Melanoma is a lethal tumor because of its severe metastatic potential, and serine/threonine-protein kinase B-raf inhibitors (BRAFi) are used in patients harboring BRAF-mutation. Unfortunately, BRAFi induce resistance. Therefore, we tested the activity of onconase (ONC), a cytotoxic RNase variant, against BRAFi-resistant cells to re-establish the efficacy of the chemotherapy. To do so, an A375 dabrafenib-resistant (A375DR) melanoma cell subpopulation was selected and its behavior compared with that of parental (A375P) cells by crystal violet, 5-Bromo-2’-deoxyuridine incorporation, and cleaved poly(ADP-ribose) polymerase 1 (PARP1) western blot measurements. Then, nuclear p65 Nuclear Factor kappaB (NF-κB) and IκB kinases-α/β (IKK) phosphorylation levels were measured. Gelatin zymography was performed to evaluate metalloproteinase 2 (MMP2) activity. In addition, assays to measure migration, invasion and soft agar colony formation were performed to examine the tumor cell dissemination propensity. ONC affected the total viability and the proliferation rate of both A375P and A375DR cell subpopulations in a dose-dependent manner and also induced apoptotic cell death. Among its pleiotropic effects, ONC reduced nuclear p65 NF-κB amount and IKK phosphorylation level, as well as MMP2 activity in both cell subpopulations. ONC decreased cell colony formation, migration, and invasion capability. Notably, it induced apoptosis and inhibited colony formation and invasiveness more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts melanoma malignancy especially in BRAFi-resistant cells and could become a tool against melanoma recurrence. Malignant melanoma is a highly aggressive skin cancer that has shown increasing incidence and mortality over recent decades . MelanomIn order to find new therapeutic strategies against drug resistance, several recent works have focused their investigations on the combination of BRAFi with synthetic compounds or with natural products ,18,19,20In the present work, we analyze the ability of ONC to affect the viability of A375 cells which have acquired resistance to dabrafenib (A375DR), in comparison with parental (A375P) cells. Therefore, the impact of ONC on cell proliferation, apoptosis, cell migration, and invasion capability, as well as on the inhibition of NF-κB nuclear translocation and of MMP2 activity, have been compared here within the two A375 cell subpopulations.25 and IC50 of 1.2 and 2.7 nM, respectively. Instead, the A375DR cell viability was not significantly affected up to 20 nM drug concentration, reaching IC25 only at 44 nM. Moreover, its relative IC50 can be only extrapolated, reaching a value that definitely overpasses 5 µM, i.e., the maximal dabrafenib concentration used in our experiments. Importantly, the differences detected by comparing the data collected from A375P and A375DR cells were statistically significant for each concentration tested .Considering that drug resistance is one of the main factors limiting the efficacy of melanoma therapy, the BRAF-mutated-A375 human melanoma cell line was cultured with gradually increasing concentrations of dabrafenib until a cell subpopulation grew in the constant presence of 5 µM of this drug. Then, the resulting dabrafenib-resistant A375DR cells were maintained for six months under a selective pressure of the drug. After this period, dabrafenib sensitivity was measured by crystal violet assay in both A375P and A375DR cell subpopulations that were treated with dabrafenib concentrations ranging from 1 to 50 nM. As shown in 50 values of 0.40 and 0.32 µM for A375P and A375DR cells, respectively. No statistically significant differences in the sensitivity to ONC emerged within the two cell subpopulations, although the mean viability reduction of A375DR cells was lower than that of parental ones for each concentration tested . Moreover, the maximal ONC dose (4 µM), tested exclusively in the NHEM cells, reduced their viability only by 14%. Hence, we conclude that ONC displays quite high cytostatic and cytotoxic effects only in melanoma cells, while not doing so in normal melanocytes.To evaluate the specificity of ONC activity against melanoma cells, we also measured the sensitivity of normal human epidermal melanocytes (NHEM) to this RNase variant. NHEM cells were incubated for 72 h with the two ONC concentrations that were the most effective against malignant cells , and also with 2 and 4 µM ONC b. By thep = 0.05, p = 0.04; 48 h, p = 0.02, p = 0.05; and 72 h, p = 0.03, n.s.). Although not visible in We performed a 5-Br-2’-deoxyuridine (BrdU) incorporation assay to identify whether in both cell subpopulations the viability reduction elicited by ONC might depend on the cell proliferation rate or, instead, on a cell mass decrease consequent to cell death. After 24, 48, and 72 h culture with ONC, an additional 4 h BrdU incubation showed a concentration-dependent reduction of its incorporation in both A375P and A375DR cells. Nevertheless, ONC-treated A375DR cells showed a smaller reduction of BrdU incorporation level than A375P ones, as is clearly visible in In the end, these data indicate that ONC decreases the proliferation rate of both A375P and A375DR cells, although this reduction is more evident in A375P, probably because these cells display a higher replication rate than the A375DR subpopulation.p = 0.01). The amount of cleaved PARP1 increased significantly in all ONC-treated cell samples if compared to each ONC-free one , thus confirming that 1 µM ONC induced the apoptotic death of A375 cells. Remarkably, ONC-treated A375DR cells showed an impressive increase of cleaved PARP1 amount in comparison with ONC-free A375DR cells, but also with ONC-treated A375P . In conclusion, although ONC-treated A375DR cells showed a lower reduction in the cell proliferation rate than ONC-treated A375P cells , suggesting that NF-κB was activated in both ONC-free subpopulations. Instead, either A375P or A375DR cells treated with 1 µM ONC showed a significantly decreased nuclear p65 level . To exclude the contamination of nuclear extracts by cytosolic proteins, WB membranes containing either the nuclear or the whole cell protein extracts were hybridized with the β-tubulin antibody. As shown in p = 0.008), while ONC decreased its amount especially in A375DR cells . Therefore, our data suggest that ONC reduces p65 NF-κB nuclear localization by inhibiting IκB kinases-α/β (IKK) phosphorylation.As measured by WB, ONC-free A375P cells showed detectable amounts of p65 NF-κB localized in the nucleus, and this expression level was even increased in ONC-free A375DR cells  qPCR in both A375P and A375DR ONC-free controls. CD133 and NANOG mRNAs showed in A375DR cells a 2.1- and 2.4-fold increases, respectively, in comparison to the relative expression levels detected in A375P, with the amount of the mRNA of TATA box binding protein (TBP) used here as the internal standard . These results demonstrate that A375DR cells display a higher gene expression level of both CD133 and NANOG markers of undifferentiation and embryonic stem cells than A375P ones. In addition, the protein amounts of the same CSC markers detected by WB were increased in A375DR cell subpopulation  pathway, as shown by the increase of ERK1/2 phosphorylation level  markers were measured in both cell subpopulations. Representative WB experiments showing a higher expression level of N-cadherin and nuclear β-catenin in ONC-free A375DR than in ONC-free A375P cell subpopulations are reported in on level b.p = 0.0008). In addition, the number of ONC-free A375DR colonies formed was very high, while the number of visible colonies approached zero upon 1 µM ONC incubation (p = 0.0001). A significant difference was found also between the two ONC-free cell subpopulations (p = 0.009). In Anchorage-independent growth measurements indicate how cells can grow independently of the presence of a solid surface support. A characteristic of stem cells is their capability of forming clones in soft agar . Hence, A wound-healing assay was performed to measure the cell migration ability of both A375P and A375DR subpopulations in the presence or absence of ONC. Notably, the time necessary to close the wound was different: In particular, A375DR cells migrated and proliferated more slowly than the A375P counterpart. Indeed, 1 µM ONC remarkably reduced the migration rate either of A375P or of A375DR cells, as is visible in p = 0.008), while a lower and not statistically significant decrease was found in ONC-treated A375P cells, as shown in In order to verify whether A375 cells can acquire an invasive phenotype, we examined the effect of ONC on the ability of A375DR cells to migrate through the Matrigel. Cells were seeded in the upper chamber of a Transwell plate, and complete Dulbecco’s Modified Eagle’s Medium (DMEM) was added to the lower chamber as a chemo-attractant. Then, the invading cells were stained and analyzed after 48 h: Both subpopulations invaded the Matrigel, and ONC-free A375DR