Mn-Co-Ni-O (MCN) spinel oxide material, a very important transition metal oxide (TMO) with the best application prospects in information and energy fields, was discovered over five decades ago, but its applications have been impeded by the quality of its films due to the magnitude of deposition challenge. Here we report that high quality of MCN nanofilms can be achieved by sputtering deposition via acetate precursors whose decomposition temperatures are matched to the initial synthesis temperature of the MCN thin films. Excellent performance of MCN nanofilms is demonstrated, combining for the first time preferred orientation, high temperature coefficient of resistance, and moderate resistivity. The film devices show an intrinsic recombination with a much faster rate of the order of a microsecond for the laser-pumped carriers, which is ~3 orders of magnitude larger compared with that of the ceramic material. The electronic structure of the thin films confirms that it is indeed of n-type nature, exhibiting appropriate electronic states consistent with the levels of metal electrodes and semiconductors. The results offer a vital avenue for depositing high performance TMO thin films for advanced oxide devices, and will have great significance for exploiting new applications in modern oxide electronics and optoelectronics. Nowadays transition metal oxides (TMO) play an increasingly important role in a variety of novel applications due to their exceptional electronic properties. TMO are utilized in a variety of applications, such as high-temperature superconductivity293 Ω·cm)−1) at room temperature. All those features make MCN thin films play an important role in oxide electronics and optoelectronics. Unfortunately, the strong electron correlations have a significant impact on the deposition quality of the MCN films. Although this ceramic material was discovered over 50 years ago, there are still no reports of realizing high performance nanoscale thin films, which restricts its prospects of successful application.Among them, Mn-Co-Ni-O (MCN) spinel oxide is considered to be the most important TMO material with extensive application prospects in the fields of information and energyIn this paper, we report the growth of highly oriented (220) MCN spinel nanofilms from suitable precursors whose decomposition temperatures are matched to the initial synthesis temperature of the MCN films during fabrication procedure. High quality MCN nanoscale films are achieved combining for the first time excellent performance of TCR and moderate resistivity. The film devices show a response time ~3 orders magnitude shorter than that of the ceramic material devices for laser-pumped carrier recombination and suitable electronic structure matched to metals and semiconductors.3O4. It has been widely used in bolometer and uncooled infrared detection because of its excellent negative temperature coefficient of resistance (TCR) characteristics and long-time stability15163+ and Mn4+ ions distributed over octahedral sites in the spinel cubic structure. To the best of our knowledge, there has been no breakthrough in the deposition of the high-quality nanometric films. Compared to other synthesis routes, such as CSD and screen printing, RF magnetron sputtering method is much more precise in controlling growth conditions, and highly efficient, as well as more suitable for large scale applications1517MCN spinel oxide develops from the precursor Mn2+ ions occupied at tetrahedral sites and of Mn3+ and Mn4+ ions distributed at octahedral sites in the spinel structure. As a consequence, high quality nanoscale MCN films are still lacking, and there is at present no solution for growing MCN nanofilms for extensive potential applicationsAs shown in 1.4Co1.0Ni0.6O4 are deposited by the target material, sintered from the stoichiometric acetate precursors of Mn(CH3COO)2·4 H2O, Co(CH3COO)2·4H2O, Ni(CH3COO)2·4H2O with an atomic ratio of 7:5:3. The thickness of the films is about 170 nm, measured by cross sectional TEM image (2O4 with the JCPDS Card No. 84 – 0542).MCN nanofilms with nominal composition of MnEM image . The latTo investigate the electrical properties of the MCN nanofilms, the temperature dependent resistivity is given by the expressionT) − 1/T0.5 plot for variable range hopping (VRH) with p. A good linear fitting relationship exists, which implies that the conduction is dominated by VRH model with a parabolic shaped density of states (DOS) around the Fermi level. To derive the material constant B of MCN, /T) − 1/T plot, we assumed approximately nearest neighbor hopping (NNH) with 0 ≈ 0.296 eV is derived by applying the formula E0 = kBB (where kB is Boltzmann constant). The advantage of MCN ceramic materials is that they have a high TCR≈–3.50%K–1 and a moderate resistivity of −1, at temperature of 295 K , ionization energy (IE), and electron affinity (EA). Ultra-violet photoemission spectroscopy (UPS) is a widely used technique that provides direct measurement of the density of electronically filled states of materials. Combined with the determination of band gap energy of the materials by optical method, the valence band (VB) and conduction band (CB) states of the films can be established.VL) of the surface can be deduced, and the density of states near the valence band edge at position 2.The UPS is measured using X-ray Photoelectron Spectroscopy . To ensure the accuracy of experimental results, the surface of the MCN thin films is etched by Ar ion-beam for 2 min before UPS characterization to eliminate surface contamination. The band gap of MCN is determined by a Fourier Transform-Infrared Spectrometer , and is found to be about 0.69 eV. F) and WF of the MCN films can be directly determined via a very standard method, i. e., WF = hv – (Ecutoff – EF), where hv is the photon energy, and Ecutoff is the cutoff energy and equal to 21.22 eV. Thus, the vacuum level position (EVL) and IE of the MCN are determined by the VB spectrum.Then the Fermi level  preferred orientation have been fabricated via RF sputtering, with well controlled oxidation states by merging the decomposition temperatures of the precursors to the initial synthesis temperature of the films. MCN nanofilms have been realized with both moderate resistivity and large absolute TCR value simultaneously for the first time. It shows a fast intrinsic response, about 3 orders shorter than the extrinsic one of the ceramic materials, observed by the recombination of photon pumped carriers. The successful breakthrough of high performance of MCN thin films ensures the opportunity for wide ranging applications. The results are very important to improve power conversion efficiency in organic electronics and energy optoelectronics by optimizing the band structures, to manufacture broadband focal plane arrays for advanced optical detection with long-term stability, and to develop high-speed TMO devices in next generation of oxide electronics and optoelectronics.1.4Co1.0Ni0.6O4 were grown starting from pressed powder of MCN target material, which was obtained by drying the chemical solution of Mn, Co, Ni acetates of Mn(CH3COO)2·4H2O, Co(CH3COO)2·4H2O, Ni(CH3COO)2·4H2O with an atomic ratio of 7:5:3 (the target ratio) and calcined at 850 °C. Then a polycrystalline MCN ceramic wafer (Φ 60 mm × 4 mm) with a cubic spinel phase and no prefer orientation, was prepared via traditional sintering method. Prior to film deposition, the base pressure was evacuated to approximately 5 × 10−3 Pa, and a working pressure of 0.4 Pa. High purity (>99.99%) argon was applied to provide the plasma. Al2O3 substrates were annealed at 750 °C for ~3 min before growth. The growth temperature was set 200 °C. MCN film sample was deposited under 100 W of RF power for 1.5 hours and post annealed at 750 °C for 90 min for crystallization. MCN bolometer was mounted to a conductive heat-sink sealed in a ceramic package. Electrical properties were measured by a Keithley 2400 and associated temperature control systems. The MCN ceramic material in the experiment was manufactured by the typical traditional method, calcined at 1100 ~ 1200 °C for 30 min and cooled to room temperature naturally. The material is polycrystalline, with a distribution of grains sizes from 1 to 3 micron, determined from SEM images. The precursor was manufactured from nitrates of Mn, Co, Ni in chemical solution of the target ratio.Thin films of MCN with nominal composition MnHow to cite this article: Huang, Z. et al. High performance of Mn-Co-Ni-O spinel nanofilms sputtered from acetate precursors. Sci. Rep.5, 10899; doi: 10.1038/srep10899 (2015).


