAP246-coDel strains.Next, we engineered s27 Fig. . The tarTetrahymena cells. The CFAP69-KO and SPEF2A-KO cells on average traveled only approximately 41% and 35%, respectively, of the distance of the wild-type cells  were less affected and swam at the rate of 81% and 74% of the wild type, respectively  and 11% (5.89 ± 0.48 µm), respectively, shorter than those assembled by wild-type cells (6.56 ± 0.5 µm)  and thus, loss of C1b could destabilize the C2b (as in Chlamydomonas14) and perhaps C1d projection.The C1b is one of the largest projections of the CA microtubules. Because C1b (and possibly C1f) are either missing or greatly reduced in the CFAP69-KO, and SPEF2A-coDel mutants by mass spectrometry (six independent samples for each cell type)  Tables , S6. MS/d Tables , S6. IntChlamydomonas, enolase and HSP70 were identified as components of the CPC1 complex21. The genome of Tetrahymena encodes three enolases, Eno1 (TTHERM_00486480), Eno2 (TTHERM_00046480), and Eno3 . Eno1 and Eno2 were detected in wild-type cilia. In the CFAP69-KO and SPEF2A-coDel cilia, the levels of Eno1 were reduced, but surprisingly, the level of Eno2 was higher in SPEF2A-coDel than in the wild-type cilia , Ssa6 (TTHERM_00171850), and Hsp110/DnaK (TTHERM_00688300). The level of Hsp70 was slightly decreased in the mutant cilia Tables .Chlamydomonas cpc1 flagella, C1b may stabilize C2b14. The levels of hydin7 and putative C2b subunits, Cfap47 and Cfap26620 were reduced in the C1b/C1f-deficient Tetrahymena cilia. Additionally, we detected a reduction of Klp/Kif9, a potential subunit of Chlamydomonas C2c/C2d complexes28  or moderately (IFT-B proteins) reduced in the C1b/C1f-deficient cilia Table . These dCFAP69-KO or SPEF2A-coDel cilia . Using qPCR and genomic DNA as a template we confirmed that similar number of transgene copies were assorted in all cell strains 30 in the SPEF2A-coDel cells expressing either Cfap246-2V5 or Adgb-2V5 (under native promoters). The overproduced HA-Cfap69 was targeted to cilia  is extended by a C-terminal epitope tag16. RSP3 forms a part of the radial spoke stem while the paralogous RSP4 and RSP631 are components of the radial spoke head32. Their C-termini are located on or above the radial spoke head upper surface that temporarily comes in contact with the CA projection(s)16. In contrast, the artificial expression of RSP4 did not rescue Chlamydomonas cpc1 mutant lacking C1b/C1f complex (Table 16).The ex Table  in16.Tetrahymena, we performed similar experiments. The Tetrahymena genome encodes three proteins orthologous to RSP4/RSP6: Rsp4/6A (TTHERM_00427590), Rsp4/6B (TTHERM_00502580) and Rsp4/6C (TTHERM_00444180). According to the Tetrahymena Functional Genomics Database (http://tfgd.ihb.ac.cn/), Rsp4/6A and Rsp4/6C are expressed at higher levels than Rsp4/6B. Thus, we expressed either Rsp4/6A-GFP or Rsp4/6C-GFP (under the control of their native promoters) in wild-type cells, SPEF2A-coDel, or CFAP69-KO mutants  was reduces by approximately 15% . When Rsp4/6A-GFP or Rsp4/6C-GFP were overexpressed  and SPEF2 (p.Arg304X36) as likely MMAF causal mutations. To better understand the outcome of these mutations, we investigated the effect of corresponding mutations in CFAP69 (Ser249X) and SPEF2A (Lys304X) on Tetrahymena cells motility. We expressed HA-Cfap69-M1-R248 protein fragment in the Tetrahymena CFAP69-KO and HA-Spef2A-M1-M303 fragment (containing the CH domain) in the SPEF2A-coDel background, both under MTT1 promoter. Western blots showed that when overexpressed both truncated proteins were present in cilia  or more frequently, multiple morphological abnormalities of the sperm flagella (MMAF) syndrome69 p.Gln25X35 and 3, approximately 60% of the identified Chlamydomonas CA proteins lack obvious orthologs in other ciliated eukaryotes including humans20. These observations raise two questions. First, what is the protein composition of the CA in cilia and flagella assembled by other species and specifically, are there additional conserved components in other lineages? Second, since patterns of cilia/flagella beating vary in different species, do lineage-specific CA components contribute to the regulation of the beating patterns?In contrast to outer doublet components, the composition of the CA is poorly characterized. Surprisingly, although the overall CA architecture is similar in cilia/flagella of diverse speciesTetrahymena-specific Tt170 are likely structural components of the C1b/C1f supercomplex in Tetrahymena and that lack of the entire C1b/C1f complex changes cilium motion from two-phase to rotatory. Besides Tt170 and Adgb, orthologs of other putative C1b/C1f proteins were also suggested to build C1b/C1f structure in Chlamydomonas20. Information concerning localization and function of the novel putative C1b/C1f components are limited. Some data link these proteins with cilia/flagella in mammals. In human and mice sperm flagella, CFAP69 is present in the midpiece and its truncation results in either mislocalization or loss of SPEF235. This agrees with our data showing that Spef2A is reduced in the CFAP69-KO cilia. Mutations in CFAP69 cause male infertility37 but the connection between CFAP69 and ultrastructural changes in sperm cells is unclear.We have shown that evolutionarily conserved Spef2A, Cfap69, Cfap246, Adgb, and 38. The OSN cilia can be divided into two segments, proximal containing a CA (9 × 2 + 2) and distal with a decreasing number of microtubules (from 9 × 1 to 4 × 1)39. It will be of interest to determine whether in the OSN cilia CFAP69 is present only in the CA-containing proximal fragment or along the entire cilia length, suggesting another intraciliary localization and likely function in these sensory cilia.Interestingly, in mice, CFAP69 is also present in the immotile olfactory cilia of the olfactory sensory neurons (OSN)40. In contrast to Tetrahymena Cfap246, Chlamydomonas FAP246/CHLRE_14g618750v5 and the mammalian LRGUK are much larger proteins. LRGUK-1, besides LRR domains, also has a guanylate kinase domain while predicted FAP246/ CHLRE_14g618750v5 contains C-terminal EF-hand domains. Thus, Cfap246 orthologs could mediate protein–protein interactions (LRR) and have lineage-specific functions that involve Ca2+ signaling and local regulation of GMP, and indirectly, cGMP.Cfap246 orthologs contain LRR domains in their N-termini. The LRR domains form a horseshoe shape which provides a scaffold for protein–protein interactionshttps://www.ncbi.nlm.nih.gov/geoprofiles) and spermatozoa and in mouse Lrguk-1 localizes to the acrosome and the sperm tail42. LRGUK-1 mutant mice either lack the sperm tail or assemble a short one41. It remains to be determined if LRGUK is indeed a C1b/C1f component in mammals.In humans, LRGUK is highly enriched in the trachea, testis 43 suggested to be a component of the C2 region of the CA44. On the other hand, FAP174 co-immunoprecipitates with FAP24619. This latter result agrees with our data showing that Cfap174 is positioned near Cfap246. Surprisingly, the level of Cfap174 was unaltered in cilia of Tetrahymena SPEF2A-coDel or CFAP69-KO mutants but slightly reduced