cells showed only a small and not significant increase in their invasion potential with respect to the ONC-free A375P subpopulation. Instead, the incubation with 1 µM ONC caused a remarkable 33% reduction in the A375DR cell-invasion capability .Both subpopulations were cultured for 48 h with or without 1 µM ONC. Thereafter, cells were serum-starved for 30 h and the conditioned media collected to measure the MMP2 activity by gelatin zymography. The results shown in CD133 and NANOG gene and protein expression levels than A375P ones  therapy of unresectable malignant mesothelioma. In this study, however, patients had a significant advantage for survival only when they were previously treated with DOX before they were given ONC . TherefoAll chemicals, solvents, and buffers were from Sigma-Aldrich , unless noted otherwise.E. coli, and extracted and refolded from inclusion bodies. Finally, it was purified following the protocol described by Notomista et al. [4, with P. aeruginosa aminopeptidase (AAP) at an AAP:ONC = 1:1000 molar ratio. The reaction was blocked after 96 h by adding 0.01 M EDTA, final concentration. Each reaction step was monitored by analyzing the products with SDS-PAGE under reducing conditions. The final chromatographic purification was performed after the removal of the Met (-1) residue with a Superdex 75 HR 10/300 SEC column attached to an ÄKTA-FPLC system  [The plasmid encoding for recombinant ONC (pcDNA-ONC) was kindly provided by Prof. D. Picone . ONC was expressed in a et al. . Met  . Then, tont, UK) ,53, its ont, UK) .2 humidified atmosphere.Normal human epidermal melanocytes and human melanoma A375 cell lines , were separately cultured at 37 °C in high glucose DMEM  supplemented with 10% fetal bovine serum , 1% Antibiotic Antimycotic Solution , and in a 5% COThe parental human melanoma A375P cell line was induced to generate dabrafenib resistance (DR) with the aim of selecting a A375DR subpopulation: To do so, A375 cells were cultured at 37 °C with concentrations of dabrafenib  that gradually increased from 1 nM up to 5 µM until a subpopulation grew in the constant presence of this maximal drug concentration. Subsequently, A375DR cells were maintained in culture with 5 µM dabrafenib for six months. Both A375P and A375DR subpopulations were mycoplasma tested (negative) every three months.3 cells/well). After 24 h culture, A375 cells were incubated with different concentrations of dabrafenib or of ONC, and harvested after 72 h. In a parallel experiment, NHEM cells were treated with ONC to compare their cell viability with the ones of both A375 subpopulations. In the experiments performed with A375DR cells, dabrafenib was removed from the culture medium one day before starting the 72 h incubation with ONC or dabrafenib. To measure cell viability, each cell sample was washed at the end of the treatment with 1× PBS solution , and stained for 5 min with 0.75% crystal violet powder dissolved in 8% formaldehyde and 50% ethanol. Crystal violet was solubilized in PBS/1% SDS solution, and OD595 measured in a plate reader . Six replicates were performed for each condition/time point.NHEM and both dabrafenib-sensitive parental, A375P, and A375 DR cell subpopulations were seeded in a 96-well plate (2.9 × 103 cells/well). After 24 h, cells were incubated with or without 0.5 or 1 µM ONC for 24, 48, or 72 h, and, subsequently, for another 4 h with BrdU . The medium was aspirated and cells were incubated with a fixing solution for 30 min. Plates were washed three times and the anti-BrdU detector antibody was added for 1 h. Plates were then washed a further three times, incubated for 30 min at room temperature with peroxidase goat anti-mouse IgG conjugate and then washed three times again. The tetramethybenzidine chromogenic peroxidase substrate was added, and plates were incubated for 30 min in the dark. Finally, a stop solution was added and the OD450 measured in a plate placed in the Tecan NanoQuant Infinite M200 Pro reader .Cell proliferation was assessed with a colorimetric immunoassay based on the measurement of BrdU incorporation during DNA synthesis. A375P or A375DR cells were seeded in 96-well plates  were plated at the bottom layer of the plate, while 0.6% agarose and 2× DMEM supplemented with 10% FBS and 1% Antibiotic Antimycotic Solution were plated on its top. ONC-treated and ONC-free (control) cells were detached, counted and seeded in six-well plates (5000 cells/well), together with the upper soft agar solution. 