Cytokines play multiple roles during neuro-inflammatory processes and several cytokines have been studied in the context of specific diseases. This study provides a comprehensive picture of cerebrospinal fluid (CSF) changes during neuro-inflammation by analyzing multiple cytokines in combination with immune cell subsets and standard CSF parameters.n = 18), secondary progressive MS (n = 8), neuro-syphilis (n = 6), Lyme neuro-borreliosis (n = 13), bacterial and viral meningitis (n = 20), and patients with non-inflammatory neurological diseases . Cytokine concentrations were correlated with CSF standard parameters and CSF immune cell subsets  quantified by flow cytometry.Using multiplex assays, we simultaneously measured 36 cytokines  in the CSF and serum of 75 subjects. Diagnoses included clinically isolated syndrome and relapsing-remitting multiple sclerosis . Within the CSF compartment, CXCL13 showed a strong association with B cells when analyzing all patients, as well as patients with an intact blood-brain barrier (BBB). NK cells positively correlated with CSF concentrations of multiple cytokines (22/36) when analyzing all patients. These correlations were maintained when looking at patients with a disrupted BBB but not detectable in patients with an intact BBB.We observed increased levels of multiple cytokines (26/36) in patients with neuro-inflammatory diseases when compared to NIND that consistently correlated with CSF cell count and QUnder conditions of neuro-inflammation, multiple CSF cytokines are regulated in parallel and most likely produced locally. A combined increase of CSF CXCL13 levels and B cells occurs under conditions of an intact BBB. Under conditions of a disrupted BBB, CSF NK cells show significantly increased values and seem to have a major contribution to overall inflammatory processes, reflected by a strong correlation with multiple cytokines. Future studies are necessary to address the exact kinetics of these cytokines during neuro-inflammation and their relation to specific diseases phenotypes. Cytokines are small proteins that are secreted by various cell types and play multiple roles during development, homeostasis, and immune regulation . AccordiSeveral studies on CSF cytokines are available in patients with multiple sclerosis (MS). CSF levels of TNF-α, IL12, CXCL9, CXCL10, CCL17, CCL21, CCL22, IL10, IL6, IL23, IL17, IL8, CXCL13, CCL19, and CCL5  have been shown to be consistently upregulated , 41. LatAlbumin), and immunoglobulin (Ig) indices. In addition, we also performed correlation analyses with different CSF immune cell subsets including CD4 and CD8 T cells, B cells, plasmablasts, NK cells, and monocytes that have been routinely quantified by flow cytometric analyses.In order to obtain a more comprehensive picture of CSF cytokine profiles in neurological diseases, we measured the concentrations of 36 cytokines in the CSF and serum of 75 patients using multiplex assays. Diagnoses included non-inflammatory controls (NIND), multiple sclerosis (RRMS) and clinically isolated syndrome (CIS), secondary progressive MS (SPMS), neuro-syphilis (Lues), Lyme neuro-borreliosis (LNB), and bacterial and viral meningitis. Cytokine values were correlated with CSF standard parameters such as CSF cell count, albumin quotient (Qn = 10), relapsing-remitting multiple sclerosis , clinically isolated syndrome , secondary progressive MS , neuro-syphilis , Lyme neuro-borreliosis (n = 13), and bacterial (n = 10) and viral meningitis (n = 10) were included in our analysis. Patients with NIND suffered from normal pressure hydrocephalus (n = 3) and pseudotumor cerebri (n = 7). In patients with bacterial meningitis (n = 10), the following specific pathogens were identified: Streptococcus pneumonia (n = 2), Haemophilus influencae (n = 2), Listeria monocytogenes (n = 1), suspicion of Mycobacterium tuberculosis (n = 3), and undetermined, presumably bacterial pathogens (n = 2). In viral meningoencephalitis (n = 10), varicella zoster virus (n = 2), herpes simplex virus (n = 3) and undetermined presumably viral pathogen (n = 5) were detected. Further details are displayed in Table All patients were recruited at the Department of Neurology of the Technical University of Munich. CSF samples were obtained for routine diagnostic work-up, and patients consented to the scientific use of their biosamples. The ethics committee of the Technische Universität München approved the scientific use of CSF biosamples. Patients with non-inflammatory diseases  to obtain CSF cell count. Total protein, albumin, IgG, IgM, and IgA concentrations in CSF and serum were determined by nephelometry according to the manufacturer’s instructions . Oligoclonal bands were investigated by isoelectric focusing followed by silver staining.+CD3+CD4+), CD8 T cells (CD45+CD3+CD8+), monocytes (CD45+CD14+), NK cells (CD45+CD56+), B cells (CD45+CD19+CD138−), and plasmablasts (CD45 CD19+CD138+).Flow cytometric analysis of immune cell subsets was performed as described previously . ShortlyIn order to measure several cytokines in parallel, we performed multiplex assays (BioRad #171304070 M (10-Plex), #171AK99MR2 (40-Plex)) according to the manufacturers’ instructions. The 40-Plex kit contained the following cytokines: CCL1, CCL11, CCL13, CCL15, CCL17, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL3, CCL7, CCL8, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, GM-CSF, IFNγ, IL10, IL16, IL1β, IL2, IL4, IL6, MIF, and TNF-α. The 10-Plex Kit included the cytokine antibodies GCSF, IL12, IL17A, IL2, IL4, IL5, IFNγ, MIB1β, CCL5 (RANTES), and TNF-α. In order to enable an optimal comparability between different patient groups within one compartment, all CSF samples were measured on a single multiplex plate; all serum samples were measured on a single multiplex plate for each kit, respectively. CSF samples were applied undiluted; serum was pre-diluted 1:4. Multiplex plates were measured on a Luminex MAGPIX®. Standard curves and values were calculated using xPONENT 4.2 software for MAGPIX®. The required amount of 50 beads per analyte was consistently detectable for all analytes in the 40-Plex Kit. Although the 10-Plex Kit was used according to the manufacturer’s instructions, less than the required 50 beads per analyte were detectable for most cytokines. For this reason, only MIB1β from the 10-Plex Kit  was used for further analyses in order to ensure good data quality. Standard curve ranges with upper and lower limits of cytokine concentrations are displayed in Additional file p values below 0.05 were considered significant. Bonferroni’s correction (correction for multiple testing of cytokine/immune cell subsets) was systematically used during statistical testing to reduce false positives.All analyses and figures were done in R (version 3.5.3), using packages ggplot2 (3.3.1) and dplyr (0.8.1). The non-parametric Mann-Whitney rank sum test was used to compare cytokine concentrations in different conditions. The Shapiro-Wilk normality test was used to test the normal distribution of the data. In case of normality, Pearson’s correlation test was used; otherwise, Spearman’s non-parametric correlation test was used to test for correlation between cytokine concentrations. Albumin), IgG, IgA, and IgM index showed disease-specific changes within expected limits .In order to obtain a more detailed picture, we also analyzed changes in percentage distributions for the different diseases, using NIND as a control group Table . An elevCSF concentrations of all cytokines were analyzed by comparing values from different neuro-inflammatory diseases with NIND. Multiple cytokines 26/36) showed significantly elevated CSF concentrations under conditions of neuro-inflammation Fig. , Table 26/36 showp value < 0.05). CXCL16 and GM-CSF showed significantly lower concentrations  whereas IL4, CCL2, CXCL5, MIF, and MIB1β values did not show significant differences. We found no evidence that gender nor freezing time had an impact on overall cytokine levels in the serum or CSF. However, MIF serum concentrations correlated with freezing time  in patients with LNB and age correlated with CCL27 CSF concentrations  in patients with CIS-RRMS.Patients with a disrupted blood-brain barrier (BBB) defined by albumin quotient ≥ 8 showed significantly higher cytokine levels within CSF for 29 out of 36 (81%) cytokines when compared to patients with albumin quotient < 8 . Interestingly, CXCL16 and GM-CSF showed a negative correlation with QAlbumin. Multiple negative correlations were observed between CSF cytokines and glucose levels (21/36), and positive correlations between CSF cytokines and lactate levels (23/36). In regard to an intrathecal immunoglobulin synthesis, IgA index significantly correlated with 29 out of 36 cytokines , and IgG with 28 out of 36 cytokines ; IgM index showed a significant correlation with 22 out of 36 cytokines   and QAlbumin < 8  into two different groups for further analyses . Under conditions of a disrupted BBB, NK cells significantly increased and seemed to have a major contribution to inflammatory processes, which was reflected by a strong correlation with multiple pro-inflammatory cytokines.Albumin (31/36), indicating that an upregulation of CSF immune cells and disruption of the blood-brain barrier are associated with numerous pro-inflammatory cytokines. However, the cytokines CCL2, CXCL5, IL4, MIF, and MIB1β did not show a significant elevation or correlation with CSF cell count and QAlbumin, so that these cytokines are most likely not involved in inflammatory CSF processes. In patients with a disrupted blood-brain barrier, only CCL27 concentrations correlated between CSF and serum. Thus, a passive exchange of cytokines across the BBB does not seem to be the major source of CSF cytokines in neuro-inflammation. Instead, a local production and parallel upregulation of cytokines seem more plausible. These results suggest that a cascade of intrathecal cytokine production and immune cell recruitment in combination with a disruption of the BBB occurs during neuro-inflammation. The chronology of cytokine expression during these neuro-inflammatory processes cannot be determined by correlation analyses, but certain hints can be drawn from further detailed analyses.Analyzing all samples together, the majority of measured cytokines (26/36) showed elevated CSF values under certain inflammatory conditions. Most of these CSF cytokines correlated with each other (31/36) and with routine CSF parameters such as CSF cell count (29/36) and QAlbumin. Possible chemotactic factors for intrathecal NK cell recruitment are CXCL8, CCL3, and CX3CL1 [Regarding the different immune cell subtypes, NK cells turned out to be a prominent cell population that might support neuro-inflammatory processes especially under conditions of a disrupted BBB. Both NK cell percentage and cytokine concentrations in the CSF were significantly higher in patients with a disrupted BBB. Consequently, NK cells showed a strong correlation with multiple CSF cytokines in those patients whereas patients with an intact BBB did not show any correlations at all. Pro-inflammatory cytokines such as TNF-α and IL6 have been shown to be involved in BBB breakdown , 24, 33 d CX3CL1 . Indeed,d CX3CL1 , 30. We Another strong association could be found between CSF B cells and CXCL13 values which is in line with previous results . In contWhen analyzing our data according to disease-specific changes, the following conclusions can be drawn for certain cytokines. The CSF milieu in multiple sclerosis has been intensively studied , and we Albumin was used as a clinically pragmatic way to define a disrupted BBB but might not accurately reflect the actual barrier for cytokines, which are small molecules and might show transfer kinetics different from albumin. Fourth, we used Bonferroni’s correction for all analyses to reduce the number of false positives at the cost of having more false negatives. This strict correction for multiple testing can possibly explain differences to previous studies that measured smaller subsets of cytokines.The following limitations of our study have to be discussed. First, we limited the total number of patients to 75, so that they fitted on 1 plate for CSF and 1 plate for serum samples, along with the standard curve, in order to avoid interplate variation for the multiplex assays that was observed in preliminary experiments with the same samples on different plates. Second, correlations between different CSF parameters do not prove causality, so that direct conclusions on functional aspects remain speculative. Thus, multiple correlations in our studies have to be interpreted at a descriptive level. Third, an elevated QWe could show that B cell activation with an upregulation of CXCL13 can occur under conditions of a grossly intact blood-brain barrier. After break down of this barrier, NK cells significantly increased and seemed to have a major contribution to inflammatory processes, which was reflected by a strong correlation with multiple cytokines. From a clinical point of view, CXCL13 was again confirmed as a reliable marker for CSF B cell recruitment and might be used as a clinical marker to predict disease activity in MS, confirming the diagnosis of LNB and CNS lymphoma . HoweverAdditional file 1: Figure S1. Boxplot diagrams of all serum cytokine concentrations including all patient groups . Diagrams are further grouped according to ranges of concentrations with 0–25 pg/mL (A), 0–100 pg/mL (B), 0–200 pg/mL (C), 0–500 pg/mL (D), 0–1000 (E) and 0–5000 pg/mL (F).Additional file 2: Figure S2. Heatmap representing correlations between different CSF cytokines among each other. Positive correlations are given in red, negative correlations in blue. Only correlations with p-value < 0.05 after Bonferroni correction are displayed.Additional file 3: Figure S3. Heatmap representing correlations between CSF and serum cytokine concentrations. Positive correlations are given in red, negative correlations in blue. Only correlations with p-value < 0.05 after Bonferroni correction are displayed.Additional file 4: Figure S4. Heatmap representing correlations between different CSF cytokines among each other for patients with Qalbumin ≥ 8. Positive correlations are given in red, negative correlations in blue.Additional file 5: Figure S5. Heatmap representing correlations between different CSF cytokines among each other for patients with Qalbumin < 8. Positive correlations are given in red, negative correlations in blue. Only correlations with p-value < 0.05 after Bonferroni correction are displayed.Additional file 6: Table S1. Measured standard curves in CSF and serum and sensitivity values provided by the manufacturer. Measured standard curves above had a wide linear range and showed very close values to the ranges provided for serum values by the manufacturer. N.A. not provided by manufacturer.Additional file 7: Table S2. Routine CSF parameters including CSF cell count, glucose, lactate, QAlbumin and Ig indices are shown for different diseases. Average values with ± standard deviations are displayed.