in Chlamydomonas cpc1 mutant20. Considering that C2b projection is partly or entirely missing in flagella of the Chlamydomonas cpc1 mutant, we speculate that FAP174/Cfap174 is positioned at the very distal end of C1b or C2b projection. This model is supported by following data: (1) Cfap174 co-immunoprecipitates with Adgb but not with Cfap69 which together with Spef2A likely docks the projection to the C1 microtubule, (2) the distal part of C1b but not the entire projection is altered in the Adgb knockdown cells, suggesting that Adgb forms a distal part of C1b, (3) Cfap174 is highly biotinylated in cells expressing Cfap246-HA-BirA* but not in cells expressing Spef2A-HA-BirA*, and thus likely positioned in close proximity to Cfap246. Alternatively, Cfap174 may have multiple axonemal docking sites as it was suggested for Chlamydomonas20.In Chlamydomonas FAP42 were found only in unicellular green algae. A detailed inspection of the FAP42 and Adgb sequences shows that both proteins contain a calpain-like domain near their N-termini. Thus, we speculate that the Adgb and FAP42 have similar position within C1b/C1f supercomplex. Importantly, in mammals androglobin is expressed at higher level in cells assembling motile cilia and its expression is co-regulated by FOXJ145.Orthologs of Chlamydomonas21, in Tetrahymena neither enolases nor Hsp70 were detected among proteins associated with C1b/C1f subunits  that makes their presence in the CA structure unlikely.The predicted molecular mass of FAP39/CHLRE_02g145100v5, a P-type ATPase, is 127 kDa that is close to the weight of the unidentified protein that co-sediments with CPC1/SPEF2me Table . One of me Table . HoweverTetrahymena and human genome databases did not reveal any orthologs of Chlamydomonas CHLREDRAFT_177061 or CHLREDRAFT_170023. The presence of the WD40 repeats in the latter rendered limited similarity to WD40-containing protein, FAP57. However, the Tetrahymena protein identified with the highest score in the blastp search was not detected as biotinylated in Spef2A-, Cfap69- or Cfap246-HA-BirA* expressing cells , pf18 or pf19 (katanin p60) mutants, the levels of the IFT and BBS proteins was significantly47 or slightly20 elevated compared to wild-type flagella, and IFT proteins and BBS4 were trapped in the lumen of the CA-less, detergent extracted axonemes47. The amount of the trapped IFT proteins was reduced when the CA re-assembled47. Thus, the reduced level of IFT and BBs proteins in C1b/C1f-less Tetrahymena cilia is an unexpected result. It would be interesting to investigate if the lack of other CA projections also affects the level of IFT and BBS proteins and cilia length in Tetrahymena.The levels of IFT and BBS proteins were not investigated in 48. In mammals, the differentiation of spermatids requires an intense transport of cargoes along the manchette microtubules and this process involves intraflagellar transport proteins49. However, how Spef2 is related to intraflagellar and IFT-related inter-manchette transport is to be determined.Interestingly, in mice, the IFT20 was identified as a Spef2 partner protein in maturing sperm cellsTetrahymena strains were obtained from the Tetrahymena Stock Center . Wild-type and other motile strains were grown in the SPP (Super Proteose Peptone) medium50 with the antibiotic–antimycotic mix at 1:100 . Mutants with greatly reduced motility were grown in MEPP (Modified Enriched Proteose Peptone) medium, on which cells take up nutrients without using oral cilia51, supplemented with the antibiotic–antimycotic mix at 1:30 . Cells were grown with moderate shaking (80 rpm) at 30 °C. Cell swimming and cilia beating patterns were analyzed as previously described53. To induce protein overexpression, cells were cultured in media with 2.5 µg/ml CdCl2 for 16–18 h.The wild-type CU428.2 and B2086.2 53. MluI and BamHI restriction endonucleases were used to insert a fragment of the coding region and PstI and XhoI were used to clone a fragment of 3’UTR  and genomic DNA purified from CU428.2. The primers used are listed in Table TR Table . For genFAP44 were removed from the Neo2-3HA-FAP44 plasmid53 and replaced by approximately 1 kb fragments of the 5’UTR and a coding region starting with the ATG codon of the ADGB gene, both amplified by PCR with primers listed in Table MTT1 promoter and the neo2 selectable cassette54. The plasmid was digested with SacII and BamHI to separate the transgene from the plasmid backbones prior to biolistic bombardment.To express Adgb with an N-terminal BirA*-HA in the native locus, the 5’UTR and the coding region of 55 or pMTT1-HA56 plasmids enabling the integration of the transgene into BTU1 locus and overexpression controlled by the Cd-inducible MTT1 promoter30. To select transformed Tetrahymena cells, based on paromomycin or blasticidin resistance, a neo254 or bsr cassette57 was inserted between 5’BTU1 and MTT1 promoter. Plasmids were digested with SacII and ApaI restriction endonucleases to separate a transgene from the plasmid backbone.To overexpress proteins as fusions with C-terminal -HA or -GFP tag, the entire coding region was amplified by PCR with the addition of MluI and BamHI sites at 5’ and 3’ ends, respectively Table  and clon54), 100 µg/ml paromomycin and 1.5 µg/ml CdCl2 (transgenes with neo4 cassette58), 200 μg/ml puromycin  and 1.5 µg/ml CdCl2 (transgenes with pPur cassette59), or 60 μg/ml blasticidin  (transgenes with bsr cassette57). The positive clones were grown in medium with decreasing concentrations of CdCl2 (to 0.05–0.1 μg/ml) and either an increasing concentration of paromomycin (up to 1 mg/ml) or blasticidin (up to 100 μg/ml), or a constant concentration of puromycin to promote phenotypic assortment.Approximately 10 µg of the digested plasmid DNA was precipitated onto DNAdel Gold Carrier Particles  according to the manufacturer’s instructions and used to biolistically transform CU428.2 cells. Positive clones were selected for 3–4 days at 30 °C on SPP supplied with (depending upon the introduced transgene) 100 µg/ml paromomycin  . When we attempted to engineer germ-line CFAP246 knockout cells, only few paromomycin-resistant clones were found among transformed exconjugants , suggesting a deletion of the introduced transgene during the rearrangement of the genome of new macronuclei. Therefore, in the case of the CFAP246 gene we could investigate only the effect of CFAP246 knockdown. At least two independent clones of the coDel and germ-line knockout strains were obtained.The Tetrahymena knockout cells, CFAP69-KO cells were transformed with a transgene enabling expression of the HA-Cfap69 from the BTU1 locus, under the control of the MTT1 promoter (cells were grown without CdCl2). Mutants obtained by the coDeletion approach were rescued with the approximately 3 kb fragment of the genomic DNA, encompassing the deleted region and about 1 kb upstream and downstream of the deletion. The rescued cells were selected based on the restored wild-type cell motility.To rescue A real-time PCR was carried out using genomic DNA or cDNA as a template and a PowerUp SYBR Green Master Mix  with Standard