In vitro, IFNα inhibited proliferation of human pulmonary artery smooth muscle cells and as well as human pulmonary artery endothelial cell proliferation and apoptosis. Together these findings demonstrate that IFNα reverses established experimental PH and provide a rationale for further exploration of the use of IFNα and other immunotherpies in PH.Pulmonary hypertension (PH) is a progressive and fatal disease with no cure. Vascular remodeling in PH involves intraluminal growth of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Cell growth in these lesions is quasi-neoplastic, with evidence of monoclonality, apoptosis resistance and cancer-like metabolic derangements. Herein we tested the effect of human interferon alpha 2b (IFNα), a pleiotropic cytokine and anti-cancer therapeutic, on the development and progression of PH in the rat SU5416/hypoxia (SUH) model and mouse hypoxia model of the disease. In both models IFNα attenuated the development of PH and reversed established PH as assessed by measuring right ventricular systolic pressure and right ventricular hypertrophy. The effect of IFNα was dependent on the type I interferon receptor (IFNAR) since mice lacking a subunit of the IFNAR were not protected by IFNα. Morphometric analysis of pulmonary aterioles from hypoxic mice or SUH rats showed that IFNα inhibited pulmonary vascular remodeling in both models and that IFNα reversed remodeling in SUH rats with established disease. Immunohistochemical staining revealed that IFNα decreased the number of PCNA and Tunel positive cells in the wall of pulmonary arterioles.  Pulmonary Hypertension (PH) is a devastating disease characterized by increased pulmonary artery pressure, right ventricular (RV) failure and death. Although the natural history of the disease is incompletely understood, the traditional view is that endothelial dysfunction and upregulation of pulmonary vasoconstrictors leads to pulmonary vasoconstriction and increased pulmonary artery (PA) pressure. In addition, several pulmonary vasoconstrictors are also smooth muscle cell (SMC) mitogens In severe disease the PAs of PH patients exhibit invasive growth of endothelial cells (EC) into the vessel lumen resulting in luminal obstruction by clusters of ECs known as plexiform lesions. EC growth in plexiform lesions is aberrant with some areas containing a solid core of ECs and others exhibiting various stages of angiogenesis Type I interferons (IFN), were identified in 1957 by Isaacs and Lindenmann based on the ability to inhibit viral replication Little is known about the effect of IFNα on the pathogenesis of pulmonary hypertension. There are case studies of patients receiving IFNα therapy for the treatment of hepatitis C or chronic myelogenous leukemia developing reversible or irreversible PH The goal of the present study was to evaluate the effect of IFNα on experimental PH. Based on the case studies demonstrating IFNα-induced PH, and our data showing activation of interferon response factor-3 in PH our original hypothesis was that IFNα would exacerbate experimental PH. Instead, we found that, in both the mouse model of chronic hypoxia and the rat model of SU5416 plus chronic hypoxia, IFNα not only attenuated the development of PH, but also reversed established disease.and the studies were approved by the University of Pittsburgh IRB. All patients gave written consent. Animal studies were approved by the University of Pittsburgh Institutional Animal Care and Use Committee .Human IPAH cells and serum samples were obtained in compliance with University of Pittsburgh Institutional Review Board (IRB) guidelines C57BL/6J mice were purchased from The Jackson Laboratory  and 129S6/SvEvTac WT mice were purchased from Taconic Farms . IFNAR-deficient mice (IFN-IR−/−129S6) were a kind gift of Akiko Iwasaki  4 IU human recombinant interferon alpha 2b . The dose of interferon alpha 2b was chosen based on a search of the literature Eight to ten week old male mice were placed into a partially ventilated Plexiglas chamber  and exposed to chronic hypoxia  for 21 or 42 days under normobaric conditions. Mice maintained in room air served as normoxic controls. For all mouse studies mice were treated with daily subcutaneous injections of 105 IU human recombinant interferon alpha 2b. This dose was chosen to approximately match the dose given to mice. The interferon was prepared and stored as described above for the chronic hypoxia mouse model.225–250 g male Sprague Dawley Rats were injected with a single dose of 20 mg/kg s.c. SU-5416 (A VEGF receptor inhibitor) or vehicle and were placed into a partially ventilated Plexiglas chamber  and exposed to chronic hypoxia  for 21 days under normobaric conditions. Rats maintained in room air served as controls. Some Rats were returned to room air on day 22, and maintained in normoxia for an additional 14 days. For rat studies animals were treated with daily subcutaneous injections of 10Right ventricular systolic pressure (RVSP) was measured essentially as described Following hemodynamic measurements the vasculature was flushed with PBS, the heart was excised and right heart hypertrophy was determined by the ratio of the weight of the RV to the left ventricle (LV) plus septum (Fulton index) or the ration of weight of the RV to body weight. The right lung was tied off, dissected and flash frozen, and the left lung was perfused with paraformaldehyde (4%) for embedding in paraffin.For mice, pulmonary vascular remodeling was assessed by counting the number of partially and fully muscularized peripheral arterioles (35–100 mm) per high-power field . For each mouse, at least 20 high-power fields were analyzed in multiple lung sections. Wall thickness % was determined by measuring the thickness at four points on pulmonary arterioles using the Java-based image-processing program ImageJ . Vascular occlusion was assessed in a blinded fashion by grading at least 50 small (<50 µm) pulmonary arterioles in at least 3 lung tissue samples per group.Serum IFNα was measured using commercially available ELISA kits .Paraffin-embedded lung sections (5 µm) were baked 60 min at 55°C, deparaffinized in xylenes and rehydrated through decreasing alcohol concentrations  followed by antigen retrieval citrate buffer by using a microwave. Smooth muscle α-actin staining was performed as described Control Human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells (HPASMC) were from Lonza. Control and IPAH HPAEC were cultured in EBM2 media and HPASMC were cultured in SBM2 (Lonza) containing the recommended serum and growth factors. Cells were used between passages 4 and 9.Briefly, HPAEC or HPASMC were serum-starved for 24 h in 12-well plates and treated with the indicated doses of IFNα with or without VEGF (50 ng/ml) or platelet-derived growth factor (PDGF)  for 24 h in the presence 0.2 µCi [3H] thymidine. Cell Proliferation was then determined by measuring [3H] incorporation as previously described 30 µg of cell lysate was separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in TBST , 5% nonfat dry milk for 30 min, followed by incubation in primary antibody overnight. Membranes were washed in TBST before incubation for 1 h with horseradish peroxidase conjugated secondary antibodies. Membranes were washed and developed using enhanced chemiluminescence substrate (Pierce). Blots were probed against p21 (#sc-397 Santa Cruz), stat3 , phospho-Stat3 , Stat1 (sc-98783 Santa Cruz), phospho-Stat1 , Phospho-Akt , Akt (61086 BD Transduction Laboratories), β-actin .P values of <0.05 were considered significant.Statistical analyses were performed by using Graphpad Prism software. Data were analyzed by one-way ANOVA and Tukey's post hoc tests. 2). These rats received daily injections of IFNα  or sterile saline (vehicle) for the duration of the experiment. For the therapeutic protocol, the SUH rats were given a single injection of SU5416, exposed to 3-weeks of hypoxia and then returned to normoxia for 2 weeks. These rats were given daily injections IFNα  or vehicle during the 2 week normoxic period. Rats maintained in normoxia served as controls. Treatment of SUH rats with IFNα using the prevention protocol attenuated the development of PH, as evidenced by decreased right ventricular systolic pressure (RVSP) and decreased right ventricular hypertrophy (RVH) compared to vehicle treated animals (–1C). More importantly, IFNα treatment of SUH rats with established PH (therapeutic protocol) decreased RVSP and RVH compared with untreated SUH rats assessed for PH at 3 or 5 weeks (–1C). Visual inspection of hearts from SUH rats further suggests that the hearts from 5-week SUH rats demonstrate increased RV dilatation compared with hearts from 3-weeks SUH rats, which was prevented by therapeutic IFNα . SUH rats were randomly assigned to a 3-week “prevention protocol” or a 5 week “therapeutic protocol” Fig. 1A.4 I.U./day, s.c.). To establish the efficacy of IFNα on established disease, mice were exposed to 6 weeks of hypoxia and treated daily with IFNα  from week 4 through week 6  we examined whether 1) human IFNα could elicit a typical type I interferon signaling response in rats and mice and 2) whether genetic deletion of a subunit of the type I interferon receptor could prevent the effect of IFNα in hypoxic mice. As expected of a type I IFN response, IFNα increased phosphorylation of STAT1 in both SUH rats  that were occluded greater than 50% was less in the 3-week SUH or 5-week SUH treated with IFNα compared with untreated SUH rats ,3D. from both control and IPAH patients.Pulmonary vascular remodeling in SUH rats is characterized by increased proliferation of vascular smooth muscle and endothelial cells. Thus, we observed increased expression of proliferating cell nuclear antigen (PCNA) in the vessel wall of both 3 week and 5 week SUH rats. Consistent with the anti-proliferative effects of IFNα we observed less PCNA staining in the wall of pulmonary arterioles from SUH rats treated with IFNα  is impaired in the SUH model and that IFNα stimulates efferocytosis. The number of apoptotic cells in a tissue is affected both by the rate of apoptosis and the rate of efferocytosis by macrophages and resident cells. Apoptotic cells that are not cleared become necrotic causing release of pro-inflammatory molecules IFNα is a pleiotropic cytokine that affects many cell types. Thus, it is likely that other cell types beyond those explored in this study are involved in the effect of IFNα in these models of PH. Of interest is the possibility that the effects of IFNα may be mediated by activation of natural killer cells. Ormiston et al. recently demonstrated impairment of natural killer cell phenotype and function in PH patients, hypoxia-induced PH in mice, and monocrotaline-induced PH in rats Despite our finding that IFNα prevents and reverses experimental pulmonary hypertension in 2 distinct models, chronic treatment with IFNα for hepatitis C or chronic myelogenous leukemia was associated with the onset of PH in humans and the Food and Drug Administration has labeled IFNα with a warning about the risk of PH with its use. Recently, Dhillon et al. In terms of a role for endogenous IFNα in the development of PH we found that despite a slight elevation in circulating levels of IFNα in mice after 21 days of CH that deletion of IFNAR1 subunit of the type I interferon receptor had no effect on the progression of PH. Interestingly this is different from what was recently reported by George et al. where they found that deletion of this subunit protected mice from the development of chronic hypoxia-induced PH. In this study they also report that IFNAR1 is upregulated in Systemic Sclerosis patients with PH and that Interferon Regulated Protein 10 (IP10) correlated positively with pulmonary hemodynamics and serum brain natriuretic peptide and negatively with 6-minute walk test and cardiac index To our knowledge this is only the second demonstration of disease reversal in the SUH rat model of PH. While case studies suggest that IFNα therapy may cause pulmonary hypertension, our data raise legitimate questions as to the role of IFNα in this process. IFNα is a pleiotropic cytokine that mediates a wide range of biological effects including, anti-proliferative, anti-angiogenic and anti-tumor activities, which theoretically could provide benefit to PH patients. The reversal of PH with an immunotherapeutic modality is novel and provides proof of principle that immunotherapy can have a positive impact on PH progression. While evidence of IFNα-induced PH in humans might mean that IFNα will never be used to treat human PH our results warrant further investigation of IFNα and other immunotherapeutics in PH.