However, the potential endocrine toxicity of SiO2 NPs has not been characterized.Silicon dioxide nanoparticles  of SiO2 NPs significantly increased blood glucose levels in mice. However, the same dose of SiO2 fine-particles (FPs) did not result in altered blood glucose. Whole-genome analysis showed that SiO2 NPs affected the expression of genes associated with reactive oxygen species (ROS) production and endoplasmic reticulum (ER) stress. In addition, we showed that SiO2 NPs activated xenobiotic metabolism, resulting in ER stress. Endoplasmic reticulum stress resulted in increased ROS production, which activated the NF-κB pathway leading to expression of inflammatory cytokines. Increased inflammatory cytokine expression resulted in serine phosphorylation of IRS1, which induced insulin resistance (IR). Furthermore these inflammatory cytokines activated the MAPK pathway, which further promoted the serine phosphorylation of IRS1. Insulin resistance resulted in elevated blood glucose. The ER stress inhibitor 4-phenylbutyric acid (4-PBA) inhibited SiO2 NP-induced ROS production. The ROS scavenger N-acetylcysteine (NAC) did not affect SiO2 NP-induced ER stress, but inhibited SiO2 NP-induced activation of the NF-κB and MAPK pathways, expression of inflammatory cytokines, SiO2 NP-induced serine phosphorylation of IRS1, and SiO2 NP-induced elevations of blood glucose.In the present study, mice were orally administered a series of doses of SiO2 FPs did not. Therefore, lifelong exposure of humans to SiO2 NPs may result in detrimental effects on blood glucose. The results of this study strongly suggested that non-nanoformed SiO2 should be used as food additives.Silicon dioxide NPs induced IR through ER stress and generation of ROS, but SiO The conventional form of amorphous silica is used as a food additive [2 NPs at 124 mg per day based on consumption of food products that contain SiO2 NPs. The major sources of SiO2 NPs are noodles, soups, rubs, and coffee creamers [2 NPs has not been addressed. In cells, exposure to SiO2 NPs resulted in glutathione depletion and DNA damage, and activation of the MAPK/ERK1/2 and Nrf2/ARE pathways [2 NPs per day for 84 days by oral administration had SiO2 NPs distributed in the lungs, kidneys, and spleen. Although no silicon was detected in the liver, histopathological analysis and gene expression studies showed evidence of liver fibrosis after 84 days of exposure [2 NPs, histopathological analysis showed lung thrombosis, cardiac wall fibrosis and calcifications, brain infarctions with necrotizing inflammatory response, retinal injuries with calcification, and focal gliosis [2 NPs [2 NPs can induce wide-ranging systemic effects. However, the effects of SiO2 NPs on the endocrine system are unknown. Diabetes is a multifactorial endocrine disease in which both genetic predisposition and environmental factors contribute to disease onset and progression [2 NPs induced cytotoxicity through excessive ROS production [2 NPs in food additives in the development and progression of diabetes is of importance. The mechanisms by which SiO2 NPs increase ROS levels in animals have not been characterized. Recent studies showed that ROS generation is closely linked to endoplasmic reticulum (ER) stress [2 NPs has not been reported. In this study, we evaluated the effects of dietary SiO2 NPs on blood glucose homeostasis. Silicon dioxide NPs were administered orally to mice, and blood glucose was measured. RNA sequencing was used to evaluate the whole-transcriptome response of the mice, and the genome-wide pathways affected by SiO2 NPs in mice were further investigated, particularly those related to blood glucose homeostasis.Particles between 1 and 100 nm in size are characterized as nanoparticles (NPs) [creamers , 4. Howepathways , 6. Induexposure . In BALBs [2 NPs . These sgression . Food adgression . Reactiv) stress , 15. ER ) stress . ER stre) stress , 17. In ) stress . However2 NPs (Fumed NPs) were obtained from Sigma Co. Ltd. The structure was amorphous (as indicated by the absence of peaks in the X-ray diffraction pattern), and 3.5–4.5 hydroxyl groups were present per square millimicron of SiO2 surface. As controls, stober SiO2 NPs (Stober NPs), fumed SiO2 fine-particles (Fumed FPs) and stober SiO2 fine-particles (Stober FPs) were also used in this study. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) were used to characterize SiO2 NPs and FPs. The SEM and TEM images showed near-spherical shape and good dispersion of the SiO2 NPs and FPs. The average diameters of the Fumed NPs, Stober NPs, Fumed FPs, and Stober FPs were 35.76 ± 4.71 nm, 31.65 ± 2.33 nm, 227.63 ± 9.85 nm, and 213.37 ± 3.04 nm, respectively . Mice were also treated simultaneously with 4-PBA (4-phenylbutyric acid) and NAC (N-acetyl-cysteine) in addition to 100 mg/kg bw Fumed NPs. Blood was collected at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h. Blood silicon levels increased and peaked at 1 h after oral administration , and not larger aggregates, were found in the livers, pancreases, spleens, kidneys, and small intestines of mice treated with 25, 50, 100, and 200 mg/kg bw Fumed NPs , and not larger aggregates were found in the livers, pancreases, spleens, kidneys, and small intestines in the Fumed NPs and Stober NPs groups, but not in the livers, pancreases, spleens, and kidneys in the Fumed FPs or Stober FPs groups  were found in the small intestines in both groups. This observation may have been due to residual SiO2 FPs in the small intestines  and other oxidants , endoplasmic reticulum (ER) stress , and the inflammatory response  . The transcriptomic analyses showed significant changes in mRNA abundance for 702 genes following oral administration of Fumed NPs, with 517 genes upregulated and 183 genes downregulated. In addition, one gene was upregulated after mice were exposed to Fumed NPs for 10 weeks, but then downregulated after 18 weeks of exposure. In contrast, one gene was downregulated after 10 weeks of exposure to SiO2 NP-induced increase in blood glucose and ROS and ER stress, mice were orally administered SiO2 NPs, 4-PBA, and NAC. The results showed that blood glucose increased from week 10 in the 100 mg/kg bw SiO2 NP group  and glutathione (GSH)  Fig. a. HoweveSH) Fig. b, c. FurSH) Fig. d. These ice Fig. a-d. Nqo1O-1 Fig. e. The ef2 NPs affected genes related to ER stress in mice  in the livers of mice starting at week six  corresponds to a 41.3 mg daily intake of a 0.03 kg mouse . A dose of 100 mg/kg bw , which increased blood glucose in mice in this study, corresponds to 2.5-fold the daily intake of humans. Whether a lifelong exposure of humans to SiO2 NPs can induce increased blood glucose should be evaluated further. Lifelong exposure of humans to SiO2 NPs likely results in accumulation of these nanoparticles, which is likely to lead to increased blood glucose. In this study, Stober SiO2 NPs were also absorbed from the digestive system, leading to increased blood glucose in mice starting at week 10. However, fumed SiO2 FPs and Stober SiO2 FPs were not absorbed from the digestive system and did not affect blood glucose levels in mice. Therefore, the main factor in SiO2 NPs effects on blood glucose was particle size. The World Health Organization uses the OGTT and ITT for diagnosis of diabetes types [2 NP-induced hyperglycemia in both the early and late stages was IR. Hyperglycemia is the main characteristic of both diabetes subtypes [2 NPs did not induce apoptosis of β-cells, and thus did not affect insulin secretion. However, the mice exhibited reduced insulin sensitivity at weeks 10 and 18. Compared with SiO2 NPs, SiO2 FPs did not affect insulin secretion and did not induce IR in mice. The liver is the classical insulin-responsive organ, and is closely associated with IR [2 NP toxicity. Our results showed that SiO2 NPs affected several GO terms associated with ROS generation  and ER stress . Reactive oxygen species production is a hallmark of oxidative damage in many diseases, and can lead to mitochondrial dysfunction, cellular aging, and apoptosis [2 NPs increase ROS levels in animals remains unclear. In this study, whole-genome network results showed that ROS generation was closely associated with ER stress. The ER is the cellular organelle responsible for synthesis and folding of secreted and membrane-bound proteins. A variety of stimuli can lead to accumulation of unfolded proteins, a condition called ER stress [2O2 to generate singlet oxygen in the ER, resulting in ER stress [2 NPs induced ER stress (from week 6), which was followed by increased ROS (from week 8), and increased blood glucose in mice (from week 10). These results indicated that ROS did not induce ER stress in this study. Whole-genome results showed that SiO2 NPs increased the expression of Cyp2b9 and Cyp2a4. Cytochrome P450 enzymes, specifically CYP families 1–3, play a pivotal role in drug and xenobiotic metabolism [2 NPs increased the gene expression of Cyp2b9 earlier than fumed SiO2 NPs induced ER stress, which indicated that fumed SiO2 NPs activated xenobiotic metabolism, resulting in ER stress. Endoplasmic reticulum stress and ROS generation were each blocked by specific inhibitors. 