cycling protocol according to the manufacturer instruction in StepOnePlus Real-Time PCR System . A genomic DNA was purified with Tissue DNA Purification Kit  using cell culture protocol provided by the manufacturer. Total RNA was isolated with Universal RNA Purification Kit  with the on-column DNAse digestion protocol provided by the manufacturer. Approximately 500 ng of purified RNA was subjected to reverse transcription with SuperScript III First-Strand Synthesis SuperMix for qRT-PCR  and oligo dT, according to manufacturer instruction. To compare the levels of PCR products between housekeeping and experimental genes, for each gene a standard curve, with the use of know amounts of DNA (either plasmid or PCR product) was generated. For each sample, the initial amount of the genomic DNA or cDNA was estimated using Step One Plus Software.60 cells were stained with anti-α-tubulin 12G10 antibodies, and confocal images were recorded with a 0.32-µm distance between z-sections. A length of cilia was measured on merges of two to four z-sections using ImageJ bundled with 64-bit Java 1.8.0_172. The cell swimming paths and cilia beating were recorded as described53.To evaluate cilia lengthTetrahymena cells were fixed and stained on coverslips as previously described61. The primary antibodies were used at the following final concentrations: monoclonal mouse anti-HA antibodies  1:300, monoclonal rabbit anti-HA antibodies  1:300, monoclonal rabbit anti-V5 antibodies  1:1600, polyclonal rabbit anti-GFP antibodies  1:6000, anti-α-tubulin 12G10 antibodies  1:300, and the secondary antibodies, anti-mouse or anti-rabbit IgG conjugated either with Alexa-488 or Alexa-555 antibodies , all in concentration of 1:300. After washing, the coverslips were mounted in Fluoromount-G  and viewed using a Zeiss LSM780  or a Leica TCS SP8  confocal microscope.For in situ protein localization, 52 and samples were viewed using a JEM 1400 transmission electron microscope .To analyze ciliary ultrastructure cells were fixed as described5 cells from the logarithmic culture were pelleted and briefly washed with 10 mM Tris–HCl buffer pH 7.4. Pelleted cells were suspended in a buffer containing 7 M urea, 2% CHAPS, 40 mM Tris–HCl, pH 7.4, and 10 × concentrated protease inhibitors , and mixed with 5 × concentrated sample buffer . For analyses of the ciliary proteins, ~ 107 cells from the logarithmic culture were pelleted, washed with 10 mM Tris–HCl, pH 7.4, and deciliated62.For analyses of the total cell proteins, 2–5 × 10Total proteins (40 µg) or ciliary proteins, either 40 µg (detection of the tagged protein) or 2 µg (tubulin detection) were run on the SDS-PAGE gel , transferred onto nitrocellulose membrane, stained with Ponceau S, and blocked for an hour with 5% non-fat milk in the TBST  at RT. The nitrocellulose membranes were incubated overnight with the primary antibodies diluted in the blocking solution as follows: mouse monoclonal anti-HA (1:2000), rabbit monoclonal anti-V5 (1:1600), and mouse monoclonal anti-α-tubulin (12G10)  at 4 °C. After washing (4 × 10 min in TBST) membranes were incubated with the appropriate HRP-conjugated secondary antibodies  for 1 h at RT, washed and proteins were detected using Westar Supernova kit .For analyses of the biotinylated ciliary protein, samples were run on the 10% SDS-PAGE gel, transferred onto nitrocellulose membrane, and blocked overnight with 3% BSA in the TBST at 4 °C. Next, the nitrocellulose membranes were incubated for 4 h at RT with the streptavidin-HRP  diluted 1:40 000 in the blocking solution, washed and biotinylated proteins were detected using Westar Supernova kit .23 engineered Tetrahymena cells expressing either Spef2A-HA-BirA*, Cfap69-HA-BirA*, Cfap246-HA-BirA*, Adgb-HA-BirA*, or BirA*-HA-Adgb were grown in SPP medium to a density of 2 × 105 cells/ml, starved overnight in 10 mM Tris–HCl buffer, pH 7.5, and incubated in the same buffer supplied with 50 µM biotin for 4 h at 30 °C (or 16 h in the case of Adgb). Next, cilia were removed and collected62, resuspended in the axoneme stabilization buffer  and treated for 5 min on ice with 0.2% NP-40  to remove the ciliary membrane. Collected axonemes  were lysed in a buffer containing 50 mM Tris–HCl, pH 7.4, 0.4% SDS, 0.5 M NaCl, 1 mM DTT, 1 × protease inhibitors for an hour at RT. After centrifugation (8000 × g at 4 °C) the supernatant was diluted with 3 volumes of 50 mM Tris–HCl, pH 7.4, and incubated overnight with 100 µl of streptavidin-coupled Dynabeads  at 4 °C. After washing  at 4 °C, resin-bound proteins were analyzed by western blot and by mass spectrometry. For mass spectrometry analysis, resin-bound proteins were reduced by 60 min incubation with 5 mM tris(2-carboxyethyl)phosphine (TCEP) and cysteines were blocked with 8 mM methyl methanethiosulfonate (MMTS). After overnight digestion with 0.5 µg of trypsin  tryptic peptides were acidified with 0.1% trifluoroacetic acid (TFA) and subjected to LC–MS/MS analysis.To identify proteins positioned in close proximity to BirA* tagged proteinsTetrahymena cells expressing Cfap69-3HA or Adgb-3HA under the control of the respective native promoters, were grown in SPP medium to a density of 2 × 105 cells/ml and washed with 10 mM Tris–HCl buffer, pH 7.5. Next, cilia were removed and collected62, and resuspended in 10 mM Tris–HCl buffer, pH 7.5, supplemented with 2 × protease inhibitors, and combined with an equal volume of 2% NP-40 and 1.2 M NaCl in 80 mM Tris–HCl buffer, pH 7.5. After 20 min of incubation on ice, the axonemes were pelleted at 21,000 × g for 20 min. The collected supernatant was diluted 4 × with 40 mM Tris–HCl, pH 7.5 (supplemented with protease inhibitors) to reduce NP-40 and NaCl concentration and incubated with anti-HA-conjugated resin  with rotation, overnight at 4 °C. Beads were washed six times (5 min each wash) with 40 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.25% NP-40, and 0.5 mM EDTA at 4 °C. Resin-bound proteins were separated on 10% SDS–PAGE gel and silver-stained63 or eluted from resin with 50 mM NaOH and analyzed by mass spectrometry as described above.To identify proteins that co-immunoprecipitate with HA-tagged Cfap69 or Adgb, engineered 7 wild-type (control) or mutant cells from logarithmic cell culture were deciliated62. Cilia were lysed with 7 M urea, 2% CHAPS, 40 mM Tris–HCl, pH 7.4 and 100 µg of protein per sample were prepared according to FASP protocol with minor modifications64. In brief, the protein sample was diluted up to 200 µl with urea buffer (8 M urea in 50 mM ammonium bicarbonate) and incubated for an hour with 50 mM TCEP at 60 °C to reduce cysteine residues. After ultrafiltration onto 10 kDa molecular weight cut-off ultrafiltration units , samples were washed twice with urea buffer and incubated with 50 mM IAA for 30 min in dark at RT. Modified proteins were washed three times with 8 M urea buffer followed by three rinses with 50 mM ammonium bicarbonate to remove denaturant. Each time the filters were centrifuged at 14,000xg at for 15 to 30 min until the membrane was dry. Digestion was carried out overnight using trypsin/LysC mix  in