Tribolium castaneum represents a well-established experimental model organism for studying questions in ecology and evolution, however, the relevance of its microbial community is still largely unknown. T. castaneum larvae orally exposed to bacterial components of the entomopathogen Bacillus thuringiensis bv. tenebrionis showed increased survival upon a subsequent challenge with spores of this bacterium. To investigate whether T. castaneum microbiota plays a role in this phenomenon, we established a protocol for raising microbe-free larvae and subsequently tested whether they differ in their ability to mount such a priming response. Here we demonstrate that larvae with significantly lowered microbial loads, show decreased survival upon secondary challenge with B. thuringiensis bv. tenebrionis spores, compared to animals that were allowed to regain their microbiota before priming. Although the exact mechanism of oral immune priming is unclear, we here suggest that microbiota plays a crucial role in oral immune priming in this species.Animals are inhabited by a diverse community of microorganisms. The relevance of such microbiota is increasingly being recognized across a broad spectrum of species, ranging from sponges to primates, revealing various beneficial roles that microbes can play. The red flour beetle  Parasites and infectious diseases impose a serious threat to the fitness of their hosts. To defend themselves, vertebrates have evolved an innate and adaptive immune system, the latter being characterized by specificity and memory . The degTenebrio molitor  larvae show increased survival after oral exposure to entomopathogenic bacterial spores of Bacillus thuringiensis subsp. morrisoni bv. tenebrionis when previously orally primed with the supernatant taken from a spore culture of the same bacterium (Bacillus thuringiensis (Berliner 1915) (Bt) is a Gram-positive, endospore forming bacterium expressing plasmid-encoded crystalline inclusions (Cry toxins) specifically toxic to various insect orders after ingestion  , stored in 25% glycerol stocks at -80°C before the experiments started.In this study we used the  in 2010 . IndividT. castaneum by bleaching eggs and raising larvae under sterile conditions. We then allowed the larvae to either be colonized with microbiota or not, by using three types of flour: “untreated,” “sterilized,” or “recolonized” (see below for details). The “untreated” and “recolonized” flour were expected to contain microbes capable of larval gut colonization. After that, we performed priming-challenge experiments with these differently treated larvae. We performed two independent experiments, the methods of which were identical unless described otherwise, however, in Experiment 2 we used eight independently replicated flour treatments for each of the three different flour types.To test for the effect of microbiota on oral immune priming, for every individual we always first removed the natural microbiota of For each experiment, a subpopulation of ca. 2,000 approximately 1-month-old adult beetles was allowed to copulate and oviposit for 24 h. The beetles were kept under standard breeding conditions in approximately 500 g of standard flour in a 4.0 L transparent plastic box  with six holes (3 cm in diameter) punctured into the lid and plugged with foam stoppers (4.2 cm in diameter) (K-TK e.K.) to allow the air to circulate. After 24 h the adult beetles were separated from the flour using a 710 μm mesh size sieve (Retsch). The flour was subsequently separated from the eggs using a 280 μm mesh size sieve.® and kept at standard breeding conditions for 4 days.The following egg bleaching and washing procedures were performed in a sterile environment. Four micro-spoons (Sigma-Aldrich) of eggs were placed in a cell strainer with a 40 μm nylon mesh (BD Biosciences). The cell strainer containing the eggs was soaked and rinsed in 6% NaClO solution for 3 min and subsequently washed with sterilized, deionized water two times for 3 min. The cell strainer containing the bleached and washed eggs was placed upside down on an open, sterilized glass Petri dish (150 × 15 mm) and left to dry in a sterile environment for 5 min. The total number of cell strainers with bleached eggs in both experiments was nine and 24, respectively. In Experiment 1 the nine cell strainers were divided into three glass Petri dishes each containing three cell strainers with the eggs, while in Experiment 2 each of the 24 cell strainers was placed into a separate Petri dish. After drying, the Petri dishes containing the cell strainers and sterilized eggs were closed and sealed with Parafilm M60Co radiation source  to kill any microbes. The “sterilized” flour was not treated in any way after irradiation. In order to reintroduce the insect-associated microbiota to the gamma sterilized flour, the “recolonized” flour was prepared using the following procedure: 100 larvae and 100 adult beetles randomly chosen from the stock population were left ad libitum per 100 g of irradiated flour for 7 days under standard breeding conditions. On the seventh day of the flour conditioning, the animals were sieved from the “recolonized” flour using a sterile 280 μm mesh size sieve.By the fifth day post oviposition (DPO) the eggs had hatched. The Petri dishes were opened in a sterile environment, the larvae were flicked from the cell strainer into the Petri dish, and placed onto one of three different flour treatment: “untreated,” “sterilized,” or “recolonized.” Each of the three flour treatments was prepared from a single mixture of standard flour. The “untreated” flour was not treated in any way, representing our control treatment. Both the “sterilized” and the “recolonized” flour were gamma irradiated with a single dose of 29 kGy using a In Experiment 1 three Petri dishes of larvae were placed into 1.7 L glass jars each containing 300 g “untreated,” “sterilized,” or “recolonized” flour, respectively. In Experiment 2 the larvae from the each of the 24 Petri dishes were placed into one of 24 0.7 L glass jars, eight replicates of either 100 g of “untreated,” “sterilized,” or “recolonized” flour, respectively. The glass jars containing the flour and the newly hatched larvae were sealed with sterile breathable sealing foil for culture plates (Kisker Biotech) and kept under standard breeding conditions for 9 days. On DPO 14 the larvae were separated from the flour using a 280 μm mesh size sieve. After the flour was removed, the remaining larvae were allowed to crawl through a system of sterile sieves with four descending mesh sizes  put on top of one another for 5 min. Uniform-sized larvae that remained in the 500 μm mesh size sieve after 5 min were used for priming.Btt culture (“primed”), BT medium (“medium control”) or phosphate buffered saline—PBS (“PBS control”). The plates for oral priming were designed in a way that each plate contained all treatments. For Experiment 2 we omitted the PBS control group. All bacterial cultures were grown at 30°C in darkness. The oral priming diet was prepared following a previously described protocol , 0.1% glucose, 0.34% KH2PO4, 0.435% K2HPO4] was supplemented with 25 μL of sterile salt solution , 6.25 μL of sterile 1 M CaCl2 × 2H2O solution, inoculated with three Btt colony forming units from the LB agar plate and incubated overnight in culture tubes (Simport) at 200 rpm. The following morning, 300 mL of BT medium were supplemented with 1.5 mL of salt solution, 375 μL 1M CaCl2 × 2H2O and inoculated with 1 mL of the overnight culture. The culture was incubated in a 2 L Erlenmeyer flask for 6 days at 200 rpm. On the third day of incubation, an additional 1.5 mL of salt solution and 375 μL 1M CaCl2 × 2H2O were added to the culture. On day six, the culture was centrifuged at 3700 × g for 10 min at room temperature (RT). After centrifugation, the supernatant was retained and transferred to a new 50 mL centrifugation tube (Sigma) and the centrifugation step was repeated. After the second centrifugation, the supernatant was transferred to a new 50 mL tube and filter-sterilized using first a 0.45 μm and subsequently a 0.2 μm cellulose acetate filter (Whatman GmbH). For the primed diet, the sterile supernatant was mixed with 0.15 g of gamma irradiated standard flour per milliliter of supernatant. For the medium control diet, we mixed 0.15 g of gamma irradiated standard flour per milliliter with unconditioned medium . For the PBS control diet, designed to control for the effects of the BT medium itself on the larval survival, we used 0.15 g of gamma irradiated standard flour per milliliter of PBS. For both experiments 30 μL of diet  were pipetted into each well of a 96-well plate, and this was replicated 15 times in Experiment 1 and 24 times in Experiment 2. After pipetting the diets, the plates were covered with a sterile breathable sealing foil for culture plates and the flour was allowed to dry for 24 h at 36°C.We produced a priming diet and two additional control diets for Experiment 1. They all contained gamma irradiated standard flour plus either supernatant from a protocol  with a fThe larvae were placed individually into the 96-well plates. In Experiment 1, 360 larvae were placed onto each of the three priming diets. In Experiment 2, 1152 larvae were primed and 1152 were used as a medium control. The plates were sealed with a transparent adhesive tape (Tesa SE) and nine holes were punctured with a needle (0.3 mm in diameter) above each well. The plates were placed in 2.6 L boxes with air circulation vents as described above. Each box contained three 96-well plates. After 24 h on the priming diet (DPO 15) all larvae were individually transferred to new 96-well plates containing only PBS diet. Note that this PBS diet step was applied to all larvae for 4 days, regardless of the previous priming treatment they received. The plates were sealed and put into new boxes with air circulation vents as described above and kept at standard breeding conditions for 4 days until challenge, i.e., DPO 19.Btt spores and the “control” diet contained no spores. The diet for the oral challenge was prepared following the same protocol as for the priming and as previously described , the supernatant was discarded, and the spore pellet was resuspended in PBS. The spore concentration of the diet was adjusted to 1 ×1010 mL-1 by adding PBS, and 0.15 g of gamma irradiated standard flour was added per milliliter of spore suspension. In both experiments 30 μL of spore containing challenge diet and 30 μL of medium control diet were pipetted into each half of the 96-well plate. Such a plate was replicated 15 times in Experiment 1 and 24 times in Experiment 2. The plates were covered, placed in 2.6 L plastic box with air circulation vents as described above and dried for 12 h at 50°C.We produced two diets: the “challenge” diet contained escribed  with a fOn DPO 19, larvae were individually transferred onto the challenge diet. The total number of larvae challenged per each of the 18 flour/priming/challenge treatments for Experiment 1 ranged between 101 and 108 (a few larvae died and/or escaped during priming), for Experiment 2, 96 larvae were used for each of the 12 flour/priming/challenge treatments. The larvae were kept on the challenge diet for 7 days and the survival was checked every 24 h.T. castaneum larvae and associated bacteria was extracted using a PowerMicrobiomeTM RNA Isolation Kit  following the manufacturer’s protocol. The concentration of the extracted RNA was measured using a Qubit® 2.0 Fluorometer and the corresponding Qubit RNA HS Assay Kit (Life Technologies GmbH) following the manufacturer’s instructions. The concentration of the extracted RNA was equalized across samples in both experiments. The cDNA was synthesized using the SuperScript® III First-Strand Synthesis System (Life Technologies GmbH) and random hexamer primers (Life Technologies GmbH) following the manufacturer’s protocol. The relative quantification of bacterial cDNA from the 16S rRNA gene was performed using a real-time quantitative PCR system LightCycler® 480 (Roche) with the KAPATM SYBR® Green I chemistry. The primers used to amplify the hypervariable region V5-V6 of the bacterial 16S rRNA cDNA were fwd: 799F-mod2 (5′ AACMGGATTAGATACCCKGGT 3′) and rev: 1114R (5′ GCAACGAGCGCAACCC 3′)  and rev: (5′ GGTAGCATGTGCTTCGTTTTG 3′)  from each jar in three replicates per jar and 500 μL of RNA later RNA Stabilization Reagent (Qiagen) were added. The larvae that had been removed from the experiment on DPO 14 were tested via real time quantitative PCR (RT-qPCR) for bacterial cDNA as an indicator of the level of bacteria in the larval guts after exposure for 9 days to one of the three flour treatments. The RNA later reagent was removed from the tubes and the larvae were homogenized under sterile conditions, on ice, using a sterile pestle. The total RNA from the ACCC 3′)  yieldingTTTG 3′) . All theLarval survival over the 7 days following challenge was analyzed using the R statistical package for Macintosh (RStudio version 0.99.441) . Within Btt spores at challenge), whether there was a significant effect of the factor “priming” ; 3. within the challenge group  whether there was a significant effect of priming. This gave us nine models in total for Experiment 1.For Experiment 1 we analyzed each of the flour treatments  separately. For each of these three flour treatments we performed three separate models, which were designed to test: 1. whether there was a significant overall effect of the factor “challenge” ; 2. within the control group  and we included jar as a random effect. The “coxme” models including jar were compared to “coxph” models (without this random effect) in order to determine whether there was a significant effect of jar. Jar was removed during model reduction as in all cases it was non-significant.In both experiments, in all cases where the Cox proportional hazards models did not fulfill the assumptions of proportional hazards over time  we also tested the model using the survival regression (“survreg”) function in R. In all cases the results from the Cox proportional hazards and survival regression were qualitatively similar, therefore we present the results of the Cox proportional hazards models.T. castaneum rp49 gene was used as the internal standard. Levels of 16S rRNA cDNA of “sterilized” and “recolonized” larvae were compared to the “untreated” larvae. Also, “sterilized” larvae were compared to “recolonized” larvae. Primer efficiency used in the analysis was 90% for the target gene and 95% for the internal standard.The results of the relative quantification of bacterial 16S rRNA across different jars were analyzed using the Relative Expression Software Tool, REST 2009 . The T. According to the German Animal Welfare Act, experimental work on insects is not a subject to any ethical approval. Nevertheless, all the experiments in this study were performed in accordance with good ethical and scientific practice.p < 0.001, Figure 1). Although the larvae raised on the “recolonized” flour also showed somewhat lower bacterial load estimates, these levels were not significantly lower than the “untreated” larvae .In both experiments, the sterilization and recolonization treatments of the flour were successful: larvae reared on the “sterilized” flour treatment, although not entirely free of bacteria or their nucleic acids that may be amplified in the qPCR, showed a significantly lower qPCR signal compared to larvae reared on both “untreated” and “recolonized” flour . In all flour treatments, as predicted, larvae challenged with Btt spores showed a significantly lower survival than control larvae, which were not exposed to any spores. For Experiment 1 the results were: “untreated” , “sterilized” , “recolonized” , while the results of Experiment 2 reflected the results from the first experiment: “untreated” , “sterilized” , “recolonized” . This confirmed that the larvae exposed to Btt spores indeed died from Btt infection.To address the question of whether different flour treatments  led to differences in priming, we analyzed each of the flour treatments in both experiments separately  and “recolonized” , only the medium control was included into Experiment 2, while the PBS control was omitted. Potential differences among the eight replicate jars for each flour treatment, which were used in Experiment 2 in order to increase the statistical power of the experiment, were initially included as a random effect into the statistical analyses, but were removed during model reduction, indicating consistency among the replicates.Since the results of the individual comparisons from Experiment 1 had shown no difference in survival between the medium and PBS control larvae regardless of the flour treatment: “untreated”  control larvae died. Among control larvae in both experiments, there was no effect of the priming treatment on survival: Experiment 1, “untreated” , “sterilized” , “recolonized” , and Experiment 2, “untreated” , “sterilized” , “recolonized” . Control larvae were thus excluded from the further analyses.As expected, for both experiments and across all treatments without χ2 = 17.49, df = 2, p < 0.001). Pairwise comparisons showed that, as predicted, challenged larvae previously primed with Btt supernatant had significantly higher survival than the medium control primed larvae , i.e., the larvae showed the expected priming effect . The same expected priming effect was observed in larvae raised on the “untreated” flour in Experiment 2: . Sterilizing the flour removed the effect of priming both in Experiment 1  and Experiment 2 . Recolonizing the sterilized flour with microbes resulted in the re-emergence of a significant priming effect in Experiment 1 . Similarly to the “untreated” flour, challenged larvae previously primed with Btt supernatant had significantly higher survival than the medium control primed larvae . The same expected priming effect was reestablished when larvae were raised on the “recolonized” flour in Experiment 2 .In contrast to the control larvae, in Experiment 1 there was a significant overall effect of priming on survival of the challenged larvae when reared on “untreated” flour , which could indicate the presence of some radiation resistant bacteria  might still be present. It is noteworthy in this context that a role of the midgut bacteria for susceptibility to B. thuringiensis was controversially discussed for lepidopteran species (T. castaneum by Btt (after non-effective priming with medium or PBS) was similar across flour treatments, with or without microbiota .In our host–pathogen system, we can envision a number of possibilities as to how oral priming could depend on the microbiota. First, reminiscent of the mosquito-malaria example , primingthe host . Althoug species . HoweverGlossina spp. modulates the host’s ability to mount an effective immune response after a challenge with trypanosome parasites (B. thuringiensis based product (Xentari) in the lepidopteran species Spodoptera exigua resulted in host lines with increased midgut microbial loads and higher antimicrobial immunity  in the gut of t health . Once ththe host .T. castaneum microbiota (Finally, it is possible that the removal of the indigenous gut microbiota is more generally detrimental for the host, especially if symbionts are needed for certain metabolic functions (reviewed, in crobiota .Whatever the mechanism, our study contributes to the still limited evidence for a role of microbes in immune priming. Importantly, this does not imply that microbes are always necessary for the host to achieve a primed state of the immune system (see e.g., MF, SA, and JK conceived and designed the experiments. MF performed the experiments. MF, SA, and JK analyzed the data, wrote and revised the manuscript and approved the final version for publishing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cardiac diseases  are associated with ventricular cellular remodeling. However, ventricular biopsies from left and right ventricles from patients with different pathologies are rare and thus little is known about disease-induced cellular remodeling in both sides of the heart and between different diseases. We hypothesized that the protein expression profiles between right and left ventricles of patients with aortic valve stenosis (AVS) and patients with coronary artery disease (CAD) are different and that the protein profile is different between the two diseases. Left and right ventricular biopsies were collected from patients with either CAD or AVS. The biopsies were processed for proteomic analysis using isobaric tandem mass tagging and analyzed by reverse phase nano-LC-MS/MS. Western blot for selected proteins showed strong correlation with proteomic analysis.all relevant ventricular biopsies. Comparison between ventricles and disease state was focused on proteins with relatively high fold (±1.2 fold difference) and significant (P < 0.05) differences. Intra-disease protein expression differences between left and right ventricles were largely structural for AVS patients and largely signaling/metabolism for CAD. Proteins commonly associated with hypertrophy were also different in the AVS group but with lower fold difference. Inter-disease differences between left ventricles of AVS and CAD were detected in 9 proteins. However, inter-disease differences between the right ventricles of CAD and AVS patients were associated with differences in 73 proteins. The majority of proteins which had a significant difference in one ventricle compared to the other pathology also had a similar trend in the adjacent ventricle.Proteomic analysis between ventricles of the same disease (intra-disease) and between ventricles of different diseases (inter-disease) identified more than 500 proteins detected in This work demonstrates for the first time that left and right ventricles have a different proteome and that the difference is dependent on the type of disease. Inter-disease differential expression was more prominent for right ventricles. The finding that a protein change in one ventricle was often associated with a similar trend in the adjacent ventricle for a large number of proteins suggests cross-talk proteome remodeling between adjacent ventricles. Studies investigating cardiac gene and protein expression profiling in disease state provide insight into pathophysiological mechanisms and improve our understanding of cardiac cellular remodeling which can help in the development of new therapeutic drugs. Although gene and protein expression profiling have been carried out in hearts of experimental models of disease, these models have significant limitations. For example non-transgenic models of coronary artery disease involve acute ischemia rather than progressive atherosclerotic coronary disease which would trigger cardiac remodeling . In contIn this novel study we hypothesized that cardiac diseases trigger different proteomic remodeling in left and right ventricles and that the extent of remodeling varies with disease type. To address this hypothesis we used proteomic analysis on tissues from the right and left ventricles of patients with either AVS or CAD.2 = 0.90) and significant (P < 0.05) correlation between the western blot analysis and the proteomic analysis for the ratio of catalase/GAPDH  expression measured using western blotting showed similar variation to those obtained using proteomic analysis. Samples from both AVS and CAD patients and from both left and right ventricles were selected for western blot analysis. The western blot for catalase and GAPDH and ratios of intensity for the selected samples are shown in Figure H Figure C. It mus2 fold difference > 0.25) in the left compared to right ventricle; lumican, vimentin, filamin-A and mitogen-activated protein kinase 14  [2 fold difference = −0.246 and P = 0.02) [Overall more than 1700 proteins were detected using this proteomic analysis. However, only 603 proteins were detected in both the right and left ventricles of all AVS patients. In 34 (5.6%) of these proteins there was a significant difference between the two ventricles Figure A. Howeve = 0.01) ,9 and my = 0.02) ,11.2 fold difference > 0.25) in left compared to right ventricle and they were all higher in the left ventricle  were significantly different between the two ventricles Figure B. Howeve2 fold difference > 0.25) which were higher in AVS left ventricle compared to CAD left ventricle, which