4-phenylbutyric acid is a low molecular weight compound that stabilizes protein conformation, improves the folding capacity of the ER, and facilitates trafficking of mutant proteins to suppress ER stress [2 NP-induced ER stress and inhibited ROS generation in mice, which suggested that fumed SiO2 NPs increased ROS production through ER stress. The transcriptional factor nuclear factor E2-related factor 2 (Nrf2), localized downstream of the PERK pathway, mediates antioxidant enzymes, resulting in augmentation of intracellular ROS production [2 NP-induced ER stress affected the Nrf2 pathway by affecting the expression of Nrf2, Nqo-1, and HO-1, resulting in increased ROS production in mice. Stober NPs also affected the expression of Cyp2b9, ER stress, and ROS generation, but Fumed FPs and Stober FPs did not induce these effects. N-acetyl cysteine is a ROS scavenger [2 NP-induced ROS production, but not ER stress. However, NAC inhibited increased blood glucose. These results showed that fumed SiO2 NPs induced ER stress, ER stress increased ROS production, and ROS induced blood glucose elevation. The major pathway of insulin receptor signal transduction is the insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (PI3K)-Akt  pathway [2 NP-induced ROS production inhibited dephosphorylation of the serine residues of IRS1 and inhibited phosphorylation of the serine residues of Akt, resulting in IR. In addition, fumed SiO2 NP-induced ROS production resulted in activation of the NF-κB and MAPK pathways, resulting in serine phosphorylation of IRS1. NF-κB is a family of transcription factors that plays a critical role in inflammation. Reactive oxygen species have been reported to induce NF-κB activation due to phosphorylation of IκB, which results in NF-κB translocation to the nucleus and phosphorylation of p65 [SiOn pastes , 2. The s 124 mg , 4. In mstration , 9. In a systems , 9. Howed spleen , 8, 19. s 2.5 m2 . Therefoes types . In thissubtypes . Type 1 subtypes . Insulinsubtypes . Insulinsubtypes , 24. In  with IR . In thispoptosis . ReactivR stress . PERK, IR stress . FurtherR stress , 25. In tabolism , 25. Ovetabolism . In thisR stress . In thisoduction , 27. In cavenger . However pathway . IRS1 is pathway , 29. In n of p65 , resultin of p65 , 29. Sim2 NPs have been widely used as food additives. In this study, SiO2 NPs induced IR through ER stress and generation of ROS, but SiO2 FPs did not . Stober SiO2 NPs were obtained from Zhongkeleiming Technology Co., Ltd. . The primary particle sizes and morphology were measured using TEM and SEM . The hydrodynamic size and zeta potential of nanoparticles in PBS were measured using DLS . Endotoxin analysis was performed using the Limulus Amoebocyte Lysate (LAL) assay and the Pyros Kinetix instrument from Associates of Cape Co., Ltd. .Fumed SiO2 NPs and FPs. After ultrasonic wave stirring for 30 min, the SiO2 NP-PBS or SiO2 FP-PBS suspension was orally administered by syringe once per day, 7 days per week, for 10 or 18 weeks to each group of mice. To explore the endocrine effects of the oral administration of SiO2 NPs and determine the dose that increased blood glucose, mice were orally administered suspensions of SiO2 NPs at the doses of 25, 50, 100, and 200 mg/kg bw. The total doses the mice received were approximately 44.5, 96.7, 177.8, and 389.6 mg after 10 weeks, or approximately 86.7, 187.4, 355.3, 750.9 mg after 18 weeks. Then, in the experiment that explored the mechanism for the effect of SiO2 NPs on blood glucose, the dose of 100 mg/kg bw SiO2 NPs was selected. In addition, in the mechanism experiment, the doses of 100 mg/kg bw 4-PBA and NAC were orally administered to mice simultaneously with the oral administration of the suspension at 100 mg/kg bw SiO2 NPs. The total dose of mice received was approximately 179.7 mg after 10 weeks. The control group mice were given an equal volume of PBS.Six-week-old ICR male mice (22.97 ± 0.33 g) were obtained from Harbin Veterinary Research Institute  and acclimated for 7 days after arrival at the study facility. Mice were housed in an animal room with controlled temperature (21–24 °C) and light cycle (12 h light/dark). Autoclaved water and rodent diet  were provided ad libitum. An ultrasonic bath  at a frequency of 40 kHz with power at 400 W was used to scatter the SiOTail vein blood was collected every 2 weeks. Before the collection, mice were fasted for 16 h. The blood glucose in the tail vein blood was measured using a glucose assay kit , and the blood insulin was measured using a mouse insulin ELISA kit . TNF-α and IL-6 were measured using TNF-α and IL-6 kits, respectively .In OGTT, mice were fasted for 16 h and then orally administered glucose (1.5 g/kg bw). In ITT, mice were fasted for 6 h and then injected with insulin (0.4 IU/kg bw). Blood was collected for blood glucose and insulin measurement from the tail vein at 0, 30, 60, 120 min. Blood glucose and insulin were measured using each kit as above described.2O2, the mixed solutions were heated at approximately 160 °C using a high-pressure reaction container in an oven chamber until the samples were completely digested. Then, the solutions were heated at 120 °C to remove the remaining nitric acid and until the solutions were colorless and clear. Last, the remaining solutions were diluted to 3 mL with 2% nitric acid. ICP-OES  was used to analyze the silicon concentration in the samples. The data are expressed as micrograms per gram fresh tissue. To assess whether SiO2 particles occurred in each tissue after the oral administration of SiO2 NPs, 0.1 g of each tissue was also weighed for SEM and EDXA. The tissues were homogenized in RIPA lysis buffer, filtered to discard cell debris using a 0.45 μm filter membrane, and centrifuged for 30 min at 18000×g to precipitate the SiO2 NPs. The precipitate was resuspended in alcohol and then spread out on an aluminum sheet, sputter-coated with platinum, and observed by SEM. The surface element was analyzed by EDXA.The livers and pancreases were removed from the mice, and 0.3 g of each tissue was weighed, digested, and analyzed for silicon content. Briefly, the tissues were digested in nitric acid (ultrapure grade) overnight. After the addition of 0.5 mL of HROS levels were assessed using the levels of total SOD (T-SOD), GSH and MDA. Livers were homogenized in nine volumes (1:10 w/v) of PBS. Homogenates were centrifuged at 750 g for 10 min at 4 °C to discard cell debris. The T-SOD, GSH and MDA of serum and liver supernatant were measured using each kit .Pancreases and livers were fixed in 4% paraformaldehyde solution for 24 h, embedded in paraffin, cut into 5 μm sections, and placed onto glass slides. The β-cell mass was identified by immunohistochemical localization of insulin with an anti-insulin antibody . For the analysis of cell apoptosis, pancreatic and liver sections were stained with the TUNEL . DNase I pretreated cells were used as the positive control.http://apps.cytoscape.org/apps/bingo) was used. The GO terms met the B H Pvalue < 0.001 were the significant GO terms.To ensure unbiased analysis of tissue response, the total RNA was isolated from random sections (10–15 mg) of liver. The RNA was isolated using TRIzol reagent  and purified using a RNeasy Plus Mini kit . Three livers of each group were sequenced, and the data were analyzed. In brief, the RNA was sequenced on one sequencing lane of an Illumina Genome Analyzer II system (Illumina). The paired-end reads were mapped to the mouse genome (version NCBIm37/mm9) using Tophat. The mapped reads were used to quantify the transcripts from the RefSeq reference database. The genes met the fold change > 2 and Benjamin Hochberg adjust Pvalue  < 0.05 were the significant differently expressed genes which were used for the following analysis. For the functional annotation analysis of genes, the BiNGO . Real-time polymerase chain reaction amplification of cDNA was performed with SYBR Premix Ex Taq™ II . Primers were listed in Table S3.Liver tissues were resuspended in RIPA lysis buffer. Lysates were centrifuged for 15 min at 12000 g and 4 °C, and protein contents of the supernatant were determined using DC protein kit . Aliquots of the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to PVDF membranes . PVDF membranes were incubated with antibodies against phospho-Eif2α, GRP78, CHOP, ATF6 (90KD), ATF6 (50KD), shear-XBP1 (XBP1-s), unshear-XBP1 (XBP1-u), Cleaved-Capsase3 (Cleaved-Casp3), phospho-IRS1 (Ser307), phospho-Akt (Ser473), IRS1, Akt, phospho-NFκB-p65, phospho-IκBα, phospho- JNK, phospho-p38-MAPK, NFκB-p65, IκBα, JNK1, p38-MAPK and β-actin. All antibodies were purchased from Cell Signaling Technology .p value less than 0.05 was considered statistically significant.Results were expressed as mean ± SEM and were analyzed using GraphPad Prism version 5.0 . All calculated significances are based on the one-way analysis of variance (ANOVA) test and the post hoc Tukey’s test. A Additional file 1: Figure S1-S5 (PDF 1327 kb)Additional file 2: Table S1. Significant differently expressed genes of RNA-seq (XLS 599 kb)Additional file 3: Table S2. GO terms Table S1 (XLS 150 kb)Additional file 4: Table S3. List of primer sequences (XLS 28 kb)


PpCAD2 was originally isolated from the ‘Wangkumbae’ pear (Pyrus pyrifolia Nakai), and it encodes for cinnamyl alcohol dehydrogenase (CAD), which is a key enzyme in the lignin biosynthesis pathway. In order to verify the function of PpCAD2, transgenic tomato (Solanum lycopersicum) ‘Micro-Tom’ plants were generated using over-expression constructs via the agrobacterium-mediated transformation method. The results showed that the PpCAD2 over-expression transgenic tomato plant had a strong growth vigor. Furthermore, these PpCAD2 over-expression transgenic tomato plants contained a higher lignin content and CAD enzymatic activity in the stem, leaf and fruit pericarp tissues, and formed a greater number of vessel elements in the stem and leaf vein, compared to wild type tomato plants. This study clearly indicated that overexpressing PpCAD2 increased the lignin deposition of transgenic tomato plants, and thus validated the function of PpCAD2 in lignin biosynthesis. Pyrus pyrifolia, ‘Whangkeumbae’, hard end is a physiological disorder of the fruit. In the hard end pear fruit, lignin deposits heavily in the pericarp and pulp, leading to the formation of rough textured flesh [During the process of plant growth and development, lignin plays a key role in supporting the plant body, water transport, and resistance against external stress factors ,2,3,4. Aed flesh .O–methyltransferase (CCoAOMT), cinnamoyl–CoA reductase (CCR), ferulate 5–hydroxylase (F5H), Caffeic acid O–methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD) [Lignin is an amorphous, complex aromatic heteropolymer, which is produced by the phenylpropanoid metabolic pathway . Lignin se (CAD) . After tse (CAD) . The monse (CAD) . Lignin se (CAD) ,11,12. Gse (CAD) .PpPAL1, PpPAL2, Pp4CL1, Pp4CL2, PpCAD1, PpCAD2, PpPOD1, PpPOD2, PpPOD3 and PpPOD4, and analyzed their expression patterns in hard end pear fruit [PpCAD2 were found to have a positive correlation with lignin accumulation in the hard end ‘Whangkeumbae’ pear; the application of calcium chloride alleviated the hard end phenomenon, while simultaneously suppressing the expression of the gene [CAD is a multifunctional enzyme that catalyses the final step in the biosynthesis of monolignols, converting cinnamaldehydes into the corresponding alcohols ,13. Relaar fruit ,15. The the gene . Studiesthe gene ,23,24,25Solanum lycopersicum) is used as a model plant of the Solanaceae family. A miniature tomato cultivar of S. lycopersicum, ‘Micro-Tom’, being an excellent model system, has some characteristics that include a small size, short generation time and a transformable quality, that make it suitable for experimental research [TOMATO AGAMOUS-LIKE 1 (TAGL1) controls the lignification of tomato fruit [TAGL1-silenced fruit pericarp, lignin pathway genes, such as PAL, 