a 1:25 enzyme-to-protein ratio at 37 °C on vortex. Peptides were collected from filters by centrifugation and two additional washes with 0.5 M NaCl and 50 mM ammonium bicarbonate, respectively. Combined eluates were acidified with trifluoroacetic acid (TFA) and vacuum-dried. Peptides were reconstituted in 0.1% TFA with 2% ACN and subjected to LC–MS/MS analysis.Approximately 5 × 10Three µg of peptides from each total cilia proteome sample and one third of proximity labeling assay and immunoprecipitation samples were analysed on an LC–MS system composed of an UPLC chromatograph  directly coupled to a Q Exactive or Elite mass spectrometer . Data acquisition for analysis of total cilia proteome was solely performed on Q Exactive system. Peptides were trapped on C18 pre-column  using water containing 0.1% FA as a mobile phase and then transferred to a nanoAcquity BEH C18 column  using acetonitrile gradient (0–35% ACN in 160 min) in the presence of 0.1% formic acid at a flow rate of 250 nl/min. Data acquisition was carried out using a data-dependent method with top 12 precursors selected for MS2 analysis after collisional induced fragmentation (CID) with an NCE of 27. Full MS scans covering the mass range of 300–2000 were acquired at a resolution of 70,000 with a maximum injection time of 60 ms and an automatic gain control (AGC) target value of 1e6. MS2 scans were acquired with a maximum injection time of 60 ms and an AGC target value of 5e5 with an isolation window of 3.0 m/z. Dynamic exclusion was set to 30 s.Tetrahymena thermophila SB210 database  for total cilia proteome samples or older version 20111116  for BioID/IP/gel samples. Search parameters were set as: enzyme—trypsin, missed cleavages—2 or 1 for BioID/IP/gel samples, fixed modifications—carbamidomethyl (C) or methylthio (C) for BioID/IP samples, variable modifications—0xidation (M), instrument—HCD. Moreover, in the total cilia proteome samples, to reduce mass errors, after a measured mass recalibration, mass tolerance settings of the peptide and fragment were established separately for individual LC–MS/MS runs65, resulting in 5 ppm (for parent) and 0.01 Da (for fragment ions) values. Data were searched with automatic decoy option.MS/MS data were pre-processed with Mascot Distiller software  and a search was performed with the Mascot Search Engine  against the NCBInr RefSeq q-values > 0.01 were removed from further analysis. A protein was regarded as confidently identified if at least two of its peptides were found. Proteins identified by a subset of peptides from another protein were excluded from analysis. The mass calibration and data filtering were carried out with MScan software, developed in-house (http://proteom.ibb.waw.pl/mscan/).The statistical significance of peptide identifications was estimated using a joined target/decoy database search approach. This procedure provided q-value estimates for each peptide spectrum match (PSM) in the dataset. All PSMs with 66. The abundance of each peptide was determined as the height of a 2-D fit to the monoisotopic peak of the tagged isotopic envelope. Quantitative values were next exported into text files, along with peptide/protein identifications, for Diffprot softwere for non-parametric statistical analysis of differential proteomics data65 Diffprot was run with the following parameters: number of random peptide sets = 106; clustering of peptide sets—only when 90% identical; normalization by LOWESS. Only proteins with q-value below 0.05 or those present in only one of two compared analytical groups were taken into consideration during further analysis.The lists of peptides that matched the acceptance criteria from the LC–MS/MS runs were merged into one common list and overlaid onto 2-D heat maps generated from the LC–MS raw files A more detailed description of the quantitative extraction procedure implemented by our in-house software is available inChlamydomonas or human proteins as baits. The sequences of Tetrahymena orthologs were obtained from Tetrahymena Genome Database . The protein amino acid sequences were aligned using the ClustalX2 program67, edited using the SeaView program68, and the similar/identical amino acid residues in the multiple sequence alignments were highlighted using the GeneDoc program69. The domain analyses were performed using InterPro (https://www.ebi.ac.uk/interpro/)71.The orthologs of Spef2, Cfap69, Cfap246, Cfap174, and Adgb were identified in the NCBI database using Blastp search and Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.Supplementary Information 12.


Escherichia coli S17-1, the model phyllosphere coloniser Pantoea eucalypti 299R, and the model pathogen E. coli O157:H7 to the recipient strain E. coli O157:H7::MRE103 (EcO157:H7red) in the phyllosphere of Arabidopsis thaliana was determined. The results suggest that short-term occurrence of a competent donor is sufficient to fix plasmids in a recipient population of E. coli O157:H7red. The spread of self-transmissible plasmids was limited after initial steep increases of transconjugants that contributed up to 10% of the total recipient population. The here-presented data of plasmid transfer will be important for future modelling approaches to estimate environmental spread of antibiotic resistance in agricultural production environments.Many antibiotic resistance genes present in human pathogenic bacteria are believed to originate from environmental bacteria. Conjugation of antibiotic resistance conferring plasmids is considered to be one of the major reasons for the increasing prevalence of antibiotic resistances. A hotspot for plasmid-based horizontal gene transfer is the phyllosphere, i.e., the surfaces of aboveground plant parts. Bacteria in the phyllosphere might serve as intermediate hosts with transfer capability to human pathogenic bacteria. In this study, the exchange of mobilisable and self-transmissible plasmids via conjugation was evaluated. The conjugation from the laboratory strain  With the introduction of penicillin in the 1940s, mankind entered the era of antibiotics (AB), which revolutionised therapeutic medicine . For theMany ABR genes present in human pathogenic bacteria are believed to originate from environmental bacteria ,9,10,11.In today’s intensive agricultural production, fertilisers are needed to replenish soil nutrients, such as nitrogen and phosphorus. They are essential for crop growth and increased crop yield. Animal manure is an excellent source for such nutrients, but it often originates from intensive animal husbandry farms, where the widespread use of AB to preventively treat animals is the rule rather than the exception . This leEscherichia coli O157:H7::MRE103 Δstx (EcO157:H7red) recipients on Arabidopsis thaliana rosettes. The exchange of the mobilisable plasmid pUC18T-mini-Tn7T-Gm-eYFP (pUC18) and self-transmissible ABR-carrying plasmids pKJK5::Plac:gfp (pKJK5) and RP4::Plac:gfp (RP4) via conjugation by filter mating and in the phyllosphere of A. thaliana was evaluated. Donors are either the model phyllosphere colonising strain Pantoea eucalypti 299R (Pe299R), the