were: sodium channel protein type 5 subunit alpha, 2-oxoisovalerate dehydrogenase subunit alpha and glycogen synthase  in the left ventricle of AVS compared to left ventricle of CAD patients: glutathione S-transferase P, myomesin-1 and 2, proactivator polypeptide, apoptotic chromatin condensation inducer in the nucleus and heterogeneous nuclear ribonucleoproteins C1/C2  were significantly different between the left ventricle of AVS and the left ventricle of CAD patients  [2 fold difference = −0.691 and P = 0.11) [In addition to these proteins that had high fold difference, the proteins that are commonly associated with hypertrophy also showed a strong fold difference but did not reach statistical significance. These include sarcoplasmic reticulum Ca = 0.07) ,9 and my = 0.11) ,11.2 fold difference > 0.25 compared to right ventricle of CAD patients; of these, 65 were higher and 8 lower in AVS compared to CAD patients. These proteins included 22 that were related to metabolism, 15 that had roles in cell signaling and 11 that were structural proteins  of proteins Figure B. In theboth the left and right ventricle of the AVS patients compared to both the left and right ventricles of CAD patients; glycogen synthase, 2-oxoisovalerate dehydrogenase subunit alpha, sodium channel protein type 5 subunit alpha and apoptotic chromatin condensation inducer in the nucleus  to compare protein levels between different ventricles from two different diseases: AVS and CAD. Successful strong correlation between proteomic analysis and western blotting for two proteins suggests this proteomic method is valid although more studies are needed before making a firm conclusion.This work shows for the first time significant intra- and inter-ventricular differences in protein profiling including; i) between left and right ventricles of patients with AVS and CAD, ii) between the left ventricles of the two pathologies, iii) between the right ventricles of the two pathologies and iv) between ventricular tissues of AVS compared to CAD, irrespective of the side of the heart. The pattern of differential abundances for a large number of proteins tended to be similar for the same disease but different between the diseases.Aortic valve stenosis is associated with left ventricular hypertrophy. Therefore it is expected that there will be significant differences in protein profiling between the hypertrophic left and the relatively normal right ventricles. Surprisingly there were only four proteins that were differentially expressed (significant and greater than 1.2 fold difference) between left and right ventricles. Three of these proteins  were structural proteins and were higher in the hypertrophic left ventricle. Lumican is a small extracellular matrix-localized proteoglycan, produced by cardiac fibroblasts and plays a role in the fibrillogenesis following insults ,13. Lumi2+ ATPase and myosin-binding protein C) confirming the presence of hypertrophy in the left ventricle of these patients; however, it is likely the hypertrophy was mild as the fold difference was relatively low.In addition, the patients in the AVS group demonstrated changes in proteins commonly associated with hypertrophy  are implicated in cardiovascular disease and their inhibition is cardioprotective in animal models ,27 and cNucleobindin 1 is a calcium-binding protein and therefore can be involved in calcium storage and signaling -33. ThisIn an earlier study we compared left ventricles of hearts with CAD with left ventricle from hearts with AVS and found that hypertrophic ventricle had significantly higher concentrations of ATP, but lower concentrations of lactate, branched-chain amino acids and alanine . These dThere were six proteins that had lower expression in hypertrophic compared to CAD left ventricle: myomesin 1 and 2, glutathione S-transferase P, proactivator polypeptide, heterogeneous nuclear ribonucleoproteins C1/C2 and apoptotic chromatin condensation inducer in the nucleus. The myomesins are elastic proteins mostly located in the center of the M-band  and may There was a large number of proteins differentially expressed (significant and greater than 1.2 fold difference) between the right ventricle of AVS and the right ventricle of CAD patients  or lower n = 1) expression in both left and right ventricles of patients with AVS compared to both ventricles of patients with CAD  of proteins are the key step responsible for regulation of protein activity and function ,49. TherThis work demonstrates for the first time that left and right ventricles of patients with either AVS or CAD have a different proteome and that the difference is dependent on the type of disease. Inter-disease differential expression was more prominent for right ventricles compared to the left ventricles. The finding that a protein change in one ventricle was often associated with a similar trend in the adjacent ventricle for a large number of proteins suggests cross-talk proteome remodeling between adjacent ventricles.Non-diabetic adult male patients (n = 8) with no prior cardiac surgery were included in the study. Patients were undergoing surgery for either aortic valve replacement (n = 4) with no significant co-existing coronary artery disease or coronary artery bypass grafting (n = 4). None of the patients had current congestive heart failure and they all had good left ventricular function. The New York Heart Association (NYHA) classifications for AVS patients were I (n = 1) and II (n = 3). The NYHA classifications for CAD patients were I (n = 2) and II (n = 2). One AVS patient had preoperative atrial fibrillation. The investigation conforms to the principles outlined in the Declaration of Helsinki. Local hospital ethical approval (ISRCTN84968882) as well as patient consent was obtained.Immediately following the institution of cardiopulmonary bypass, myocardial tissue biopsy specimens were collected from the apex of the right and the left ventricles using a 14 Ga. TW’11.4 cm cannula Trucut needle . Each specimen was immediately snap frozen (less than 5 s) in liquid nitrogen and stored at −80°C until processing for protein extraction and quantification.TM PARISTM RNA and protein purification kit . Following extraction, samples were incubated on ice for 30 min and then centrifuged at 10,000 × g for 10 min. The supernatant was collected and stored at −80°C. Protein content was determined using the Bradford assay and samples were diluted to 2 mg · ml−1. A pooled sample was prepared by mixing an equal volume of each sample and was used as an internal standard for normalization of each proteomics analysis.Proteins were extracted from ventricular samples using mirVanaAnalysis of proteins in ventricular tissues was performed by the University of Bristol Proteomics Facility using tandem mass tags (TMTs) .Aliquots of each sample (100 μg) were reduced with 10 mM TCEP (incubated at 55°C for 1), alkylated with 17 mM iodoacetamide (incubated at room temperature for 30 min) and then digested with trypsin  and the resulting peptides labeled with TMT sixplex reagents, all according to the manufacturer’s protocol . Each sample was labeled with a different isobaric tag  and the tagged samples were then combined.−1. Solvent A was 0.1% formic acid and solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionized by nano-electrospray ionization at 2.0 kV using a stainless steel emitter with an internal diameter of 30 μm  and a capillary temperature of 250°C. Tandem mass spectra were acquired using an LTQ-Orbitrap Velos mass spectrometer controlled by Xcalibur 2.1 software  and operated in data-dependent acquisition mode. The Orbitrap was set to analyze the survey scans at 60,000 resolution (at m/z 400) in the mass range m/z 300 to 1800 and the top ten multiply charged ions in each duty cycle selected for MS/MS fragmentation using higher-energy collisional dissociation with normalized collision energy of 45%, activation time of 0.1 ms and at a resolution of 7500 within the Orbitrap. Charge state filtering, where unassigned precursor ions were not selected for fragmentation, and dynamic exclusion  were used.The combined sample was fractionated using a Dionex Ultimate 3000 nano high-performance liquid chromatography system in line with an LTQ-Orbitrap Velos mass spectrometer . In brief, peptides in 1% (v/v) formic acid were injected onto an Acclaim PepMap C18 nano-trap column . After washing with 0.5% (v/v) acetonitrile 0.1% (v/v) formic acid peptides were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse phase analytical column  over a 150 min organic gradient, using 7 gradient segments  with a flow rate of 300 nL · minThe raw data files were processed and quantified using Proteome Discoverer software v1.2  and searched against the reviewed Swiss-Prot human database  using the SEQUEST (Ver. 28 Rev. 13) algorithm. Peptide precursor mass tolerance was set at 10 ppm, and MS/MS tolerance was set at 20mmu. Search criteria included oxidation of methionine (+15.9949) as a variable modification and carbamidomethylation of cysteine (+57.0214) and the addition of the TMT sixplex mass tag (+229.163 Da) to peptide N-termini and lysine as fixed modifications. Searches were performed with full tryptic digestion and a maximum of one missed cleavage was allowed. The reverse database search option was enabled and all peptide data was filtered to satisfy false discovery rate (FDR) of 5%. The Proteome Discoverer software generates a reverse “decoy” database from the same protein database and any peptides passing the initial filtering parameters that were derived from this decoy database are defined as false positive identifications. The minimum cross-correlation factor filter was readjusted for each individual charge state separately to optimally meet the predetermined target FDR of 5% based on the number of random false positive matches from the reverse decoy database. Thus each data set has its own passing parameters. Quantitation was done using a peak integration window tolerance setting of 0.0075 Da with the integration method set as the most confident centroid.Extracted proteins (10 μg) were separated using SDS-polyacrylamide gel electrophoresis under reducing and denaturing conditions and transferred to a 0.45 μm polyvinylidene difluoride membrane. The membranes were blocked with tris-buffered saline (TBS)-Tween containing 10% (w/v) skimmed milk powder before incubation overnight (4°C) with a primary antibody diluted in TBS-Tween containing 5% (w/v) BSA. Primary antibodies used included catalase  and GAPDH . Membranes were then incubated with an anti-rabbit horseradish peroxidase conjugated secondary antibody  and proteins were visualized using the enhanced chemiluminescence system (Amersham). Protein bands were quantified by densitometry with ImageJ 1.46r software.2 (fold difference) on the horizontal axis and –log10  on the vertical axis [2 fold difference of more than 0.25 (±1.2 fold difference) was considered as high-fold difference and was used as the main focus for presenting and discussing the data. We opted for the relatively low cutoff as the majority of the highly significant changes could be accommodated at around 1.2 fold higher (or lower). Other proteomic studies have also used similar cut-off values [For proteomics analysis protein ratios represent the median of the measured peptide ratio(s) for each. Using this quantitative proteomic approach, the data analysis was set so that any missing reporter ions (TMT tags) in each MS/MS spectra are replaced with a minimum intensity value, such that a protein ratio value is generated even if that protein is not detected in one of the samples in a single run (sixplex) of proteomic analysis. In the current study the maximum fold change was set to 100, meaning that any protein showing a protein ratio of 100 was not detected in one of the samples under comparison. No such proteins were identified. Values for each identified protein were presented as a ratio to the internal standard and then normalized to GAPDH measured using proteomics. Volcano plots were created by plotting logcal axis . A log2 f values -54. OtheAVS: Aortic valve stenosis; CAD: Coronary artery disease; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LC-MS/MS: Liquid chromatography mass spectrometry/mass spectrometry; MAO-A: Monoamine oxidase type A; PTM: Post-translational modifications; NYHA: New York Heart Association; TMT: Tandem mass tags; FDR: False discovery rate; TBS: Tris-buffered saline; SEM: Standard error of the mean.The authors declare that they have no competing interests.BL, KH: carried out the work and processed the data. MSS, GDA, BL conceived and designed the work, MSS, BL wrote the manuscript. All authors read and approved the final manuscript.Proteins differentially expressed between the ventricles of AVS and CAD patients.Click here for file