100 µL/well of fresh medium were added twice a week and cell colonies were stained with crystal violet after 21 days. Semi-solid cultures were performed in triplicate and images were captured with the EVOS FL Auto Cell Imaging System .5 cells/well) and, upon reaching confluence, the monolayer was scratched with a sterile 200 µL pipette tip. A375DR cells were seeded in six-well plates (4 × 105 cells/well) but their monolayer was scratched with a sterile 10 µL pipette tip when they reached confluence. To remove detached cells, wells were washed with complete medium and afterwards refilled with fresh medium containing 10% FBS. 1 µM ONC was added to this culture and samples were compared with the ONC-free ones. A375P cells were incubated for 17 h (1020 min), while A375DR cells for 54 h (3240 min), and the behavior of both cell subpopulations was monitored with EVOS FL Auto Cell Imaging System. Cells were kept at 37 °C in humidified atmosphere with 5% CO2 in an EVOS Onstage Incubator . The A375P cells movement frames were captured every 30 min over the aforementioned 17 h with a 5× magnification, while the A375DR cells frames every 2 h with a 10× magnification over the aforementioned 54 h, to create time-lapse videos. The relative images were quantitatively analyzed with the Image-J computing software and the MRI Wound Healing Tool . In the graphics, the mean value relative to each point calculated from four independent experiments is reported ± SD.A375P cells were seeded in six-well plates , according to the manufacturer instructions. 0.5 mL of complete medium were added as chemoattractant to the lower chamber well, while 0.3 mL of cell suspension consisting of 1 × 106 cells/mL solubilized in DMEM serum-free were seeded on the upper chamber. Invasion capability was analyzed after 48 h: Non-invading cells were removed by cotton swab scrubbing, and the invading cells treated with the staining solution. Finally, the OD450 was measured in the Tecan NanoQuant Infinite M200 Pro plate reader .Both A375P and A375DR cell subpopulations were incubated for 72 h in the presence or absence of 1 µM ONC. Afterwards, cells were detached with trypsin, counted and seeded in a Transwell composed of polycarbonate membrane inserts of 8 µm pore size, and coated with a uniform layer of a dried basement membrane matrix solution. Cell invasion was measured with the CytoSelect2, and 1 µM ZnCl2 supplemented with 2.5% Triton X-100 . It was then kept in the incubation buffer  for 24 h to allow the digestion by the MMP2 gelatinase. This buffer contained the cofactors necessary to maintain the MMP2 gelatinase activity, so that it could degrade the gelatin present in the gel. The gel was stained with 0.5% Coomassie Brilliant Blue R-250 in 20% ethanol/10% acetic acid, and the gelatinase activity was detected by evaluating presence and intensity of clear bands on the blue background.Both cell subpopulations were cultured in the presence of absence of ONC for 48 h, and then they were serum-starved for 30 h. The conditioned medium was collected and 10× concentrated with an Amicon Ultra-2 Centrifugal Filter Unit . Protein concentrations were determined with Coomassie  and samples treated with a non-reducing sample buffer. In order to separate proteins, a 7.5% SDS-PAGE was performed. The gel contained 4 mg/mL gelatin and was washed with 50 mM Tris-HCl pH 7.5, 5 mM CaClIn order to purify the total protein content, cell pellets were re-suspended in ~50–100 μL of RIPA lysis buffer , in the presence of protease and phosphatase inhibitors . Samples were incubated on ice for 20 min and centrifuged at 25,000× g for 25 min, at 4 °C. The supernatant, containing soluble proteins, was collected and maintained at −80 °C until use. Nuclear extracts were prepared as described in  in the pOD relative to protein concentrations were measured at 595 nm by the Coomassie brillant blue G-250 Bradford assay in a Jasco V-650 spectrophotometer , Italy). In order to prepare samples for WB analysis, a quarter of the reducing buffer volume  and water were added to 40 μg of total protein extract or to 15 μg of nuclear protein extract. Samples were then boiled for 3 min and placed on ice to aid in protein denaturation.RNA was extracted with the Pure link RNA kit , quantified with the Nanodrop UV-vis spectrophotometer , and its quality checked by agarose gel electrophoresis. Reverse transcription was performed (500 ng RNA in 10 µL) by using the SuperScript VILO cDNA synthesis kit , according to the manufacturer