non-pathogenic laboratory strain E. coli S17-1 (EcS17-1), or E. coli O157:H7 Δstx (EcO157:H7)  . The assEcO157:H7red (Pe299R (pKJK5), all donors were able to transfer plasmids to EcO157:H7red. In the case of EcS17-1 being the donor, all plasmids were transferred at high rates, and the transconjugant frequency was between 100 and 6.31 × 10−2 per recipient cell depending on the transmitted plasmid (transconjugant frequencies pKJK5 < pUC18 < RP4). When Pe299R was donor of RP4 or pKJK5, transconjugants were on average detected at frequencies of 6.82 × 10−5 or below the limit of detection, which was 9.62 × 10−6 per recipient cell, respectively. EcO157:H7 donors transferred the plasmids pKJK5 and RP4 with the highest efficiency to EcO157:H7red with transconjugants being detected at frequencies of 3.16 × 101 and 7.94 × 101 (transconjugant frequencies RP4 < pKJK5). Since, in contrast to EcS17-1, Pe299R and EcO157:H7 are not able to mobilise plasmids, they were not tested as donors of pUC18.Matings on nitrocellulose filters were performed to determine transconjugant frequencies of the recipient 57:H7red . BesidesA. thaliana, we inoculated EcS17-1, Pe299R, and EcO157:H7red  onto axenic plants. When grown individually, all bacterial strains, including the auxotrophic laboratory strain EcS17-1, were able to grow to high densities on A. thaliana, reaching CFU counts of 108−1010 bacteria per gram plant material after 4 and 12 days post-inoculation (d.p.i.) , the EcO157:H7red population reached similar densities as grown on A. thaliana only, i.e., EcO157:H7red multiplied to densities of >108 CFU g−1 and maintained those densities until the end of the experiment. In contrast, EcS17-1 (pKJK5) increased to a maximum of >106 CFU g−1 after 24 h, but then the population steadily declined below 106 CFU g−1 until 7 d.p.i. (EcO157:H7red was co-cultured with EcS17-1 (RP4), the initial population size of EcS17-1 (RP4) was 107 CFU g−1 and the population did not further increase and steadily declined during the experiment (EcO157:H7red population increased by two magnitudes to 5 × 108 CFU g−1 and remained stable. When in co-culture with EcS17-1 (pUC18), EcO157:H7red showed a strong initial increase and reached a maximum density of >109 CFU g−1, 7 days after inoculation (EcS17-1 (pUC18) reached a maximum density of ≈105 CFU g−1, 1 day after inoculation. Afterwards, the density remained stable and started declining 7 days after inoculation.To determine the ability of the different strains to colonise (d.p.i.) A; similaferently B,C. When7 d.p.i. B, top. Wperiment B, middleculation B, bottomPe299R (pKJK5), the EcO157:H7 population increased by one magnitude to 107 CFU g−1 and never reached densities of above 108 CFU g−1, while Pe299R (pKJK5) increased from 106 CFU g−1 to 109 and remained stable for the duration of the experiment (Pe299R (RP4), both EcO157:H7red and Pe299R (RP4) reached densities of approximately 108 CFU g−1 and remained stable thereafter  reached cell numbers above 108 CFU g−1  or EcS17-1 (RP4), on average, more than 103 EcO157:H7red (pKJK5) and ≈102 EcO157:H7red (RP4) transconjugants g−1 plant were detected  and EcO157:H7red. After co-inoculation of Pe299R (RP4) and EcO157:H7red, 5 × 102 EcO157:H7red transconjugants were detected 7 days after inoculation , on average, 103 EcO157:H7red (pUC18) transconjugants g−1 plant were detected, reaching a maximum of ≈105 transconjugants g−1 plant 7 d.p.i. (EcO157:H7red (pUC18) transconjugants reached 10−4 per recipient cell after 7 days and did not decrease within 14 days, despite the lack of selective agents  and a potential fitness cost of the plasmid  reached a maximum density at day 1 after inoculation and then started declining. The donor was outcompeted by the recipient during the experiment  donor, and EcO157:H7red, we inoculated recipient ratios 1:10, 1:5, 1:2, 2:1, 5:1, and 10:1 onto A. thaliana plants. All ratio mixtures yielded >104 transconjugants per gram of plant after 24 h, and the transconjugant population levelled off between 106 and 107 transconjugants per gram of plant , all donor strains tested were able to transfer plasmids in measurable rates to the model human pathogenic EcO157:H7red after being co-inoculated onto nitrocellulose filters, although Pe299R did so at a much lower frequency compared to EcS17-1. The reason for this transfer barrier [Pe299R was a competent recipient of the self-transmissible plasmid, that EcO157:H7red had no issue with receiving the same plasmids from EcS17-1, and that both donor and recipient are members of the family Enterobacteriaceae.To study the probability of horizontal gene transfer towards pathogenic bacteria on plant leaf surfaces, we established a model system for the exchange of self-transmissible- and non-self-transmissible but mobilisable plasmids. The model system provided insights into the conjugation between Enterobacteriaceae in the phyllosphere of  barrier ,32 is cuEcO157:H7red outgrew EcS17-1 and thereby outcompeted it. This is not unexpected, since both strains of E. coli should have a close to identical resource demand and EcS17-1 is an auxotroph, lab-adapted strain [Pe299R carrying plasmid pKJK5, EcO157:H7red did not reach the same high densities as in a monoculture, and the cell density was decreased to less than 107 CFU on average. Potentially, Pe299R is outcompeting EcO157:H7red due to nutritional competition [Pe299R produces antibiotics which inhibit the growth of EcO157:H7red [Pe299R genome [Pe299R colonies on agar plates indicative for growth inhibition of EcO157:H7red (data not shown).In planta, d strain , which ipetition . There i57:H7red  as no anR genome  and no gEcS17-1 containing different self-transmissible and mobilisable plasmids with EcO157:H7red as recipient, transconjugants could be detected after 24 h . To determine total colony forming units (CFU) of E. coli after conjugation experiments, M9 minimal medium agar containing lactose as sole carbon source , 2 mL 1 M MgSO4, 1 mL 0.1 M CaCl, 40 mL 10% w/v lactose solution) or LB supplemented with rifampicin were employed. E. coli colonies were assessed after 7 days of incubation at room temperature, Pe299R colonies on the same agar plates after additional 7 days of incubation. To select for EcO157:H7red transconjugants, we employed M9 minimal medium agar containing lactose as sole carbon source and appropriate antibiotics. EcS17-1 CFU were determined by plating on LB agar containing streptomycin. To select for EcO157:H7 (RP4) donor cells, we used LB containing kanamycin . Where appropriate, antibiotics were used in the following concentrations: kanamycin 50 µg mL−1, gentamicin 15 µg mL−1, streptomycin 100 µg mL−1, and rifampicin 100 µg mL−1.Strains and plasmids used in this study and their abbreviations are listed in E. coli [tra operon, which EcS17-1 contains in its chromosome.The plasmids employed in this study  are the E. coli . For horAn overview of the performed conjugation experiments is shown in −1 NaCl, 0.24 g L−1 KCl, 1.42 g L−1 Na2HPO4, 0.24 g L−1 KH2PO4) by vortexing and pipetting, washed twice by centrifugation at 3500× g, and resuspended