We previously demonstrated that Citrus sinensis contains six putative PAO genes, but their functions are not well understood. In this work, we reported functional elucidation of CsPAO4 in polyamine catabolism and salt stress response. CsPAO4 was localized to the apoplast and used both spermidine (Spd) and spermine (Spm) as substrates for terminal catabolism. Transgenic plants overexpressing CsPAO4 displayed prominent increase in PAO activity, concurrent with marked decrease of Spm and Spd and elevation of H2O2. Seeds of transgenic lines displayed better germination when compared with wild type (WT) under salt stress. However, both vegetative growth and root elongation of the transgenic lines were prominently inhibited under salt stress, accompanied by higher level of H2O2 and more conspicuous programmed cell death (PCD). Exogenous supply of catalase (CAT), a H2O2 scavenger, partially recovered the vegetative growth and root elongation. In addition, spermine inhibited root growth of transgenic plants. Taken together, these data demonstrated that CsPAO4 accounts for production of H2O2 causing oxidative damages under salt stress and that down-regulation of a PAO gene involved in polyamine terminal catabolism may be an alternative approach for improving salt stress tolerance.Polyamine oxidase (PAO) is a key enzyme catalyzing polyamine catabolism leading to H It is conceivable that inhibition of excessive H2O2 accumulation might act as an effective approach to alleviate ROS-associated injury, leading to improved stress tolerance2O2 may provide valuable knowledge for genetically improving stress tolerance via modulation of cellular ROS levels. Several enzymatic reactions are responsible for generation of H2O2 under normal and stress conditions, one of which is mediated by oxidation of polyamines347Spinacia oleracea, Lactuca sativa, Cucumis melo and Oryza sativa10Plants are constantly challenged by a variety of abiotic stresses, such as extreme temperatures, salt and drought, that impose negative influence on plant growth, development, and crop productivity. One common feature shared by these environmental stresses is the elevated generation of reactive oxygen species (ROS). The ROS, if not scavenged timely and efficiently, are toxic to plant cells as they can cause oxidative damages to proteins, DNA and lipids, implying that reduction of ROS levels is crucial for cellular survival under environmental stresses12O2 and ammonia2O2, and N-(3-aminopropyl)-4 -aminobutanal (Spm catabolism) or 4-aminobutanal (Spd catabolism)152O2 concomitantly. Arabidopsis thaliana genome contains five PAOs (AtPAO1-AtPAO5), and all of them catalyze back conversion of PAs2122232426PAO genes involved in back conversion reactions of PAs were identified in cotton and citrus28The catabolism of PAs is catalyzed by two classes of amine enzymes, copper-containing amine oxidases (CuAOs) and FAD-dependent polyamine oxidases (PAOs)et al.et al.2O2 from Spd oxidation can signal Ca2+ influx and regulate pollen tube growth. Perturbation of PAs catabolism in tobacco strongly affected root development and xylem differentiationet al.AtPAO5 loss-of-function mutant (pao5) presented delayed transition from vegetative to reproductive growth. In addition, PAO is also associated with abiotic stress response. Moschou et al.2O2, which further triggered additional protective stress responses or PCD. Furthermore, overexpression of ZmPAO in tobacco led to plant growth inhibition due to PCD induced by overproduction of H2O2 under salt stress.Increasing studies suggest that PAs catabolism plays a critical role in many plant physiological progresses, such as plant development and stress response2O2 can be produced from PA oxidation via back conversion and terminal catabolism, so far knowledge concerning the role of H2O2 derived from PA terminal catabolism in stress tolerance remains poorly understood. In an earlier work, we identified six putative PAO genes in sweet orange (Citrus sinensis), and analyzed their expression profiles in response to abiotic stressesCsPAO4, was postulated to be possibly involved in PAs terminal catabolism based on the expression pattern, but whether this postulation is right remains unanswered. Here, we first investigated subcellular localization of CsPAO4, and then generated transgenic plants overexpressing CsPAO4 to examine its role in PAs catabolism and salt stress response. Our results showed CsPAO4 was localized in apoplast and played a role in terminal catabolism of PAs. Overexpression of CsPAO4 promoted seed germination, but inhibited plant growth under salt stress, which is largely ascribed to PCD caused by excess production of PAO-derived H2O2. Taken together, our work demonstrates that PAO genes involved in terminal catabolism are critical for H2O2 accumulation under salt stress, which suggests that it can be considered as a target to be down-regulated for genetically improving salt stress tolerance.Although HCsPAO4 with other plant PAOs that have been reported to be implicated in PAs terminal catabolism. The result showed CsPAO4 shared low degree of sequence identity with other PAOs although some conserved regions existed among the examined PAOs. Different from the PAOs in other plants, CsPAO4 contained an transmembrane domain (TD), but no signal peptide (SP) was detected , the transgenic seeds germinated in a quicker manner than the WT within the first week, but the difference was not observed from the 8periment . Signifiperiment . At the  and GA3 .2O2 scavenger, was added along with 100 mM NaCl to MS medium, the root growth of transgenic lines was restored, and the growth was comparable to the level under control conditions. By contrast, when 20 μM guazatine was added, the root growth inhibition was not restored, but slightly mitigated in comparison with the growth observed without the inhibitor treatment (data not shown). The H2O2 content in the root tips under salt stress was also examined using the specific dye under fluorescence microscopy. In the absence of salt stress, green fluorescence was comparable in WT and the transgenic plants. When 4-d-cold seedlings were exposed to 200 mM NaCl for 4 h, both WT and transgenic plants displayed enhanced fluorescence in comparison with the normal growth conditions, whereas the fluorescence intensity in the transgenic seedlings was much greater than in the WT. When CAT was added to the seedlings treated with NaCl, the H2O2-associated fluorescence of both WT and transgenic plants was substantially decreased, but the transgenic lines still showed stronger fluorescence intensity  of the aerial parts in these plants compare to the WT . Under t2O2 scavenger CAT was added along with Spm. The inhibition of root growth in transgenic plants under Spm treatment was greatly alleviated by CAT, as the root length was only negligibly smaller than or equivalent to that of WT , ABA synthetic gene (NtNCED3), late embryogenesis abundant protein gene (NtLEA5) and salt stress response gene (NtSOS1). Under control conditions, the expression levels of these genes in WT were much higher than in the transgenic lines  that is crucial for protein localization2020252526CsPAO4. Spd and Spm were decreased in the two transgenic lines, concurrent with elevation of Dap, compared to those of WT, which is consistent with the terminal catabolism of Spd and Spm. Surprisingly, putrescine levels in the transgenic lines were also slightly decreased relative to the WT. However, this result is not unique, as Moschou et al.Zmpao, in tobacco led to reduction of putrescine level in the transgenic plants. One explanation is that the PAO-mediated Spd and Spm catabolism caused a feedback on the activation of synthesis of these two compounds from the first precursor in the PAs pathway.It has been well documented that PA catabolism plays important roles in plant development and stress response. PAs catabolism is catalyzed by PAOs that are involved in either terminal catabolism or back-conversion reactions depending on the end products; these two groups of PAOs have been demonstrated to exist in different plant species up to now. However, previous studies primarily focus on PAOs of model plants or herbaceous plants, such as Arabidopsis and rice have been well studied, and their roles in either terminal catabolism or back conversion are elucidated. Up to now, all of the PAO members in Arabidopsis PAO family are proposed to catalyze the PAs back conversion reactions212223242026Arabidopsis, but this hypothesis needs to be verified in the future.Current researches support that PAOs of monocots catalyze PAs terminal catabolism, while PAOs of dicots are involved in PAs back-conversion reactions172O2 derived from PAO-mediated PAs oxidation takes part in many physiological processes in higher plants, including development, biotic and abiotic stresses response3839CsPAO4 was overexpressed in the transgenic plants, which had higher PAO activity and increased levels of H2O2 in comparison with the WT under normal conditions. On the other hand, the transgenic lines displayed enhanced accumulation of H2O2 compared to the WT when they were treated with exogenous Spd and Spm, but the H2O2 accumulation was indistinguishable from each other when PAO inhibitor was used along with Spd and Spm. All of these results indicate that PAO-mediated PA catabolism accounted for the increased amount of H2O2 in the transgenic plants. In this study, we found that the transgenic plants were more sensitive to salt stress compared to the WT at seedling stage, as manifested by shorter root, reduced fresh weight, decreased chlorophyll content and increased electrolyte leakage and MDA content. This result is in agreement with an earlier study by Moschou et al.PAO gene led to repression of vegetative growth under salt stress. One explanation for this phenomenon is that under salt stress Spd or Spm was increasingly accumulated and then secreted into apoplast where they are oxidized by PAO to generate abundant amount of H2O2. It is known that H2O2 plays a dual role in plant stress response; at low concentration, H2O2 acts as a signaling molecule to trigger the expression of stress-responsive genes to fight against various environmental stresses, whereas at high concentration it causes oxidative stress and induces PCD412O2 in the CsPAO4-overexpressing plants may accumulate to a threshold value, beyond which it acts as a toxic compound to induce either oxidative stress or PCD. We thus speculate that the PAO-derived H2O2 was associated with the observed salt sensitive response in the transgenic lines. This speculation is supported by the following findings. First, salt susceptibility of transgenic plants is concurrent with higher level of H2O2 relative to WT under salt stress. Second, H2O2 scavenger application dramatically decreased H2O2 level in the transgenic plants and resumed the root growth under salt stress, whereas PAO inhibitor application partly abolished the negative effect of salt stress on root growth. Third, PCD in the transgenic plants was stimulated under salt stress, whereas application of CAT disrupted PCD. Lastly, exogenous supply of Spm mimicked the salt stress-derived root growth retardation, which was resumed to normal situations in the presence of CAT.It has been well documented that HPAO gene in the transgenic plants. It has been suggested that a high ratio of PA anabolism to catabolism may result in PA accumulation, while a low ratio suggests that more PAs are used for PAO-mediated oxidizationCsPAO4 might break this balance, and PA catabolism predominated over PA anabolism in the transgenic