4CL and CAD, are up-regulated, and the lignin content is also increased [Genetic transformation is a key technology for gene functional verification. For perennial fruit trees, it is hard to carry on the transgenic technology due to the lengthy generation cycles and the difficulty in regenerating in vitro . The agrresearch ,30,31. Rresearch ,33,34. Iresearch . A MADS-to fruit . In TAGLncreased .PpCAD2 constructs were generated via the agrobacterium-mediated transformation method. In transgenic tomato plants, the physiological properties and the expression level of PpCAD2 were analyzed to clarify the role of PpCAD2 in lignin synthesis.In our current study, transgenic tomato ’Micro-Tom’ plants harboring PpCAD2, we isolated a 978-bp predicted opening reading frame, encoding a protein of 325 amino acids with a cinnamyl-alcohol dehydrogenase domain  plant, the three PpCAD2--ox  lines exhibited a higher expression of PpCAD2  lines were chosen for the functional analysis  ‘Micro-Tom’ seeds were disinfected by submerging in 75% ethanol for 2 min followed by 10% sodium hypochlorite for 10 min. After three rinses with sterile water and being blotted dry of the excess water on the seed surface, the seeds were inoculated on a Murashige and Skoog (MS) medium. The cultures were kept in the dark and transferred to light conditions when cotyledons emerged [Tomato  3’, reverse: 5’ CCGGATCCAAGGGAAGCCGGAGTTTA (BamH I) 3’. The full-length cDNA of PpCAD2 was ligated into a cloning vector pMD19-T. To prepare the PpCAD2 over-expression (35S::PpCAD2) constructs, plasmids were isolated and digested with the restriction enzymes BamH I and Xba I. The insert fragment was isolated and then cloned onto a vector, pBI121, under a 35S promoter. These constructs were transformed into Agrobacterium tumefaciens EHA105 through the freeze-thaw method separately [The primers with restriction enzymes sites were designed using DNAStar software (Lasergene). parately . 2, and one hundred cotyledon explants were dipped into a bacterial suspension (OD600 = 0.3–0.4) of A. tumefaciens EHA105 harboring the transformation constructs. After 5 min, the explants were blotted dry with autoclaved filter paper. The explants were placed back onto the co-cultivation medium and incubated in the dark for 1.5 d under 28 °C. The explants were transferred to a callus induction/selection medium containing MS salts, 3% sucrose, 1.5 mg·L−1 zeatin, 50 mg·L−1 kanamycin and 500 mg·L−1 Cef, and 0.7% agar at pH 5.8 [−1 6–Benzylaminopurine (6–BA), 0.1 mg·L−1 Indole-3–Butytric acid (IBA) and 0.7% agar. The shoots forming a few leaves were transferred to a rooting medium containing MS salts, 3% sucrose, 0.2 mg·L−1 IBA and 0.7% agar. The rooted plants were transplanted to pots and grown in a growth chamber that was programmed at a constant temperature of 21 °C and a 16-h-light/8-h-dark cycle. Wild type (WT) plants were propagated concurrent to the transgenic plants. For the overexpression, cotyledons of ‘Micro-Tom’ were transformed by an Agrobacterium-mediated transformation. The cotyledons of ‘Micro-Tom’ were sectioned to 3 mmt pH 5.8 . The expPpCAD2 forward: 5’-CGTCTAGAAGTAGAGCAGCGGAGCAG-3’, and PpCAD2 reverse: 5’-CCGGATCCAAGGGAAGCCGGAGTTTA-3’. Genomic DNA was extracted from ‘Micro-Tom’ leaves using a plant total DNA extract kit . The PCR amplification used primer pairs of http://bioinfo.ut.ee/primer3-0.4.0/). The relative gene expression level was calculated using the 2-ΔΔCt method [Total RNA was extracted from the ‘Micro-Tom’ leaf tissue using EASYspin Plant RNA Kit  according to the manufacturer’s instructions. Genomic DNA was removed by DNase. cDNA was synthesized with reverse transcription using the Prime Script™ RT reagent Kit  according to the manufacturer’s instruction and was used as the template for the qPCR analysis. qPCR was performed on a Light Cycler® 480 instrument . The procedure included annealing at 94 °C for 5 min, followed by 40 cycles of 94 °C for 15 s, 60 °C for 1 min, and 72 °C for 30 s. A tomato actin gene was used for normalization. The primers used for the qPCR analysis, listed in t method . Three b2HPO4/KH2PO4, 0.5% Triton X-100, 0.5% PVP, pH 7.8) for 30 min. After being centrifuged, the pellet was washed twice (30 min) in 100% MeOH. Then, the resulting pellet was dried in 80 °C overnight. The dried pellet was added with 1 mL 2M HCl and 0.1 mL thioglycolic acid, then put into boiling water for 4 hours. The obtained end product was dissolved in 1 mL 1M NaOH. The diluted samples were assayed for absorbance at 280 nm, and NaOH was used as a blank. All measurements were performed in biological triplicates.The lignin content of the stem, leaf and fruit pericarp in the three-month-old ‘Micro-Tom’ tomato was measured according to the method described by Dyckmans . Frozen The plant height of the three-month-old ‘Micro-Tom’ tomato was measured with a ruler. Additionally, the stem diameter and mature fruit diameter were measured with a vernier caliper. Three biological replications were determined.−1 HCl was added to terminate the reaction. The samples were assayed for absorbance at 400 nm. The data were expressed on a protein basis, and the analysis was biologically repeated three times.The stem and fruit CAD enzymatic activity was measured according to the method described by Cai . The samThe transverse section of the leaf veins and stem were used by Weisner staining and microscopy. Weisner reagent (phloroglucinol/HCl) was used to stain the plant tissue for 5 min before it was visualized for lignification under a microscope . The ligPpCAD2 in transgenic tomato plants increased the plant height and stem diameter. Furthermore, overexpression of PpCAD2 increased the lignin content in the stems, leaves and fruit pericarp tissues, partially by increasing the CAD enzyme activity, which was an important enzyme in the biosynthesis of monolignols. Overexpression of PpCAD2 also increased the size of the vessel element in the xylem tissues. Our data suggest that the PpCAD2 functions by positively regulating the degree of lignification. In this study, overexpression of


The dynamics and coordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required the earliest, followed by auto-phosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps, whereas ULK1 and ULK2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way, suggesting multiple initiation events. Targeted ubiquitinated mitochondria are cradled by endoplasmic reticulum (ER) strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands, providing platforms for formation of the mitophagosomes.  •Mitophagy requires coordination of machineries for target recognition and engulfment•IVM-induced mitophagy depends on TRAF2/CIAP1/CIAP2 for ubiquitination of mitochondria•Ubiquitinated mitochondria within ER regions facilitate targeting and autophagy•ULK complex is required, but not for initiation, with FIP200 acting before ATG13 Zachari et al. use genetic and imaging experiments to define the ordered steps of mitophagy induction in response to the drug ivermectin. Their findings suggest that damaged mitochondria are ubiquitinated and dynamically encased in ER strands that then provide the platform for the formation of mitophagosomes. Autophagy is a conserved pathway for nutrient supply during periods of starvation, classified as non-selective autophagy, or for degradation of intracellular large structures that are pathogenic or have become damaged, classified as selective autophagy . In bothThe pathway of autophagosome formation in response to starvation is now well understood, although the exact origin of the autophagosomal membrane is still a matter of debate . For autThe process of selective autophagy requires a set of proteins connecting the targeted cargo to the autophagic machinery and a signal on the cargo to mark it for sequestration . SelectiThe pathway of mitophagy has been extensively studied since it was first described . The “eain vivo, the exact signals that trigger it at the organismal level are still relatively obscure  so as to avoid extremely strong, potentially non-physiological activation of this pathway. The protonophore CCCP  and, morWe carried out additional experiments to characterize IVM, which is a well-known anti-parasitic compound , in the Because treatment with IVM causes fragmentation of mitochondria and induces ubiquitination, we determined if its mechanism of action was similar to other known mitophagy inducers such as OA that modulate mitochondrial respiration during mitophagy. Indeed, oxygen consumption rate (OCR) was significantly reduced by IVM at the concentration range used in our assays E–1G. We To delineate the mechanism of mitophagy, we used inhibitors and MEFs deleted in various autophagy and mitophagy genes. A set of antibodies to endogenous reporter proteins allowed differentiation of mitophagy induction from non-selective autophagy A. These We then used MEFs deleted in early autophagy genes to delineate this pathway with respect to the autophagic machinery. In the absence of FIP200, a component of the ULK complex, translocation of ubiquitin and phospho-TBK1 was evident, but that of optineurin and WIPI2 was inhibited B. FormatBased on the MEF data, we derived the preliminary pathway shown in For PINK1-Parkin-dependent mitophagy, only optineurin and NDP-52 among the receptors are essential for engulfment and delivery . MEFs exWe next examined the dynamics of the mitophagy response using a combination of live imaging, super resolution, and EM and related this to the genetic and pharmacological analysis above. To assess engulfment, we imaged cells expressing the omegasome reporter DFCP1, the autophagy protein LC3, and a mitochondrial marker. Complete engulfment of mitochondria fragments by LC3 was seen in those cells, accompanied by translocation of DFCP1 and dynamic interaction with LC3 during the process A. Of not(A) Triple imaging of cells expressing GFP-DFCP1, CFP-LC3, and mCherry-MITO, showing one engulfment event during IVM treatment.(B) Triple imaging of cells expressing GFP-DFCP1, CFP-LC3, and mCherry-MITO, showing one engulfment event but with multiple initiation points.(A) Triple imaging of cells expressing GFP-ATG13, CFP-LC3, and mCherry-MITO, showing one engulfment event during IVM treatment.(B) Double imaging of cells expressing GFP-ATG13 and mCherry-MITO, showing one engulfment event but with multiple ATG13 translocations.The initial dynamics of optineurin translocation to forming mitophagosomes resembled those of ATG13 in being discontinuous and jerky, although eventually the optineurin structures completely engulfed the mitochondrial fragments and stayed associated with them A. Intere(A) Triple imaging of cells expressing GFP-ATG13, mCherry-optineurin, and CFP-MITO, showing an engulfment event.