in 10 mL 1 × PBS. Optical density at 600 nm was determined for the cell suspensions, and 109 CFU of donors and recipients, i.e., 1 mL of OD600nm = 1 were mixed and concentrated by centrifugation. The mixtures were resuspended in 100 µL 1 × PBS, pipetted onto a nitrocellulose filter , placed on top of LB agar plates, and incubated at 30 °C. Bacteria were harvested after 24 h by placing the filter in an Eppendorf vial containing 1 mL 1 × PBS. The vial was vortexed until the complete bacterial biomass was dislodged and resuspended. From this suspension, a serial dilution was prepared up to 10−11, and 3 µL droplets were plated onto M9 lactose agar containing appropriate antibiotics to select for transconjugant EcO157:H7red. Conjugation frequency was calculated by the ratio of EcH157:H7red transconjugants to the total EcO157:H7red population. Conjugation data are known to be log-normal distributed; thus, transconjugant and donor CFU numbers were log10 transformed before mean values were calculated.To determine in vitro conjugation rates, we grew donors and recipients as described above. To prepare conjugation mixtures, we harvested a loop-full of cell material from freshly grown bacterial lawns on agar plates. Each individual strain was resuspended in 1 mL 1 × PBS , 0.83 mL ddH2O, 20 µL 20% Triton × 100). The seeds were then incubated under constant agitation for five minutes before they were collected by centrifugation at 1500× g for 1 min. To remove residual sterilisation solution, we washed the seeds five times by adding 1 mL sterile water, centrifuging them, and then discarding the supernatant, after which we added 1 mL of sterile water. For stratification, seeds were stored at 4 °C for four days.−1 MS powder including vitamins , 10 g L−1 sucrose, 5.5 g L−1 plant agar (Duchefa), pH adjusted to 5.8), after which the plates were exposed to UV light in a laminar flow for 15 min [® and placed in a translucent plastic bag. Plants were then grown in a plant growth chamber  at long day conditions . Plants were grown 3 to 3.5 weeks and developed between six to eight leaves before they were inoculated with bacteria.For plant cultivation, all wells of 24-well microtiter plates were filled with 1 mL ½ strength Murashige and Skoog (MS) agar , 2:1 (OD600nm 0.1/0.05), 5:1 (OD600nm0.25/0.05), or 10:1 (OD600nm 0.5/0.05). For experiments described in 600nm 0.05/0.1), 1:5 (OD600nm 0.05/0.25), 1:10 (OD600nm 0.05/0.5), 2:1 (OD600nm 0.1/0.05), 5:1 (OD600nm 0.25/0.05), or 10:1 (OD600nm 0.5/0.05). The inoculated plants were further incubated at standard growth conditions . Plants were harvested at different time points, and bacteria were washed off to determine the CFU of each strain and transconjugants. To that end, 3 individual plants per treatment were individually processed. Plants were harvested using sterile forceps and the roots cut from the plants on a sterile surface with a sterile scalpel. Plants were transferred to pre-weighed 2 mL tubes and their weight was determined. To dislodge bacteria from plants, we added 1 mL 1 × PBS to a tube, vortexed it for 15 s, and after 7 min of sonication vortexed it again for 15 s. A total of 100 µL of the wash was spread on M9lactose + appropriate antibiotic to select for transconjugants when EcS17-1 or Pe299R were used as donors. When EcO157:H7 was used as a donor, transconjugants were selected on LBrif + appropriate antibiotic. To extend the range of transconjugants detection, we performed a 10-times dilution series from the leaf wash and placed 3 µL droplets on appropriate agar selective for transconjugants.Bacterial strains were grown overnight on LB agar plates containing appropriate antibiotics. Freshly grown colonies of each bacterial strain were harvested using an inoculation loop, and the bacteria were resuspended in 10 mL 1 × PBS, washed twice by centrifugation at 3500× Data were statistically analysed using the software Prism 7 . All CFU were log-transformed before plotting or statistical tests were performed. To accommodate values below the limit of detection, we added a 1 to all values. To compare the difference of the mean between treatments, we performed a one-way ANOVA using Kruskal–Wallis test with Dunn’s correction for multiple comparisons.


This study investigated the efficacy of a novel surgical method that relies on the transient fixation of L4 in Lenke Type 5C and 6C adolescent idiopathic scoliosis. Thirty-six transient surgically treated L4 fixation patients were retrospectively evaluated. The first surgery involved mechanical correction of scoliosis; the lowest instrumented vertebra (LIV) was L4. After an average of 1.3 years , the second surgery to remove transient L4 pedicle screws was performed. Radiographic parameters and SRS-22 scores were measured. Cobb’s angle, coronal balance, LIV tilting angle, and LIV coronal disc angle clearly improved after the first surgery (p < 0.01). After the second surgery, the corrected Cobb angle (p = 0.446) and coronal balance were maintained (p = 0.271). Although L3/S1 lumbar lordosis decreased after the first surgery (p < 0.01), after removal of transient L4 pedicle screws, it recovered slightly (p = 0.03). Similarly, the preoperative L3/4 lateral disc mobility eventually recovered after transient L4 screw removal (p < 0.01). The function domain of the SRS-22 showed better scores after removal of transient L4 screws (p = 0.04). L4 transient fixation surgery is beneficial for Lenke Type 5C and 6C scolioses that do not fully satisfy LIV (L3) criteria. It preserves L3/4 disc motion, increases functional outcomes, and maintains spinal correction and coronal balance. Since then, many studies have been conducted investigating the nature of the idiopathic scoliosis curve. In 1962, the development of a metal vertebral fixation device by Harrington significantly altered scoliosis treatment 2. Subsequently, the King and Moe classification system was used to identify spine curvature types and allowed surgeons to determine a fusion range during spinal surgery 4. In the 1980s, following an introduction of thoracic pedicle screw system, a new classification for idiopathic scoliosis was developed by Lenke et al. 5, which attempted to standardize and minimize fusion levels. However, for Lenke Types 5C and 6C scolioses, the optimal distal fixation level remains debatable.The surgical treatment of scoliosis was first reported in 1924 by Russell Hibbs 6. However, it is too complicated to apply these six criteria, so other authors emphasized only two of them: (1) the degree of L3 rotation is below the Nash-Moe Grade II, and (2) L3 is the stable vertebra on bending radiographs 7. Wang et al. 8 suggested that L3 should be a distal fusion level; (1) if L3 presents < 28-mm translation and (2) if L3 exhibits < 25° tilt. These overwhelming variations in L3 fixation criteria negatively impact the decision-making process of the distal fusion level in surgical treatment for idiopathic scoliosis in actual clinical practice. This study aimed to introduce a novel surgical method of transient fixation up to the L4 vertebrae for non-canonical scoliosis