instructions. The expression levels of specific genes were determined with RT-qPCR by using the SensiFAST Sybr No-ROX kit . In each sample, the gene expression level was normalized to the one of the TATA-box binding protein mRNA. PCR reactions were performed in triplicate in a Rotorgene Q , with the following amplification primers:CD133:(Rev) 5’CGCCTTGTCCTTGGTAGTGT-3’(Fw) 5’GCATTGGCATCTTCTATGGTT3’,NANOG:(Rev) 5’TGCTGGAGGCTGAGGTATTTCTGTCTC-3’(Fw) 5’AGTCCCAAAGGCAAACAACCCAGTTC3’,TBP:(Rev) 5’-ATGATTACCGCAGCAAACC-3’(Fw) 5’TGTATCCACAGTGAATCTTGG-3’,A comparative quantification of gene expression levels was performed with a Pfaffl’s efficiency corrected calculation .Protein extracts were electrophoresed by a 5–10% polyacrylamide SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane . Membranes were blocked at RT with TBST buffer  for 1 h. Then, they were incubated overnight on a shaker, at 4 °C, with a 5% BSA solution containing the primary antibodies for: Cleaved PARP1 , p65 NF-κB , p-IKKα/β (Ser176/180) , pERK1/2  , β-catenin , N-cadherin , NANOG , CD133  , and β-tubulin . Then, membranes were washed three times with TBST buffer for 30 min, then incubated for 1 h with horseradish peroxidase-conjugated secondary antibody , and washed with TBST three times again. All protein extracts were normalized with β-actin protein antibody . Immuno-detection was carried out with the ECL kit  and chemiluminescence signals were detected with ChemiDoc .p values were determined using unpaired, two-tailed Student’s t test. For each type of experiment, a minimum of three independent biological replicates were performed.All the results are reported as a mean value ± SD. Unless noted otherwise,


Over the past two decades, the pig has gained attention as a potential model for human drug metabolism. Cytochrome P450 enzymes (CYP450), a superfamily of biotransformation enzymes, are pivotal in drug metabolism. Porcine CYP450 has been demonstrated to convert typical substrates of human CYP450. Nevertheless, knowledge and insight into porcine CYP450 quantity and substrate selectivity is scant, especially regarding intestinal CYP450. The current study aimed to map the quantities of hepatic and intestinal CYP450 in the conventional pig by using a proteomic approach. Moreover, the selectivity of the six most common used probe substrates  for drug metabolizing enzyme subfamilies , was investigated. Hepatic relative quantities were 4% (CYP1A), 31% (CYP2A), 14% (CYP3A), 10% (CYP2C), 28% (CYP2D) and 13% (CYP2E), whereas for the intestine only duodenal CYP450 could be determined with 88% for CYP3A and 12% for CYP2C. Furthermore, the results indicate that coumarin (CYP2A), midazolam (CYP3A), tolbutamide (CYP2C), and dextromethorphan (CYP2D) are as selective for porcine as for human CYP450. However, phenacetin (CYP1A2) and chlorzoxazone (CYP2E1) are less selective for the specific enzyme, despite similarities in selectivity towards the different enzymes involved compared to humans. The resulting compounds are often more water soluble and available for further phase II conjugation4. Regarding xenobiotic metabolism, members belonging to the CYP1, CYP2 and CYP3 family are crucial, and are responsible for 70 to 80% of CYP450 mediated drug metabolism5. Hence, CYP450 biotransformation is investigated early on in the development of new drugs.Up to 55% of the available drugs in human medicine are metabolized by cytochrome P450 (CYP450) enzymes. These enzymes are major contributors to phase I drug metabolism and catalyze oxidative, reductive, and hydrolytic reactions of endogenous and xenobiotic compounds6. However, frequently used animals such as the mouse, rat and rabbit, are often not ideal for modelling human drug metabolism, possibly due to differences in enzyme expression and/or selectivity10. In the past two decades the pig has gained attention as a more appropriate animal model for humans due to similar anatomy and physiology14. In pigs, orthologues of human CYP1A2, CYP2A6, CYP3A4/5, CYP2C9/19, CYP2D6, and CYP2E1 have been isolated and identified with homologies in amino acid sequence, ranging from 62 to 87.5%15. Sequence identity does not guarantee similar activity/selectivity though. Single nucleotide polymorphisms have been shown to significantly alter CYP450 expression, stability, and function17. For example, recent insights in the two CYP2C9 polymorphisms show differences in side chain interaction, ultimately leading to different binding of the substrate losartan18.The