(B) Triple imaging of cells expressing GFP-optineurin, CFP-LC3, and mCherry-MITO, showing an engulfment event.For non-selective autophagy via omegasome intermediates, the ER provides a cradle for assembly of autophagosomal membranes. For selective autophagy, the role of the ER is unknown. We observed that ATG13-enriched autophagosomal structures engulfing mitochondrial fragments showed a strong coincidence with the underlying ER  for longExamples of tight association between the ER and forming mitophagosomes are evident in some previous publications using Parkin overexpression see , suggestTo examine the spatial relationship between mitophagosomes and the ER at higher resolution, we used a combination of live microscopy followed by EM  first deThe last few frames are a reconstruction of a bigger area showing the various components  in sequence of appearance.With respect to the ubiquitination step, super-resolution microscopy revealed a close association of the ubiquitin signal with the targeted mitochondria and with both autophagy and mitophagy components B. SeveraDuring ubiquitin-dependent mitophagy, several protein complexes participate in the targeting of damaged mitochondria for degradation: ubiquitination machinery, receptors that recognize damaged mitochondria, and the autophagic machinery creating the double membrane to engulf them . How theMechanistically, IVM reduces oxygen consumption in agreement with previous work  and fragThe ubiquitination step is devoid of any discernible dynamics during live imaging: fragments appear ubiquitin-free and, within 10 s, become ubiquitin positive. Ubiquitinated mitochondrial fragments move in association with ER strands but are not restricted by them until they appear to become entrapped within the strands. This step does not require any of the known downstream machinery such as mitophagy receptors or autophagy proteins.After ubiquitinated mitochondria are ER restricted, we observed auto-phosphorylation of TBK1 and translocation to the mitochondria-ER site. In MEFs, this occurs before recruitment of the autophagy machinery or of optineurin, the mitophagy receptor. Recent work in PINK1/Parkin-dependent mitophagy also showed TBK1 activation to be an early event, but in that case, optineurin function was required for TBK1 activation in a positive feedback loop . The impDownstream of TBK1 activation, we mapped the activity of FIP200 that was genetically uncoupled from ATG13 and ULK1 and ULK2, two other components of the ULK complex. In the absence of FIP200, only P-TBK1 puncta were visible upon IVM induction, but mitophagy adaptors such as optineurin and other autophagy proteins such as WIPI2 did not translocate to the mitophagy sites. In contrast, without ATG13, optineurin-positive puncta still formed. Consistent with this, FIP200 puncta were formed during IVM induction in the ATG13 knockouts. The primacy of FIP200 in the mitophagic response was noted before, and ATG9-positive structures were seen to translocate to “autophagosome formation sites for mitophagy” . Based oThe dynamics of the engulfment process were extensively studied here, and some were unexpected C. InteraWhat ensures such tight fit and efficiency? We have documented using live imaging, super-resolution microscopy, and EM that the ER is intimately associated with the formation of the mitophagosome. Importantly, we did not observe ER strands threading in and out of the autophagic structure—as is the case for non-selective autophagy—but rather, ER strands appeared as a continuation and a cradle for the forming mitophagosomes C. This Enicholas.ktistakis@babraham.ac.uk).Further information and requests for reagents may be directed to, and will be fulfilled by, the Lead Author, Dr. Nicholas T. Ktistakis  were generously donated by the following colleagues: ATG5 KO and ATG13 KO, Professor Noboru Mizushima; ATG3 and ATG7 KO, Professor Masaaki Komatsu; ULK1/ULK2 KO and ATG9 KO, Dr Sharon Tooze. The OPTN knockout MEFs were developed from OPTN deficient mice  as previPP242, IVM, PYR-41, and antimycin A were purchased from Sigma-Aldrich (now Merck). BX-795, oligomycin, and CCCP were purchased from Tocris Biosciences. VPS34 INH1 was a kind gift of Dr. Ian Ganley. All compounds were dissolved in DMSO as 1000X stocks and were used at these final concentrations: PP242 1μM; IVM 20 μM ; PYR-41 30 μM; BX-795 5 μM, CCCP 4 μM, oligomycin 10 μM, antimycin A 10 μM.Protocols have been extensively described recently . In brieA Nikon N-SIM system was used, comprising Nikon Ti-E microscope, Nikon 1.49 N.A. objective, Andor iXon 897 EM-CCD camera, Nikon SIM illuminator, Nikon LU5A laser bed and controlled using Nikon Elements software. Raw 3D-SIM images were acquired  typically using 100 ms exposure, 5.1 conversion gain and 150 EM gain. Image stacks were acquired with a 120 nm step interval and reconstructed into super resolved images using the volumetric reconstruction algorithm in the Nikon software. Excitation/emission for the different fluorescent labels was as follows: mTurquoise 405 nm ex, 447/60 em; GFP 488 nm ex, 525/50 em; mCherry 561 nm exn 607/36 em; Alexa Fluor 647 643 nm ex, 692/40 em.Two wide-field imaging systems were used to capture images of live cells: a Nikon Ti-E-based system and an Olympus cellSens. Details of our live imaging protocols were described in HEK-293 cells stably expressing GFP-ATG13 were transfected with dsRED MITO for 24 hr and were then replated onto poly lysine-coated, 35 mm gridded MatTek glass bottom dishes to reach 50% confluency on the next day. The cells were treated with 20 μM IVM for 35 min before imaging. After setting the samples on the stage of the Nikon Ti-E microscope at 37°C 5% CO2, they were imaged for an additional 20 min using a 100x 1.4 NA objective and frames were acquired every 10 sec. Before the last imaging frame, 4% paraformaldehyde in 0.2 M Hepes, pH 7.4, was added to make the final concentrations of 2% paraformaldehyde and 0.1 M M Hepes. After 10 min incubation, the fixative was replaced with 0.2M Hepes buffer to allow capturing a confocal z stack (100x 1.4 NA objective) and the grid co-ordinates were imaged by 10x10 montage bright field imaging (100x 1.4 NA objective). A second round of fixation followed in 2% glutaraldehyde (Sigma EM grade) in 0.2M Hepes (pH 7.4) for 2h. Cells were then osmicated for 1 hr at room temperature with a solution containing 1% OsO4, 0.1M CH32AsO2Na (cacodylate), pH 7.4, 15mg/ml K4 supplemented with 10 % 50mM NaF, 0.1% 10mg/ml leupeptin and 0.5% 0.2M Sodium orthovanadate. Lysates were collected and centrifuged for 10 min at 14,000 rpm at 4°C. The supernatant was collected in fresh tubes on ice. Protein concentration of the lysates was determined using the BCA protein assay kit (Thermo Scientific Pearce). For electrophoresis, samples were combined with 2 x Laemmli sample buffer  in a 1:1 ratio. Samples were then heated for 90 seconds at 95°C before loading. Gels were wet-transferred overnight to 0.45μm Immobilon-P transfer membranes (Millipore). Incubations with primary and secondary antibodies, and signal development using ECL  followed standard protocols. For immunoblots, the following antibodies at the indicated dilutions were used: rabbit anti-ATG9 , 1:1500; rabbit anti-ATG13 (Sigma), 1:1000; rabbit anti-FIP200 (ProteinTech), 1:1000; mouse anti-GAPDH (Biogenesis), 1:100,000; mouse anti-OPTN (Santa Cruz), 1:500; rabbit anti-phospho(S172)TBK1 , 1:1000; rabbit anti-phospho(S757)ULK1 , 1:1000 rabbit anti-TAX1BP1 (homemade), 1:3000; mouse anti-TBK1 (SantaCruz), 1:500, rabbit anti-TRAF2 (Abcam) 1:1000; rabbit anti-CIAP1 (Proteintech); rabbit anti-CIAP2 (Proteintech).HEK-293 cells were treated with 15 μM IVM for 45 min and lysed in 0.3% CHAPS buffer containing 40 mM HEPES-Cl pH 7.4, 120 mM NaCl, 2 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM sodium fluoride, 1.5 mM sodium vanadate and one tablet EDTA-free protease inhibitors per 50 ml. At the same time, agarose beads crosslinked to anti-ubiquitin antibody FK2  were washed in the same lysis buffer and were added to the cleared lysates for binding at 4°C for 60 min. At the end of incubation, beads were washed 4 times with lysis buffer and once with PBS before mass spectrometry or SDS-PAGE and immunoblotting.WT and OPTN KO MEFs were seeded in 6-well plates to reach 70%–80% confluency 24 h later. Transfections were performed using TransMessenger transfection reagent kit (Qiagen) with SmartPool siRNA oligos against non-targetting and TAX1BP1 (Dharmacon) for 72 hr.Downregulation of CIAP1, CIAP2 and TRAF2 simultaneously was done as follows: Cells were plated in the morning and transfected for the first time in the afternoon with 80 pmol of each siRNA SmartPool. The cells were re-transfected two days later with the same amount of siRNA, and examined for the various assays two days later.4 cells/well density 24hrs prior to the experiment. On the day of the experiment the cells were pre-incubated in DMEM supplemented with 5-mM Glucose, 2mM Glutamine and 5mM HEPES-HCl pH 7.4, for 60 min. IVM was titrated to 1, 5 or 10μM and equivalent volume of DMSO was added in the control samples. All the conditions were in quadruplicates. OCR was measured every 6 min to determine basal respiration, ATP synthase activity and proton leak (using 1 μM Oligomycin), and non-mitochondrial respiration (using 1 μM Rotenone and 2 μM Antimycin). For determination of percentage OCR inhibition rate compared to the basal, the following equation was used: [1 – [/]] × 100.For oxygen consumption rate (OCR) determination, experiments were carried out using a Seahorse XF24 analyzer (Agilent). HEK-293 cells were plated in a 24 well Seahorse cell culture microplate in 3.5 x 10In a single experiment, 10 images  were selected for each treatment condition and quantified using the ImageJ cell counter plugin. Values were then represented as puncta per cell using GraphPad Prism. Data from biological repeats were combined, log-transformed, and a two-way ANOVA was performed to obtain statistical significance and finally plotted as Mean±SD. All statistical analysis was checked by Dr Anne Segonds-Pichon, the statistician of the Babraham Institute.The live imaging data in this manuscript are derived from over 400 videos, and what is shown has been reproducibly observed multiple times by more than one scientist.The published article includes all datasets generated and analyzed during this study.