patients who do not fully meet one of the aforementioned criteria in Lenke Types 5C and 6C.Scoliosis requires a relatively long level of fusion. Thus, excessive pressure is applied to the distal level disc. Reduction of one distal fusion level in the lumbar spine might be crucial for better long-term outcomes. However, in Lenke Types 5C and 6C, the distal fusion level is set to L3 or L4, despite the lack of accepted standards. Lenke insisted that if patients met the following criteria, only L3 could be fixed: (1) less than grade I rotation of L3, (2) L3 tilt < 30° and L4 tilt < 20°, (3) bisected L4 vertebral body from the center of the sacrum vertical line (CSVL), (4) apical disc located above L1-L2, (5) parallel L3-L4 opening or opposite of the L4-L5 disc level, and (6) centered location of L3 Thirty-six patients were included in the study  Fig. . The aveThe mean preoperative Cobb angle for the thoracolumbar/lumbar region was 50.2° ± 7.8°. It was decreased to 11.7 ± 4.6° after correction surgery. As shown in Table The preoperative L3/4 lateral disc mobility was 6.6 ± 1.8°. After the first surgery, including the fixation with L4 pedicle screws, the L3/4 lateral disc motion angle could not be measured because the L4 vertebra was fixed. However, at the final follow-up after the second surgery, despite the angle being significantly reduced to 4.3 ± 1.7° , L3/4 lateral disc mobility was eventually restored after the removal of the L4 transient pedicle screws. Table . The repAmong the SRS-22 scores consisting of four domains , only self-image improved (p < 0.01) after the first surgery. While, following the removal of the L4 transient screws, patients reported better scores only in the function domain of the SRS-22 (p = 0.04) Table .Table 3CIn the earlier L4 transient fixation procedures, we had recommended the removal of the L4 screws about 1 or 2 years following the first surgery due to the possibility of the worsening of the scoliotic curve. However, we did not experience worsening of cases; we tried shortening the interval period for the restoration of segment lumbar motion to 3 months after the first surgery. There were no cases of correction loss or the adding-on phenomenon requiring revisional surgery. However, for one patient, the breakage of L4 pedicle screws was identified 2 years after the first surgery. In contrast, one patient who had the L4 screws removed 4-months after the first surgery exhibited an increase in the LIV (L3/4) coronal disc angle to 9.5°. In the first case, we just removed the broken screws and cut the rods. In the latter case, the LIV (L3/4) coronal angle increased by 9.5°, which was not considered as a marked loss of coronal balance. Therefore, we observed the patient, and it was maintained without more aggravation. The average period for the second surgery, L4 screws removal, was 1.2 ± 0.3 h, and the estimated blood loss was 102 ± 23.5 ml. No deep wound infections were noted in the patients during the first and second surgery (data not shown).14. A 40-year follow-up study by Lander et al. 12 indicated that there was a higher risk of additional surgery and poorer functional outcome after L4 or below L4 fixation in idiopathic scoliosis surgery. In a 19-year follow-up study by Bartie et al. 15, pain intensity for non-scoliosis controls was equivalent to L2 and L3 fusion patients; L4 fusion patients experienced increased levels of pain. However, a small study with 30 subjects and a 2-year follow-up reported no differences in clinical results between the L3 fixed group and the L4 fixed group 16. Despite these issues, many researchers have attempted to preserve the L4 vertebra by setting the L3 vertebra as the LIV in patients with Lenke 5C or 6C type scoliosis 17. However, we often encounter cases that partially fulfill the criteria for L3 LIV. In our study, if one of the L3 selection criteria was violated, then L4 transient fixation was performed. Therefore, 36 cases (61%) comprised the transient L4 group.Despite advances in scoliosis surgeries, there is still a major controversy regarding the distal fusion level for idiopathic scoliosis surgery 18. After removal of the L4 screws in the second surgery, correction loss did not occur. In our study, the coronal balance correction ratio from transient L4 fixation procedures was approximately 54.6%. Comparing this ratio with the one from Ding et al.'s study 16, this correction ratio was arithmetically superior compared to the 50% (6.5 ± 5.5/13.0 ± 9.0 mm) correction ratio observed when L3 was fixed. However, it was inferior to 65% (7.2 ± 6.8/21.1 ± 11.1 mm) when L4 was fixed. Recently, Shu et al. 11 reported a coronal balance correction ratio of 22% in a distal adding-on group when the LIV presented L3 in Lenke Type 5C scoliosis; however, it was 57% in patients not exhibiting adding-on phenomenon 19. Considering results from previous studies, we could assume that the transient L4 fixation might correct coronal balance effectively even without fusion up to the L4 and might lower the potential for adding-on phenomenon.The correction rate of the main thoracolumbar/lumbar curve because of our transient L4 fixation surgery was 76.6%, which was similar to previous studies 9 reported an increased LIV tilting angle of 11.0 ± 5.3° when the LIV was L3 in patients with L3 rotation above those of Nash-Moe Grade II and the L3 did not cross the CSVL on the bending film. Whereas, in the group in which L3 crosses the mid-sacral line without rotation inferior to grade II, the LIV tilting angle was 3.3 ± 2.9°. The former group satisfied two of the three criteria for transient L4 fixation in this study. Similarly, Shu et al. 11 reported worse results with a LIV tilting angle of 11.0 ± 3.5° in patients with adding-on phenomenon than the group without adding-on phenomenon with LIV (L3) in Lenke Type 5C scoliosis. In this study, the final LIV (L3) tilting angle was 4.7 ± 2.2°. The LIV (L3/4) coronal disc angle at the final follow -up (2.5 ± 1.6°) was not significantly different from values after first surgery . Additionally, similar to the LIV tilting angle, Chang et al. 7 reported worsening of LIV (L3/4) coronal disc angles of 3.8 ± 2.2° in the L3 rotation group than in Nash-Moe Grade II or in cases where L3 did not cross the CSVL on bending film compared to the group of L3 cases crossing the mid-sacral line with no rotation of below grade II . These results indicated that transient L4 fixation had the advantages of maintaining the LIV tilting angle and LIV coronal disc angle even in patients presenting severe grade of L3 rotation and translation without adding-on phenomenon.The LIV tilting angle and LIV coronal disc angle are important radiographic factors for evaluating the results of lumbar type C scoliosis surgery. Kim et al. When performing L4 transient fixation, there were two major concerns. First, if the fixed period is more than a few years, the L3/4 disc may or may not move after L4 screw removal. Second, however, if the fixed period is only a few months, the corrected deformity might reappear. After the second surgery, the L3/4 lateral disc mobility