lines. As a consequence, they accumulated less PAs in the presence of salt stress in comparison with the WT, which is largely true based on the analysis of endogenous PA levels. PAs have been suggested to act as important stress molecules and function to promote plant tolerance to various abiotic stresses through stabilizing membranes and promoting ROS scavenging4344However, it is worth mentioning that growth inhibition of the transgenic seedlings was possibly caused by the disruption of a dynamic balance between PA anabolism and catabolism due to the overexpression of a CsPAO4 led to salt susceptibility at the vegetative growth stage, we found that the transgenic plants exhibited better salt tolerance than WT at germination stage, as shown by faster germination time and higher germination rate. It suggests that plants responses to a given stressor may vary depending on the developmental stages, which has been also documented in earlier studieset al.PAO gene was impaired in the presence of salt stress. The underlying reason for these contrasting results in different studies remains unclear. Based on our data it is assumed that the overproduction of H2O2 contributes to the accelerated seed germination under salt stress. H2O2 has been demonstrated to act as a priming factor to play a key role in coordination of seed germination47482O2 was useful to overcome germination delay and functions to prevent salt stress-derived negative effects. These findings, together with ours, point to the positive role of H2O2 in promoting seed germination under abiotic stress. Seed germination is a highly complex process that is regulated by an array of endogenous and exogenous factors, of which ABA has been shown to act as a major signaling phytohormone. ABA has been shown to inhibit seed germination of different plants512O2, to relay the relevant signals to downstream targets. ABA is also verified to act as a key signal to induce oxidation of PAs and regulates the generation of H2O2 under environmental stress2O2 in the transgenic plants overexpressing CsPAO4 may have a feedback inhibitory effect on the ABA signaling associated with inhibition of seed germination. Therefore, high level of H2O2 in transgenic lines might reverse the inhibitory effect of ABA on seed germination under salt stress.Although overexpression of CsPAO4 functions in polyamine terminal catabolism to produce H2O2. Overproduction of H2O2 in the transgenic plants has been shown to be associated with the contrasting tolerance to salt stress at germination and seedling stage. To our knowledge, this is first report on elucidating a PAO gene involved in terminal catabolism from a perennial plant, and the finding from this study provides knowledge for modulating salt stress tolerance via genetic manipulation of a PAO gene.Taken together, in this study we functionally characterized CsPAO4 of sweet orange. Transgenic approach demonstrates that 3, respectively, and cultured under above conditions. The samples were collected at 0, 6, 12, 24, 48 and 72 h after treatment. All the samples were frozen immediately in liquid nitrogen and stored at −80 °C until using for RNA extraction.Six-year-old sweet orange plants grown in greenhouse were used to examine the gene expression patterns under different treatments. Uniform and healthy shoots were selected from the plants and inserted in a flask containing distilled water, which were kept for 1 d in a growth chamber at 26 °C with 16-h light/8-h dark photoperiod. For hormone treatment, the shoots were transfered into distilled water containing 100 μM ABA or 100 mg/L GA® First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. The primers used for qPCR were listed in 2O. qPCR was performed on a QuantStudio 7 Flex system  with the following cycling conditions: 50 °C for 2 min, 95 °C for 5 min and 40 cycles of 95 °C for 10 s, 56–63 °C (depending on the primers) for 30 s and 72 °C for 20 s. The expression level of each sample was calculated with 2−∆∆CT method (Livak and Schmittgen)β-actin gene, which was used as an internal control. Four replicates were used for each sample. The relative expression levels were log2 transformed to generate heat map representation using Cluster 3.0 software.Total RNA was extracted with RNAiso Plus  and treated with DNase I  according to the manufacturer’s recommendations. The quality of total RNA was detected with a spectrophotometer . About 1 μg of total RNA was used to synthesize first-strand cDNA with PrimeScriptCsPAO4 was PCR amplified with primers ACsPAO4F/R  and Genedoc (version 2.7).Six putative polyamine oxidase genes were previously identified in sweet orange genomeCsPAO4 ORF was amplified using the primer LCsPAO4 F/R with the StuI and MluI restriction sites, and ligated to the N terminal of a GFP gene in the pCAMBIA1302 vector that was linearized with StuI and MluI, yielding 35S::CsPAO4-GFP. The resulting construct was transferred into onion epidermal cells by Agrobacterium tumefaciens (strain GV3101)-mediated infection. The control construct  was used as control. After infection, the onion epidermal cells were cultured on 1/2 MS basal medium at 26 °C in dark for 72 h and then observed with a fluorescence microscope . For plasmolysis, the onion epidermal cells were treated with 0.5 M mannitol for 10 min.The CsPAO4 ORF was amplified using the primer YCsPAO4F/R with the SalI and PstI restriction sites, and cloned into the 6 × His tag of the pCold-I vector (TAKARA BIO INC) resulting in pCold-CsPAO4. After confirming the cloned fragments by DNA sequencing, the pCold-CsPAO4 was transformed into E. coli (BL21) cells and recombinant CsPAO4 protein tagged with 6 × His was produced according to the manufacturer’s instructions (TAKARA BIO INC). The transformant was inoculated in the medium including 100 μg/ml of ampicillin and cultured at 37 °C with shaking. At OD600 0.4–0.5 the culture solution was refrigerated at 15 °C and kept still for 30 min. Then IPTG was added to the final concentration of 0.1–1.0 mM, and the culture was shaken gently for 24 h at 15 °C. Subsequently, the cells were collected to confirm the expression of target protein in soluble and insoluble fractions with SDS-PAGE. Protein purification was then performed according to the Ni-NTA SefinoseKit instructions .The et al. (2006)The catalytic activities of recombinant CsPAO4 protein for the oxidation of Put, Spd and Spm were determined as described by Tavladoraki et al.18 column and an UV detector (230 nm). The programmed methanol-water step gradient was applied with a flow rate of 0.7 ml/min .To determine the reaction products of PAs oxidation, 10 μg of purified CsPAO4 protein was incubated with 150 mM Spd in 100 mM phosphate buffer (pH 7.0) at 37 °C or with 150 mM Spm in 100 mM phosphate buffer (pH 7.0) at 30 °C for different time points. The reaction products were extracted by 5% (v/v) perchloric acid (PCA) as described by Flores and GalstonCsPAO4 was amplified with primers CCsPAO4F/R including SmaI and SacI restriction sites. The sequenced PCR product was fused with the binary pBI121 expression vector under the control of the 35S cauliflower mosaic virus (CaMV35S) promoter. The pBI121 vector containing CsPAO4 cDNA was introduced into A. tumefaciens strain GV3101. Agrobacterium-mediated leaf disc transformation was used to generate the stable transgenic tobaccoF/R including forward CaMV35S promoter primer and reverse gene specific primer for CsPAO4. Semi-quantitative RT-PCR method was used to analyze the expression level of CsPAO4 in transgenic lines with the primers QCsPAO4F/R. Ubiqutin gene was used as an internal control using specific primers. Two transgenic lines (#25 and #27) of T2 generation with high expression levels of CsPAO4 were selected for further study. Three-week-old seedlings of wide type (WT) and transgenic lines under normal growth conditions were selected for PAO protein activity measurement, PAs analysis and ROS detection.The full length of cDNA (1710 bp) encoding 2 generation and wild type (WT) plants were used. For germination rate analysis, WT and transgenic seeds were surface-sterilized and sown on MS medium with 0, 100, 200 and 300 mM NaCl. For root elongation assessment under salt stress, the 4-d-old seedlings of uniform size were grown on MS medium containing NaCl  or Spm . In another set of work, 4-d-old seedlings were grown on MS medium containing 20 μM guazatine or 100 U/ml catalase (CAT), respectively before they were treated with 100 mM NaCl. Root length was measured after 10 d of treatment on the relevant media. For vegetative growth observation under salt stress, 4-d-old seedlings of uniform size were grown on MS medium containing NaCl (0 and 200 mM). Samples of WT and transgenic plants were collected after 30 d of treatments for measurement of fresh weight (FW), PAs contents, ROS level and DNA fragmentation.For salt stress treatment, transgenic lines of TThe extraction, derivation and benzoylation of free PAs and Dap were performed as described above. The PAO activity was detected in the leaf samples with plant PAO assay kit  according to the manufacturer’s instruction. Three replicates were performed with independent samples. The protein concentration was detected based on coomassie brilliant blue staining as described by BradfordAbout 0.1 g of the leaf samples was homogenized in 900 μl of 100 mM phosphate buffer (pH 7.0) on ice, followed by centrifugation at 10000 rpm for 10 min. Subsequently, 10% of the supernatant was used for measurement of activities of antioxidant enzyme, including peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) was measured with detection kits designed for these enzymes  according to the manufacture’s introductions, respectively. Three replicates were performed with independent samples. The protein concentration was detected with the method of coomassie brilliant blue staining as described above.In situ accumulation of H2O2 was detected by histochemical staining with 3, 3′-diamino-benzidine (DAB) as described by Shi et al.2O2 and O2•−, nearly 0.1 g of the leaf samples was homogenized in 0.9 ml of 100 mM phosphate buffer (pH 7.0) on ice, and then the homogenate was centrifuged at 10000 rpm for 10 min. Subsequently, 10% of the supernatant was used for measurement of H2O2 and O2•− with detection kits  according to introductions provided by the supplier. The protein concentration was detected as described above.In situ localization of H2O2 was performed using the highly sensitive, cell-permeable probe 2′, 7′-dichlo-rodihydrofluorescein diacetate (DCFH2-DA) as described by Fu et al.2O , 200 mM NaCl and 200 mM NaCl + 100 U/ml CAT, respectively, at 26 °C in light for 4 h. Then the seedlings were incubated in 50 μM DCFH2-DA dissolved in Tris buffer  in darkness on a rotary shaker (40 rpm) for 20 min. After incubation the seedlings were washed with Tris buffer  to remove excessive dye and observed with a fluorescence microscope .et al.2O, while tube contained only 20 mL of ddH2O was used as a control. The tubes were shaken slowly for 90 min on a rotary shaker at room temperature. Initial conductivity of the samples (Cs0) and control (Cc0) was measured using a conductivity meter . Subsequently, the samples were boiled for 10 min, then cooled down to room temperature. The final conductivity (Cs1 and Cc1) of the sample and control was measured as described above. EL, represented by the relative conductance, was calculated using the formula: C(%) = (Cs0–Cc0)/(Cs1–Cc1) × 100. Chlorophyll content was measured as described by Fu et al.Electrolyte leakage (EL) was detected according to Liu et al.Total DNA from tobacco seedlings after exposure to salt treatments was extracted by cetyltrimethyl ammonium bromide (CTAB) methodP < 0.05 (*), P < 0.01 (**), and P < 0.001 (***), respectively.Three replicates were performed with independent samples for each treatment, which was repeated at least twice. Statistical differences were analyzed using ANOVA  based on Duncan’s multiple range test, taking the significance levels at How to cite this article: Wang, W. and Liu, J.-H. CsPAO4 of Citrus sinensis functions in polyamine terminal catabolism and inhibits plant growth under salt stress. Sci. Rep.6, 31384; doi: 10.1038/srep31384 (2016).


Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified  and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above.Coded by a single gene ( There are, however, several differences between Kv and BK channels that make BK channels unique. First, BK channels are encoded by a single gene (Slo1). Second, they contain seven transmembrane domains and, hence, the N-terminus is in contact with the cell external milieu  produce BK channel-mediated spontaneous transient outward currents (STOCs), thus hyperpolarizing the membrane and producing muscle relaxation  have been identified Figure  and thei2+ sensitivity and alters its gating kinetics, but the existence of 31 more amino acid residues in the N terminal has shown to promote an inactivation process  trimethine oxonol), nitric oxide (NO) and tetrandrine, whose effects are determined by β subunits and have not been studied in detail will be also described.BK channels can be modulated by diverse molecules that may induce either an increase or decrease in channel activity. The effects of these molecules can be exerted through direct interactions with the α subunit and, in some cases, by the expression of β subunits, which would modify such effects. Nevertheless, some of these molecules need β subunits to modulate BK channel activity. The effects of various molecules that have been extensively studied as BK channel modulators and whose activity has been proposed to be related to β subunit expression will be described here Table . Among tButhus tamulus , iberiotoxin (IbTX), slotoxin (SloTX), and martentoxin Figure . Toxins + ions entering the selectivity filter from the internal side when the membrane is depolarized, hence inducing a repulsion force that unbounds the toxin from the channel  of the BK channel and a lessening of the toxin association rate by 250–1000-fold. To explain the mechanism that may determine the differences in effects induced by the β1 and β4 subunits on ChTX binding to BK channels, the smooth muscle β1 subunit extracellular loop was exchanged with the neuronal β4 subunit loop. The results showed that the β subunit extracellular loop determined the affinity of this toxin to the BK channel. The β4 subunit external loop contains more basic residues  than the β1 subunits, and their neutralization, or charge reversals  greatly increase toxin association and dissociation rates. These results suggest that positively charged residues impose an electrostatic shield on ChTX action  in dissociation rates. The end result of this is that although α + β1 BK channels show less sensitivity to IbTX, binding is essentially irreversible  , which has 76% of identity to IbTX and 54% to ChTX  conductance-voltage dependence, thus indicating that this toxin does not interact with the voltage sensor, unlike β1  on the biophysical properties of BK channels. For instance, BK channel activation by epoxyeicosatrienoic acids (EET), which are arachidonic acid metabolites, has been observed to result in concentration-dependent coronary artery relaxation , oleic (OA), linoleic (LA), linolenic (ALA), arachidonic (AA), and eicosapentaenoic acids (EPA)  without affecting channel voltage sensitivity or unitary conductance  produced a decrease in N0P. Increases in 0P result from decreases in mean closed time (τc) without modifying mean open time (τo)   has been shown to interact with the β2 subunit in inhibiting BK currents  is evident at Ca2+ concentrations less than 20 μM. On the other hand, rises in Ca2+ concentrations diminish potentiation and concentrations' over 30 μM result in inhibition. The latter response denotes that basal Ca2+ concentrations promote BK channel potentiation, whereas high concentrations (>10 μM) induce inhibition. Ethanol does not generate changes in channel unitary current amplitude, which implies that its effect on cbv1 channels is limited to modifying gating. Finally, the effect of ethanol depends on the intracellular Ca2+ levels sensed by the α subunit . These results indicate that β1 subunit effect on ethanol activity is mediated with an allosteric mechanism where β1 acts like a transducer that couples changes in Ca2+ concentration to BK channel activation   Figure  has beenAlthough E2 can activate the BK channel when the β1 subunit is present, the exact binding site has not yet been identified. E2 activation can be induced when E2 is conjugated to a protein that is impermeable to the membrane, but only when it is applied to the outside of the cell, suggesting that the binding site is located in the extracellular region of the β1 subunit , acts as a BK channel activator only in channels formed by α + β1  studies have led to the identification of a binding site for lithocolic acid in the β1 subunit TM2 domain  Figure  isolated1/2, but reductions in current amplitude are seen only at higher concentrations. V1/2 declines are calcium-dependent and are only seen in the presence of β1 or β4 subunits, but not in channels containing β2 or β3 subunits -dibutylbarbituric acid) trimethine oxonol), which is a voltage-sensitive fluorescent dye that significantly increases whole-cell BK α + β1 currents in HEK293 cells and in native channels from urinary bladder smooth muscle cells. DiBAC4 shifts the activation voltage to hyperpolarizing potentials without causing changes in single-channel conductance. Morimoto et al. (4 activates whole-cell rBKα + β1 and rBKα + β4 currents partially blocking rBKα + β2 currents with no effects on channels where the α subunit is alone (Morimoto et al., 4 binding site should be in the α subunit, since DiBAC4 selectively enhances BK channel activity in the presence or absence of the β1 subunit (Scornik et al., 4 can increase deactivation time constants without changing the voltage dependence of deactivation. Although the latter effects do not necessarily require the presence of β1 subunits, these subunits enhance BK channel response to DiBAC4. It has also been established that leftward shifts in conductance-voltage curves induced by DiBAC4 are significantly larger in channels expressing α + β1 than in those expressing α subunits alone. Moreover, β1 subunits promote a four-fold decrease in the dK of DiBAC4. Such findings could envision a new scope for the role β1 subunits in cardiovascular pharmacology (Bosch et al., Another BK channel activator is DiBACo et al.  reportedhslo current and its action as a BK inhibitor is potentiated in channels coexpressing α and β subunits (Dworetzky et al., P0 (Wu et al., Additionally, NO, being an endogenous vasodilatation gas, can rapidly, reversibly, and significantly increase BK open channel probability from both sides of the membrane (Bolotina et al., BK channels are related to different pathophysiological processes, such as changes in vascular tone regulation, diabetes, kidney, and nervous system diseases. The expression of auxiliary β subunits plays an important role in these processes because of the modulatory effects of these subunits on BK channel activity, inducing changes in their biophysical properties. Furthermore, β subunits act like binding sites for various molecules that regulate BK channel activity, prompting an increase or decrease in the effects induced by drugs. This knowledge would be important in drug design, since it may be possible to find molecules that induce specific modulatory effects on BK channel properties, depending on the tissue types where they are expressed. That is particularly important in smooth muscles and in the nervous system, where β1 and β4 are highly expressed and have been found to significantly regulate the biophysical properties of BK channels.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sulfonyl-derived functional groups populate a broad range of useful molecules and materials, and despite a variety of preparative methods being available, processes which introduce the most basic sulfonyl building block, sulfur dioxide, using catalytic methods, are rare. Described herein is a simple reaction system consisting of the sulfur dioxide surrogate DABSO, triethylamine, and a palladium(0) catalyst for effective convertion of a broad range of aryl and heteroaryl halides into the corresponding ammonium sulfinates. Key features of this gas- and reductant-free reaction include the low loadings of palladium (1 mol %) and ligand (1.5 mol %) which can be employed, and the use of isopropyl alcohol as both a solvent and formal reductant. The ammonium sulfinate products are converted in situ into a variety of sulfonyl-containing functional groups, including sulfones, sulfonyl chlorides, and sulfonamides. Sulfonyl-derived functional groups such as sulfones, sulfonamides, and sulfinic acids feature in a myriad of applications ranging from active ingredients in pharmaceuticals and agro2- arrangement of atoms, thus suggesting that a conceptually simple route to these molecules is one that involves the direct catalytic introduction of SO2. Despite the ready availability of SO2 as a bulk feedstock, its use in transition-metal catalysis is severely limited. Pioneering reports from Pelzer and Keim established that the palladium-catalyzed introduction of SO2 was feasible, although these early reports of sulfinic acid synthesis described reactions of poor scope and required high pressures of SO2 and hydrogen, and the use of inconvenient aryl diazonium substrates [Eq. The fundamental feature that links all sulfonyl-derived functional groups is the -SOates Eq. .9]2- arran2 charge-transfer complex DABSO [DABCO⋅(SO2)2] as a convenient and easy-to-handle solid which functions as a surrogate for SO2 gas in a number of known transformations,[2S2O5 as the SO2 source,[We have introduced the amine–SOrmations,,11 and armations, Wu et al2 source, and also2 source, All of t2 source,2S2O5. However, the process requires high ligand loadings (30 mol %), the use of an excess of inorganic reductant, as well as a stoichiometric additive (TBAB).[Rather than focus on the synthesis of a single class of sulfonyl derivative, a catalytic synthesis of sulfinates would potentially allow access to a host of sulfonyl-derived functional groups through established reactions. Althoughe (TBAB). Herein w1 a) into the corresponding sulfinate (2 a) as a model transformation , and the optimal ligand proved to be PdAd2Bu[iPrOH, used as solvent, could also function as a reductant in palladium catalysis,[2) delivered the optimized reaction conditions (entry 11). The catalyst loading could be reduced further, for example, using only 0.5 mol % palladium and 0.75 % ligand led to a 91 % conversion into the sulfinate (entry 12).We selected the conversion of 4-iodotoluene  a into te PdAd2Bu . Notable examples include the mixed sulfide-sulfone 3 d, a product difficult to prepare using traditional sulfone methodology, and sulfones 3 e and 3 f, featuring unprotected OH and NH2 substituents, respectively. A selection of electron-neutral substrates were employed without incident (3 g–j). Electron-withdrawing functional groups were likewise compatible with the reaction conditions, with ester, ketone, amide, nitrile, and trifluoromethyl-substituted products being isolated in good yields (3 k–o). The greater reactivity of aryl iodide substrates allowed sulfones featuring bromo, chloro, and fluoro substituents to be prepared (3 p–r). Finally, although aryl iodide substrates proved to be most efficient, it was possible to employ aryl bromide starting materials, with the sulfones 3 k and 3 o being prepared from both aryl halide substrates.Sulfinates are versatile intermediates, but they are rarely the desired final product of a transformation. To evaluate the scope of the developed reaction we elected to derivatize the newly formed sulfinates in situ. Using α-bromo ts Table . In gene3 s–x). Several alkenyl iodides were also investigated, and proved to be compatible with the reaction .We next explored the use of the heteroaryl halides as substrates Table . Example3 g being isolated in 96 % yield from a reaction employing 1 mol % of palladium and 1.5 mol % of ligand (Scheme 1). Although we have not scaled the reaction further, the use of isopropyl alcohol as both the solvent and formal reductant holds promise in this perspective, as it has been identified as a sustainable solvent suitable for process development.[For pragmatic reasons all of the reactions shown in Tables elopment.2 g, are shown in Scheme 2. Alkylation with a range of alkyl halides was possible and was achieved by adding a DMF solution of the electrophile to the crude sulfinate salt, and examples employing α-bromo tert-butyl acetate and benzyl bromide are shown (3 g and 4). Cyclohexene oxide was efficiently opened to deliver the corresponding β-hydroxysulfone (5).[2 g with diphenyliodonium chloride provided the diarylsulfone (6). Aryl(heteroaryl)sufones were available using an SNAr-type process.[2 g with 2-chlorobenzothiazole provided the corresponding heterocyclic sulfone (7). Treatment of the ammonium sulfinate with sulfuryl chloride resulted in oxidative chlorination and allowed isolation of the derived sulfonyl chloride (8).[9 was isolated in good yield. Finally, reduction using a Sn/HCl mixture allowed isolation of the corresponding disulfide (10).[At the outset of our investigation an efficient catalytic synthesis of sulfinates was targeted because of the wide range of established reactivity, for additional transformations, known for these molecules. In situ derivatization of sulfinates is particularly important as their purification is hampered because of their propensity to disproportionate in the presence of acid. However, the majority of these proven derivatization reactions had been developed on metal sulfinate salts. To validate our sulfinate synthesis we needed to ascertain the utility of the corresponding ammonium salts. Our initial findings, employing the ammonium sulfinate fone (5). Diarylsufone (5). and trea process. Accordinride (8). The moderide (8). For examide (10). In gener2 surrogate DABSO, partnered with isopropyl alcohol as the reductant, we have been able to develop a simple and efficient process for the palladium-catalyzed conversion of aryl and heteroaryl halides into the corresponding ammonium sulfinates. This effective sulfination reaction employs low catalyst loadings, is operationally simple to perform, and has broad substrate scope. The ability to employ a simple alcohol as both reaction solvent and reductant significantly streamlines the process. The ammonium sulfinate products display good levels of reactivity and can be smoothly converted into a variety of sulfonyl derivatives. Further studies to develop related transformations, and to elucidate the mechanism of this transformation, are underway.In summary, by employing the SO