Aldefluor assay and cell proliferation assay revealed that PEITC treatments resulted in a reduced number of ALDHhi hCSCs in a concentration-dependent manner (p < 0.05). In the ROS assay, PEITC promoted oxidative stress in hCSCs (p ≤ 0.001). Using immunoblotting and flow cytometry techniques, we reported that PEITC suppressed the cancer-associated transcription factor (Sp1) and a downstream multidrug resistance protein (P-glycoprotein) . Furthermore, PEITC-treatment of hCSCs, prior to xenotransplantation in mice, lowered the in vivo tumor-initiating potential of hCSCs. In summary, PEITC treatment suppressed the proliferation of ALDH1 expressing cancer stem cells as well as key factors that are involved with drug-resistance, while promoting oxidative stress and apoptosis in hCSCs.Aldehyde dehydrogenase 1 (ALDH1) is a cytosolic marker of cancer stem cells (CSCs), which are a sub-population within heterogeneous tumor cells. CSCs associate with therapy-resistance, self-renewal, malignancy, tumor-relapse, and reduced patient-survival window. ALDH1-mediated aldehyde scavenging helps CSCs to survive a higher level of oxidative stress than regular cancer cells. Cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) selectively induces reactive oxygen species (ROS), leading to apoptosis of cancer cells, but not healthy cells. However, this pro-oxidant role of PEITC in CSCs is poorly understood and is investigated here. In a HeLa CSCs model (hCSCs), the sphere-culture and tumorsphere assay showed significantly enriched ALDH While these surface markers are common across various tumor types [The heterogeneity of the cell population in solid tumors frequently precludes the complete elimination of malignancy after conventional treatments and it can contribute to cancer recurrence. Cancer stem cells (CSCs), which are a subset of tumor cells 0.1–30%) with self-renewal and differentiation potential, are associated with tumor initiation, maintenance, metastasis, and recurrence of a number of cancer types –30% with. CSCs caor types ,6, head or types , melanomor types , hepaticor types , pancreaor types , colon [or types , lung [1or types , prostraor types , bladderor types , ovarianor types , endometor types , and ceror types ,18 canceor types .CSCs-targeted therapy is of growing interest, as conventional chemo- or radiation-therapy does not eradicate drug-resistant CSCs . ActivitThe ability of CSCs to survive a significantly higher oxidative stress in comparison to other types of cancer cells or healthy cells also contribute to therapy resistance. Rapidly growing cancer cells produce higher levels of reactive oxygen species (ROS) than normal healthy cells due to increased oxidative metabolism and the faster depletion of nutrient supply . Many caHigh/CD24Low CSCs markers, when compared to the other SiHa sphere cells [Phenethyl isothiocyanate (PEITC) is a biologically active dietary compound that is derived from cruciferous vegetables, such as watercress, broccoli, cabbage, and cauliflower ,29. We are cells . Since wre cells , here we5 cells/mL produced the optimum signal intensity when compared to 5 × 105 and 1 × 106 cells/mL. After growing the cells in serum free sphere-culture medium, 25.40% of ALDHhi in HeLa cells at day 0 significantly enriched (p < 0.001) to 57.75 % at day 10  control , while using diethylaminobenzaldehyde (DEAB) as a negative control for Aldefluor reagent (p < 0.01), using disulfiram as a positive control (hi cells by 20% (p < 0.01), whereas 5 and 10 μM PEITC reduced ALDHhi cells by around 40% and 65% (p < 0.001), respectively , which was comparable to the ROS levels that were induced by 50 µM of hydrogen peroxide (H2O2), a prooxidant positive control (p < 0.001) , leading to ROS-induction in cells . Since C control . Further< 0.001) , supportp < 0.05) and 10 μM (p < 0.001), respectively (p < 0.01). Together, these observations indicate the pro-apoptotic and anti-proliferative properties of PEITC in hCSCs  with 10 μM PEITC pre-treatment (10 µM PEITC-treated hCSCs) developed lower tumor load when compared to untreated control group (hCSCs) at weeks 2 and 4 (3 vs. 0.01 mm3 and 6.04 mm3 vs. 4.3 mm3) at week 2 and 4, respectively.The higher tumor grafting potential of hCSCs and its attenuation by PEITC pretreatment of hCSCs was further evaluated in a small-scale pilot study using a xenotransplantation NOD-SCID mouse model with two test groups (hCSCs/untreated control and 10 µM PEITC-treated hCSCs). Previously, we reported in the same model a higher tumorigenicity of hCSCs when compared to parental HeLa cells . Consist 2 and 4 . FurtherIn the absence of rigorous screening programs in developing countries, cervical cancer remains the second-most-fatal cancer in women worldwide . We and Two decades ago, Lindros et al. described a role for PEITC as an inhibitor of mitochondrial ALDH in the liver in the context of alcohol consumption, with similar effects to that of a known ALDH inhibitor, disulfiram . Intereshi hCSCs tumorsphere formation.It is important to note that this study did not aim to test the efficacy of PEITC on diminishing the tumor burden in mice, which would require tumor transplantation, followed by an oral or parenteral PEITC treatment. The animal study aimed to support in vitro observations showing the suppression of hCSCs upon PEITC-treatments. Thus, it was hypothesized that PEITC-pretreatment prior to xenotransplantation in mice would lower cancer initiating or tumor grafting potential of hCSCs that we observed. Our observation is consistent with a prior study that showed in vitro pretreatment of human breast cancer HMLER-shEcad cells with salinomycin, not paclitaxel, resulted in an inhibition of HMLER-shEcad-induced tumorsphere formation . SimilarAlthough clinical studies are needed to verify the proposed role of PEITC in targeting CSCs that are associated with a range of cancer types, our observation of PEITC attenuating hCSCs-spheres in the context of cervical cancer resonate with a previous report regarding the inhibitory effects of PEITC in colorectal CSCs spheroids . Additio2-humidified atmosphere at 37 °C. HeLa cells were trypsinized with TrypLE  and then sub-cultured (1:5 splitting ratio) when the cells were 80–90% confluent. From the parental HeLa cells, CSCs (hCSCs) were cultured following a modified protocol that was described by Gu et al. [4) were seeded into a 100-mm ultra-low attachment (ULA) petri dish  containing 8 mL of serum-free mammary epithelial basal medium , supplemented with 20 ng/mL hEGF, 20 ng/mL hFGF , 1x B27 , and 4 μg/mL heparin . After an initial five-day culture in suspension at 37 °C, an additional 9 mL of sphere culture medium was added for another five days of culture. On day 10, the spheres were harvested, centrifuged at 500× g for 3 min, washed with phosphate-buffered saline (PBS), trypsinized with TrypLE for 10 min at 37 °C, centrifuged at 500× g for 3 min, resuspended in 5 mL of hCSCs culture medium, and counted with a hemocytometer. The hCSCs were used for flow cytometry and cell proliferation experiments.Following our previous work , human cu et al. ,37. Brie5 cells/mL. Activated Aldefluor reagent (300 μM) was prepared following the manufacturer’s instructions. PEITC-treated cells were incubated with activated Aldefluor reagent and its control reagent, diethylaminobenzaldehyde (DEAB) at 37 °C for 45 min, with intermittent shaking after every 15 min. Following incubation, all of the tubes were centrifuged at 250× g for 5 min at 4 °C. Aldefluor-stained cells were washed once in Aldefluor buffer and maintained in 4 °C until acquired through FL1 channel in flow cytometer  as ALDH high (ALDHhi) and side scatter low phenotypes.The Aldefluor Kit  was used to determine the levels of ALDH1 in hCSCs. 24 h prior to cell harvesting, hCSCs were treated with PEITC  or DMSO . The cells were trypsinized, washed with 1× PBS, and counted using a hemocytometer. Cells were then resuspended in Aldefluor buffer at a concentration of 2 × 105 hCSCs per sample were aliquoted into a six-well plate and then incubated with 10 μM PEITC or DMSO control for 3 h at 37 °C. Glutathione  was used as an inhibitor of ROS production and a pro-oxidant hydrogen peroxide (H2O2) was used as a positive control in the assay. The read signal was detected at Ex485 nm/Em535 nm using a flow cytometer.The cells were harvested, trypsinized, washed with 1× PBS, and counted using a hemocytometer before collecting in conical test tube. Using Cellular ROS Detection Assay Kit , the cells were stained with 20 μM 2′,7′-dichlorofluorescin diacetate (DCFDA) in 1 × Buffer supplied for 30 min at 37 °C following the manufacturer’s protocol. Individual batches of 2 × 106 cells/mL, followed by immunostaining. The cells were immunostained with anti-CD24–FITC  or anti-CD44–FITC  antibodies for 1 h at room temperature. Immunofluorescence was measured using FACSCalibur cell analyzer , with approximately 10,000 events in each sample. Propidium iodide/annexin V staining was performed according to the manufacturer’s instructions. Briefly, the 5 × 105 cells were centrifuged and resuspended in 100 μL of 1 × binding buffer . The cells were treated with 10 μM PEITC or control (DMSO) for a total of 24 h. The cells were then incubated with 5 μL of annexin V–FITC  and 5 μL of propidium iodide  at room temperature for 5 min in the dark before acquiring in a flow cytometer.Following our previous study , the celThe hCSCs were enriched in spheres in serum-free medium, as described previously . Briefly4 cells per well. Cells were cultured in DMEM that was supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 5% heat-inactivated FBS, and 50 µM 2mercaptoethanol. hCSCs were treated with three concentrations of PEITC . After 24 h of incubation, 20 µL of CellTiter reagent was added directly to the cell-culture wells and incubated for 1 h at 37 °C, followed by cell viability assessment using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit , containing MTS. The manufacturer’s instructions were followed and the treatments were compared with control (DMSO-treated cells) at 490 nm in a BioTek Synergy H4 multimode plate reader .3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to determine the number of viable cells, which were seeded into 96-well microplates at a density of 2 × 106 per well) were seeded in a six-well plate and incubated overnight at incubator . Old culture media were replaced by culture medium containing 1.25, 2.5, 5, or 10-μM concentrations of PEITC for 3 h. Cell harvesting and immunoblotting were carried out as we previously reported [g for 15 min at 4 °C. Equal amounts of cell lysate proteins were separated by SDS-PAGE through a 12% separating gel, transferred to nitrocellulose membranes that were blocked with 5% non-fat dry milk, and probed overnight at 4 °C with mouse anti-human Sp1 . Rabbit anti-human β-actin  antibody was used as a housekeeping protein. Blots were then washed in PBS and further incubated with secondary antibodies, goat anti-mouse horseradish peroxidase , and goat anti-rabbit horseradish peroxidase , for 1 h at room temperature. Finally, after rinsing in PBS with 0.1% Tween20, the blots were incubated for 5 min in enhanced chemiluminescent substrate for horseradish peroxidase  and imaged with a ChemiDoc XRS+ Imaging System , followed by a mean fluorescence intensity analysis.The hCSCs (1 × 10reported . BrieflyPrkdcscid/J) mice  were randomly grouped into two groups (four mice per group) in a specific pathogen-free (SPF) housing at a constant temperature of 24–26 °C, with a 12-h:12-h light/dark cycle. All of the mice were provided with autoclaved standard natural ingredients chow diet  and autoclaved water ad libitum, starting from one-week of acclimatization period prior to the treatment. In the meantime, hCSCs were cultured, trypsinized, washed, pre-treated with 10 µM PEITC for 24 h, and resuspended in PBS at the concentration of 1 × 107 cells/mL prior to injection. Each mouse was subcutaneously injected at both flank regions, with one injection of PBS , hCSCs (1 × 106), or hCSCs (1 × 106) pretreated with 10 µM PEITC. The cell number in each injection was consistent with the study that was previously carried out by Gu et al. [2 inhalation 28 days post-treatment and the average tumor number per injection was calculated in each group.Following the guidelines and approval of the Institutional Animal Care and Use Committee (IACUC), South Dakota State University , eight female (five-week old) non-obese diabetic, severe combined immunodeficient  was used for the statistical analyses. Analysis of variance (ANOVA), followed by a Dunnett’s post hoc test, was used to compare multiple means, while statistical significance was assessed by Student’s PEITC treatments suppressed the proliferation of hCSCs expressing higher levels of intracellular CSCs marker ALDH1. An increase in oxidative stress and early apoptotic cells in PEITC-treated hCSCs was observed. The in vitro observations of hCSCs-targeting effects of PEITC associated with a lower tumorigenic potential of PEITC-pretreated hCSCs in vivo. In addition, PEITC treatments attenuated the transcription factor Sp1 and its downstream target, a drug efflux transporter protein P-gp, which further supports a potential role for PEITC in targeting therapy-resistant CSCs in the context of cervical cancer. Vegetable derived PEITC may be further investigated for its use as a complementary intervention for the mitigation of cancer relapse and the alleviation of associated healthcare burden.


Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [σs]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress. Sinorhizobium meliloti carries genes for 11 ECF-like σs (RpoE1 to -E10 and FecI). We hypothesized that some of these play a role in mediating the interaction between the bacterium and its plant symbiotic partner. The bacterium senses changes in its immediate environment as it establishes contact with the plant root, initiates invasion of the plant as the root nodule is formed, traverses several root cell layers, and enters plant cortical cells via endocytosis. We used genetics, transcriptomics, and functionality to characterize the entire S. meliloti cohort of ECF σs. We discovered new targets for individual σs, confirmed others by overexpressing individual ECF σs, and identified or confirmed putative promoter motifs for nine of them. We constructed precise deletions of each ECF σ gene and its demonstrated or putative anti-σ gene and also a strain in which all 11 ECF σ and anti-σ genes were deleted. This all-ECF σ deletion strain showed no major defects in free-living growth, in Biolog Phenotype MicroArray assays, or in response to multiple stresses. None of the ECF σs were required for symbiosis on the host plants Medicago sativa and Medicago truncatula: the strain deleted for all ECF σ and anti-σ genes was symbiotically normal.Bacteria must sense alterations in their environment and respond with changes in function and/or structure in order to cope. Extracytoplasmic function sigma factors (ECF σs) modulate transcription in response to cellular and environmental signals. The symbiotic nitrogen-fixing alphaproteobacterium IMPORTANCE Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [σs]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress. Sinorhizobium meliloti, a Gram-negative alphaproteobacterium, can live as a heterotrophic soil saprophyte or in symbiosis with a host plant such as Medicago sativa or Medicago truncatula  genes (nif and fix genes) in bacteroids in response to low levels of free oxygen in infected plant cells  genes , 8, whicd) genes . Anothernt cells .70 is encoded by rpoD. Alternative σs in Escherichia coli include RpoH (σ32), RpoS (σ38/S), RpoE (σ24/E), FecI (σFec), FliA (σ28/F), and, in some strains, RpoN (σ54/N). All E. coli σs except RpoN belong to the σ70 family, whose members contain up to four conserved structural domains (σ1 to σ4) , a member of the extracytoplasmic stress function (ECF) σ family, responds to cell envelope stresses such as periplasmic protein unfolding and outer membrane disruption  sigma factor (σ) subunits control global transcription by determining promoter specificity , 10. The1 to σ4) ; each di1 to σ4) . In E. csruption . The antsruption . In othesruption . ECF σs sruption . At leassruption , 17.S. meliloti, genomic annotation discloses genes coding for the housekeeping σ, RpoD (σ70), RpoN, and two RpoH σs . In this work, we systematically studied all 11 ECF-like σs. We used global transcription analyses to identify ECF σ target genes and putative ECF σ promoter motifs. Construction of mutants deleted for these ECF σs showed they have no major effects on free-living growth besides a slight decrease in growth rate. None of the ECF σs were required for symbiosis: a strain deleted for all ECF σ and anti-σ genes was symbiotically normal.The 1 to σ4)  . S. meli2 and σ4 , 27. The10.1128/mSphereDirect.00454-18.1FIG S1Sinorhizobium meliloti sigma factors (σs) and anti-σs. (A) Genomic distribution of σ genes by replicon. Locations of ECF σ genes are indicated by blue triangles, RpoH σ genes by orange triangles, the RpoN σ gene by a red triangle, the RpoD housekeeping σ gene by a green triangle, and the RsiA2 orphan anti-σ gene by a yellow triangle. (B) Map of the rpoE5 region on replicon pSymB showing two candidate anti-σ genes. Downstream of rpoE5, SMb21687 is annotated in the S. meliloti Rm1021 genome, while SMb23398, encoding an RsiA-like anti-σ protein, was additionally annotated in the S. meliloti Rm2011 genome (rsiA-like ORF is located at positions 1405878 to 1406054 of pSymB. Transcription start sites (TSSs) identified by our 5′-RACE mapping and RNA-Seq . Using ECFfinder, we classified S. meliloti ECF σs into six of the 94 groups based on protein domain architecture, sequence similarity, genomic context, putative promoter motifs, and anti-σ features  compared to a control strain carrying the empty vector. We used S. meliloti CL150, a wild-type (WT) Rm1021 derivative corrected for nonfunctional ecfR1 and pstC genes  show high constitutive expression of RpoE1 target genes in agreement with our data from overexpressing RpoE1 via an exogenous promoter  are related to phosphate metabolism, as expected from previous studies  mapping  are arrangements widely conserved among other group ECF26 σs . Th. Th33). ECF26 σs . In contbacteria . All foutivation ; thus, tsorT-sorU-azu2 operon, whose proteins likely detoxify sulfite and contribute to sulfite respiration during stationary growth , confirming the findings of Bastiat et al.  and activate expression of the y growth , 34–36. t et al. , as wellt et al. . The sorregulons  , but most (21 of 27) showed lower expression when activated by RpoE6. The basis for this overlap is unknown since RpoE2 does not appear to activate rpoE6 expression. Expression of the downstream anti-σ gene, ecfR6, increased only when we overexpressed RpoE6, arguing for autogenous regulation of the rpoE6-ecfR6 operon; however, besides being cotranscribed with rpoE6, ecfR6 may also be transcribed from an RpoE6-dependent promoter located within the rpoE6 open reading frame (ORF)  . The mosme (ORF) . RpoE6 obut most  of 27 shOur Affymetrix GeneChip also contains probe sets corresponding to tiled intergenic regions (IGRs) that are ≥150 bp . PreviouGGAATA/−10 GT) determined earlier  sequesters σ from interacting with RNAP, until an anti-anti-σ (RsiB1/RsiB2) is activated by phosphorylation of its receiver domain  and two-component systems (exsF/exsG and SMa0113/SM0114); the latter pair is important for succinate-mediated catabolite repression  S. meliloti strains such as Rm41 and the closely related species Sinorhizobium medicae lack rpoE7-ecfR7 and SMb20527-SMb20530. However, orthologous proteins that are proposed to be involved in response to oxidative stress and heavy metals  are present in the alphaproteobacterium Caulobacter crescentus  , CdCl2 (50 and 100 µM), or K2CrO4 (50 and 100 µM) (data not shown). While the exact activation mechanism for group ECF16 σs is unknown, two cysteine residues in the C. crescentus NrsF anti-σ are required for its inactivation, leading to subsequent release of its partner SigF; these residues are conserved in S. meliloti RpoE7 (Bradyrhizobium diazoefficiens (EcfF and EcfS) have also been shown to be important for oxidative stress response and symbiosis , 31rpoE7S. melilotirpoE8 is located upstream of genes encoding a putative outer membrane protein (SMb20593) and a blue copper-like protein (AmcY) that may be involved in intermolecular electron transfer reactions  (rpoE8-SMb20593 intergenic region (Despite overexpressing 8 target . BecauseCATCCAA)  31), bu, burpoE8CATCCAA) , despitec region . Since orpoE9, it is unlikely to function as an anti-σ . We. WerpoE9r domain  upstreamECF41 σs    .rpoE10 are predicted to be cotranscribed  tha tharpoE1nscribed , 31. We nscribed . The facS. meliloti RpoE10 is similar to that seen in Pseudomonas putida ECF10, the only other group ECF42 σ characterized to date; it deals with antibiotic stress resistance and biofilm formation , which include Pseudomonas aeruginosa PvdS and Pseudomonas fluorescens PbrA, involved in iron uptake , 17findemitation , consistmitation , or when cascade .fecR and SMc04205, but could not identify any promoter motifs upstream of those genes . Because (i) some of the insertions conferred polar effects on adjacent genes, (ii) when more than one plasmid insertion integrates into the genome, it allows their similar DNA sequences to promote genome rearrangements, and (iii) limited availability of antibiotic resistance markers precluded construction of a strain carrying more than a few ECF σ gene mutations, we switched to a precise deletion strategy. We constructed 12 strains: 11 each lacked a different ECF σ gene and its adjacent presumptive anti-σ gene, and the 12th lacked the orphan and fecR .10.1128/mSphereDirect.00454-18.4TABLE S1ecfRx, fecR, and rsiAx in the first column have in silico or experimental evidence suggesting that they encode anti-σ factors; those designated with SM identifiers were deleted based solely on their close proximity to a putative ECF σ gene. rsiA2 encodes an orphan anti-σ gene not located near any σ gene. S. meliloti possesses three replicons: SMc, chromosome; SMa, pSymA megaplasmid; SMb, pSymB megaplasmid. The S. meliloti genome annotation is available at NCBI  and https://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi. Download Table S1, DOCX file, 0.1 MB.Deletions of individual ECF σ and putative anti-σ genes. Genes designated Copyright © 2018 Lang et al.2018Lang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. meliloti plant hosts: M. sativa  and M. truncatula (barrel medic). Nodulation was assessed by counting root nodules at 7 and 21 days postinoculation (dpi). Nitrogen fixation was assessed by nodule color and seedling appearance at 21 dpi. Nitrogen-fixing nodules are distinctly pink in color due to the presence of the oxygen-sequestering protein leghemoglobin  cycle gene. We corrected the mdh defect, creating RFF625c, and sequenced it and its CL150 WT parent. Our sequencing confirmed all expected deletions and corrections , and also revealed two spontaneous nonsynonymous sequence variants not present in S. meliloti Rm1021  are important for growth, stress response, or symbiosis. Correction of these variants would be required to confirm this assertion.Because none of the ECF σ/anti-σ pairs appeared essential for symbiosis when deleted singly or doubly, we constructed a strain deleted for all ECF σs and presumptive anti-σs . It remains possible that in different environments, or facing other challenges, differences in competitiveness or fitness might be found.We assayed RFF625c, the all-ECF σ deletion strain, for symbiosis as for the single and double mutants. Surprisingly, RFF625c behaved like the WT: it formed