was maintained at 65% of the original value, and the L3/S1 lordosis slightly improved after the removal of L4 screws, suggesting that a transient fixation of the L3/4 facets maintains function in most cases without correction loss. Therefore, removing the L4 screws after a certain period had no impact on the results of the lumbar C scoliosis surgery. Nonetheless, the screw broke 2 years after the first surgery in one case, and the LIV (L3/4) coronal angle increased in one case in which the transient L4 screws were removed 4 months after the first surgery. These results suggested that the timing for removing the screw requires careful attention.24, SRS-22 analysis revealed a statistically significant difference in the self-image domain comparing pre- and post-operation evaluations. However, the change in self-image domain was not large, which may be due to a large proportion of patients indicating a maximum score in the questionnaire. Interestingly, despite minimal changes, the function analysis from the SRS-22 indicated a statistically significant difference between the first and second surgery. This main difference was due to one question which assesses back function limitations. According to Carreon et al., 25 the minimum clinically important difference in function domain in SRS-22 scores after surgical correction of AIS is 0.08. Therefore, the statistically significant increase of 0.2 in the function domain in this study is sufficient to produce a significant clinical improvement.Similar to other studies This study has a few limitations. First, it was a retrospective study. Second, the patients included in this study were relatively small. Third, the optimal timing of L4 transient screw removal was not adjusted because this study only investigated the data retrospectively, and the interval became shorter when we applied the surgical method. However, it should be noted that the new concept of the surgical method could be applied in patients with Lenke Type 5C and 6C scoliosis. Fourth, the real effect of transient L4 fixation on L3/4 facet joints was not evaluated because postoperative computed tomography scan was not performed routinely in all patients. With more patients and a prospective study design, the optimal interval and real effect on facet joints of transient L4 screw fixation is recommended for future evaluation.In conclusion, the L4 transient fixation surgery method preserves L3/4 disc motion and increases functional outcome while obtaining equally improved correction and coronal balance outcomes in Lenke Type 5C and 6C scoliosis cases, which did not fully satisfy the L3 LIV criteria.This study was approved by the institutional review board of Gangnam severance hospital (IRB No. 3–2020-0119). All investigations were carried out in accordance with relevant guidelines and regulations of our licensing committee. The requirement of informed consent was waived by the institutional review board of Gangnam severance hospital because this was a retrospective study.We reviewed 549 cases of patients with adolescent idiopathic scoliosis (AIS) who underwent surgical treatment between March 1996 and December 2017 in our hospital. Of these, 78 cases were classified as Lenke Type 5C or 6C. Among 78 cases, 16 cases lacking suitable data relative to serial follow-up whole spine X-ray, and three cases lost follow-up were excluded from the study. Based on the lowest instrumented vertebra (LIV), 20 cases in which LIV was L3 or L5 were excluded. We also excluded three cases with a permanent fixation of L4. Overall, 36 Lenke 5C or 6C cases treated with L4 transient fixation were analyzed  L3 tilt of < 25°, (2) L3 rotation less than Nash-Moe Grade II on bending film, and (3) L3 crossing the CSVL on bending film 9. If all three criteria were satisfied, L3 was determined as the distal LIV. If none of the criteria were present, L4 or L5 permanent fixation was performed. When one or two of the three criteria were not satisfied, L4 transient fixation was performed. The bilateral facet capsule of L3-4 was preserved during the exposure, and heads of L4 pedicle screws were placed slightly above the capsule, which was meant not to violate the capsule and facet joints. At the second surgery, a small incision was made on the distal part of the preoperative scar, and L4 pedicle screws were exposed. The caps of pedicle screws were untightened. The distal rods were cut using a rod cutter and removed. Then, bilateral L4 pedicle screws were removed.All 36 patients underwent instrumented spinal fusion using CD Horizon spinal instrumentation . We used a slightly modified Lenke criteria set In the early phase of this study, because we removed the transient L4 screws 1 or 2 years later, we recommended the application of a hard brace for 3 months after the first surgery and a return to low-level sports activity, such as jogging. After the second surgery, we applied the hard brace only for 2 weeks and did not limit activity. In the later phase of this study, we recommended only activities of daily living with the 3-month hard brace application after the first surgery. Then, we removed the L4 transient screws. Afterward, we extended the application of the hard brace for an additional 2 weeks. After that period, we gradually permitted low-level sports activities.The patients’ age and Risser stage on preoperative pelvis anteroposterior (AP) X-ray were investigated. All radiologic parameters were measured at three-time points; prior to the first surgery, before the second surgery of L4 screw removal, and at the final follow-up after removal of the L4 screw. Consequential radiologic changes were assessed after the removal of transient L4 pedicle screws. The following radiographic parameters were investigated; (1) Cobb angle (Thoracolumbar/lumbar and thoracic), 2) shoulder balance (coracoid head difference (CHD)), (3) sagittal vertical axis, (4) coronal balance, (5) LIV (L3) tilting angle, (6) LIV (L3/4) coronal disc angle on standing whole spine posteroanterior (PA) images, (7) L3-S1 lordosis on standing whole spine lateral images, and (8) L3/4 lateral disc mobility on full flexion–extension dynamogram of the lumbar spine. Methods used for the measurement of angles are shown in Fig.  shoulderData from SRS-22 scores were also collected at the three-time points to assess patient-centered outcomes, including pain, self-image, function, and mental health.Radiographic parameters and each SRS-22 domain were compared by calculating the mean and standard deviation (SD). A paired t-test was used to compare radiographic parameters and SRS-22 scores between those at the baseline, after the first surgery, and after the second surgery. Intra- and inter-observer agreements were assessed using the intraclass correlation coefficient (ICC). The ICC with 95% confidence interval (CI) was also calculated, comparing the mean of all two trials for the two observers. ICCs < 0.40 indicate poor; 0.40–0.75, fair or good; and 0.75–1.00, excellent reliability. A p-value < 0.05 was considered to be statistically significant. SAS v9.3  was used for the statistical analyses.