nitrogen-fixing nodules on both host plants, with nodulation efficiency similar to that of the WT . We alsoM. truncatula nodules. We also analyzed gene expression in the rpoE3-SMc02714 and rpoE8-SMb20593 deletion mutants, because previous transcriptome analyses showed that expression of rpoE3 and rpoE8 was enhanced in nitrogen-fixing nodules , and RFF465 (ΔrpoE8-SMb20593 [green circle]). We used a 1.1-fold cutoff for gene expression changes because <20 genes in the combined data sets showed expression increases of ≥1.5-fold , RFF716 . In contrast, WT CL150 and RFF625c showed similar growth curves in both LB and M9 sucrose liquid media  and M9 sucrose+Sm and incubated them at 30 and 37°C. Our usual growth temperature for 10.1128/mSphereDirect.00454-18.3FIG S3FIG S3, JPG file, 0.2 MB.Growth curves for WT strain CL150 and the all-ECF σ deletion mutant RFF625c. Strains were grown in LB and M9 sucrose liquid media . Download Copyright © 2018 Lang et al.2018Lang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Because ECF σs often mediate response to external stresses, we compared how RFF625c copes, relative to WT, with agents or conditions that provoke various stresses . We expo2O2 (1 mM for 30 min) as previously described  and similarly saw little difference in survival . Our ΔrpoE2 stationary-phase results are consistent with those of Flechard et al. , which is critical for symbiosis. In both cases, RFF625c swam as well as the WT, and produced an indistinguishable amount of exopolysaccharide .n = 2) means the opm analysis was prone to errors but allowed discovery of potential phenotypes that would be validated upon further experimental testing. RFF625c showed 23 subtle differences compared to WT   to compaed to WT . CL150 slog data  to G.10.1128/mSphereDirect.00454-18.9DATA SET S4Data Set S4, XLSX file, 0.2 MB.Biolog Phenotype MicroArray data, analyzed by the opm R package , as descCopyright © 2018 Lang et al.2018Lang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. melilotirpoE2 mutant was reduced in comparison to WT Rm1021 at 0.5% NaCl, and Sauviac et al. (rpoE2 mutant at up to 2.5 M (14.6%) NaCl. The Biolog system did not detect differences between WT CL150 and RFF625c at NaCl concentrations up to 10%. While the Biolog assay of RFF625c did not appear to have the same phenotype as the Rm1021-derived rpoE2 mutant analyzed by Flechard et al., we did observe subtle respiratory defects with the osmolytes NaNO3 (100 mM) and urea (3%)  , which mS. meliloti ECF σs would be retained to carry out narrow functions for adaptation to specific environmental conditions and that closely related Sinorhizobium species would differ in composition, regulation, and genomic contexts of ECF σs. In this study, we explored the S. meliloti ECF σ landscape using transcriptome analyses, TSS mapping, in silico analyses, and phenotypic tests of ECF σ mutants.Rhizobia are known for the large size, complexity, and plasticity of their genomes ; thus, iS. meliloti, while the other ECF σs perform accessory roles that confer growth advantages in certain situations, but not for growth or symbiosis under laboratory conditions. A recent transposon sequencing (Tn-Seq) insertion study identified genes required for fitness during growth in rich and defined media (fecI was the only ECF σ gene insertion that conferred moderate growth impairment (and only in rich medium). This is consistent with our results, where a mutant deleted for all ECF σ genes was symbiotically normal and grew well under most conditions tested, and supports our conclusion that S. meliloti ECF σs are mostly dispensable.Our work shows that, except for RpoE2, which directly or indirectly alters expression of >300 genes , 22, 48,ed media : insertiRickettsia, Wolbachia, and Liberibacter lack ECF σs , LB (5 g/liter NaCl), or TY (tryptone-yeast extract) medium at 30°C, as described previously (E. coli strains were grown in LB medium at 37°C. Antibiotics were used at the following concentrations: ampicillin (Ap), 50 to 100 µg ml−1; chloramphenicol (Cm), 50 µg ml−1; gentamicin (Gm), 5 µg ml−1 for E. coli and 25 to 50 µg ml−1 for S. meliloti; hygromycin (Hy), 50 µg ml−1; kanamycin (Km), 25 to 50 µg ml−1 for E. coli; neomycin (Nm), 50 to 100 µg ml−1 for S. meliloti; spectinomycin (Sp), 50 µg ml−1 for E. coli and 50 to 100 µg ml−1 for S. meliloti; streptomycin (Sm), 500 µg ml−1 for S. meliloti; and tetracycline (Tc), 10 µg ml−1. Triparental conjugation transferred both replicative and nonreplicative plasmids to S. meliloti. Marked insertions and deletions were transferred between S. meliloti strains using N3 phage transduction (eviously . E. colisduction . We usedS. meliloti ECF σs into pCAP11 (rpoE6 and rpoE7) use GTG as their native start codon; this was changed to ATG for purposes of uniformity. Specific downstream primers contained an AvrII sequence and 17 to 20 nt of sequence complementary to and mostly downstream of the ORF termination codon.A nested-PCR approach was used to clone each of the 11 o pCAP11  so that 10.1128/mSphereDirect.00454-18.10DATA SET S5Data Set S5, XLSX file, 0.0 MB.Oligonucleotide primers used in this study. Sheet 1, PCR primers for plasmid and strain construction; Sheet 2, primers for 5′-RACE mapping. Download Copyright © 2018 Lang et al.2018Lang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Amplification of each σ gene was initiated using low levels (0.1 µM) of primers for 20 cycles; a universal pCAP11 primer  and the specific downstream primer were then added , and amplification was continued for another 20 cycles. Purified PCR products were digested with AvrII and cloned into AvrII-digested pCAP11 to create a complete set of plasmids, each carrying a distinct ECF σ under the control of the melibiose-inducible promoter.melA) plasmids described above. Each plasmid was conjugated into the S. meliloti WT strain CL150, an Rm1021-derived strain corrected for mutations in pstC and ecfR1 (ecfR1), and Rm1021/pCAP11.To identify genes whose expression was dependent on each of the 11 ECF σs, we used strains overexpressing ECF σs via the melibiose-inducible promoter (Pnd ecfR1  22). We. WemelA)S. meliloti. S. meliloti carrying ECF σ overexpression constructs grew well in M9 minimal medium with either 0.4% glycerol  or 0.4% succinate (to an OD600 of ∼1.7). We induced the melA promoter of the PmelA-rpoE2 strain with 0.4% melibiose when M9 glycerol-grown cells reached an OD600 of 0.5, and used real-time quantitative PCR (RT-qPCR), as previously described  cv. Jemalong on buffered nodulation medium agar plates and spot inoculated them essentially as described previously (rpoE8-SMb20593 mutant (RFF465), the ΔrpoE3-ecfR3 mutant (RFF716), or the ΔrpoH1 mutant (RFF157). We inoculated 22 plants for each of four replicates, and harvested nodules 25 days after inoculation. Nodule RNA purification, cDNA synthesis, cDNA amplification, and hybridization of labeled amplified RNA to our custom Affymetrix Symbiosis Chip were performed as described previously  for ECF σs and their target genes were identified by performing 5′-RACE (5′ rapid amplification of cDNA ends) on a subset of ECF σ-dependent genes as described previously  and minihttps://weblogo.berkeley.edu) (Promoter consensus motifs for putative target genes of each ECF σ were generated by subjecting sequences upstream of the TSS to MEME (Multiple Em for Motif Elicitation) analyses , as descley.edu) , and varley.edu) .sacB vector, pJQ200SK, and sucrose counterselection  showed that RFF625 carried a point mutation in ymbiosis . To corrWe sequenced the complete genomes of Rm1021, CL150, and RFF625c via Illumina MiSeq technology at the Stanford Protein and Nucleic Acid Facility. Using Nextera kits (Illumina), we prepared sequencing libraries from genomic DNA purified with DNeasy blood and tissue kits (Qiagen). We used CLC Genomics Workbench software (Qiagen) to map paired-end sequence reads to the Rm1021 reference genome and identify single-nucleotide and structural variants. Since the published reference sequence contains errors, only some of which have been identified , we compS. meliloti plant hosts, M. sativa  and M. truncatula [Gaertn.] cv. Jemalong (barrel medic), with CL150 as the WT control strain and CL309 (nifD::Tn5-233) as a non-nitrogen-fixing control strain. CL309 was made by transducing the Tn5-233 from Rm1312-Sp (600 of 0.05. Nodulation was assessed by counting root nodules on each plant at 7 and 21 days postinoculation (dpi). Nitrogen fixation was assessed by nodule color and seedling appearance at 21 dpi. Nitrogen-fixing nodules are distinctly pink in color due to the presence of the oxygen-sequestering protein leghemoglobin  sensitivity by spotting serial dilutions of log-phase MgSO4-washed cells onto LB+Sm agar plates containing 0.1% DOC and incubating them at 30°C, as previously described . P values were calculated using the R aov  function. Because we assayed only two replicates of each strain, statistical analysis is error prone, yet provided a method to identify potential conditions under which growth levels of the WT and RFF625c strains are most likely to differ.Biolog  ran Phenotype MicroArrays on our WT CL150 strain and all-ECF σ deletion (RFF625c) strains. Briefly, fresh colonies from LB plates were resuspended in proprietary Biolog media and dispensed into 96-well Biolog PM plates (PM1 to -20) to test ∼1,900 cultivation conditions, including sources of carbon, nitrogen, phosphorous, or sulfur, and challenge with osmolytes, pH, or chemical stresses. Strains were cultivated in duplicate using an OmniLog incubator. In addition to data generated by the Biolog OmniLog software, we received data as a .csv file, which we analyzed using the do_aggr (bootstrap = 100) and opm_mcp function of the opm package for R  water-alamarBlue (Thermo Scientific) mixture was added. Fluorescence was measured at an excitation of 544 nm and emission of 590 nm immediately after addition of alamarBlue and after 60 min of incubation at 30°C. Viability was calculated by subtracting the zero time point fluorescent readings from the 60-min readings and normalized to the untreated WT control.To validate some of the Biolog results, we inoculated four cultures of CL150 and RFF625c in M9 medium plus 0.4% glycerol to an ODGSE116680.The Affymetrix GeneChip data have been deposited in the Gene Expression Omnibus (GEO) database under Superseries accession no.