establishment of a preclinical animal model that correlates well with humans, is a valuable tool in drug development and safety testing22. The detailed comparison and distinction between the two types of pigs is further complicated by a lack of quantitative data of pig and minipig CYP450 expression at the protein level. The few reports available indicate that differences in activity between conventional and minipigs primarily originate from differences in immunochemically quantified CYP450 enzymes22, stressing the importance of comparing activities from a quantitative perspective. Furthermore, CYP450 amino acid sequence in conventional and minipigs seems to be almost identical. Specifically there is only one amino acid difference in the CYP2E1 enzyme and a predicted difference of 10 amino acids between minipig CYP2D21 and conventional porcine CYP2D2523, however the latter has not been verified at the protein level.In previous research both pigs and minipigs have been used as a model for humans. Unfortunately, very few studies consistently compared minipig CYP450 expression (and activity) vs. conventional pig CYP450, hence the use of the word ‘(mini)pig’ in the text belowWhen comparing porcine and human CYP450, three main areas of interest can be distinguished: mRNA expression, enzyme quantity, and CYP450 activities.35. Although mRNA expression data can be indicative of the amount of translated protein, mRNA levels are not predictive for protein quantity and activity. This can be due to mRNA instability, protein degradation, and translational or posttranslational modifications. Consequently, direct comparison between mRNA amounts are of limited use36.To date, mRNA expression of (mini)pig CYP450 has been determined in the liver, intestine, lung, kidney, heart, and even brain38. Although Western blotting has proven to be a valuable tool, it has the limitation that a selection of enzymes of interest has to be made prior to the experiments. Contrarily, a mass spectrometry based method does not impose such prior restrictions and allows for absolute quantification more easily. Such an approach was used by Achour et al. (2011) to quantify hepatic CYP450 in conventional (Suffolk White) adult pigs by a state-of-the-art high resolution mass spectrometry (HRMS) method39. Unfortunately, the study was performed on just two pigs and did not include quantification of intestinal CYP450 enzymes. Moreover, intestinal quantitative data are restricted to CYP1A1 and CYP3A enzymes41 of which none included the entire small intestine, i.e. duodenum, jejunum, and ileum. Nonetheless, this information is pivotal for in vitro-in vivo correlations and to successfully determine the contribution of each enzyme to specific biotransformation reactions42.Cytochrome P450 proteins have usually been quantified by Western blotting, resulting in relative expressions of the investigated enzymes43. These substrates are also metabolized by CYP450 in the (mini)pig50. Although similarities and differences in biotransformation rates between humans and pigs have been discussed51, less attention has been devoted to the apparent selectivity of the substrates used. It has been shown that chlorzoxazone is metabolized by porcine recombinant CYP2A, CYP1A, and CYP2C enzymes, although it is a typical CYP2E1 substrate52. Furthermore, it has been proposed that dextromethorphan-O-demethylation may be catalyzed by CYP2B in the pig rather than CYP2D20, even though no CYP2B22 was found in conventional pigs39.In the past, typical probe substrates for human CYP450 have been used to measure CYP450 activity in the (mini)pig. Phenacetin (CYP1A), coumarin (CYP2A), midazolam (CYP3A), tolbutamide (CYP2C), dextromethorphan (CYP2D), and chlorzoxazone (CYP2E) are commonly used substrates in human drug researchTherefore, the goals of the current study were (1) to consistently map and characterize the hepatic and intestinal CYP450 enzymes in 16 conventional, 12 weeks old pigs, to allow a more rational comparison between pigs and minipigs in future research, (2) to assess the apparent selectivity of the most commonly used probe substrates towards six important CYP450 enzymes for drug metabolism, which are phenacetin (CYP1A), coumarin (CYP2A), midazolam (CYP3A), tolbutamide (CYP2C), dextromethorphan (CYP2D), and chlorzoxazone (CYP2E), and 3) to compare porcine and human CYP450 with respect to substrate selectivity and activity.13C6 (OH-CZ-13C6) was obtained from Alsachim . Potassium chloride, potassium dihydrogenphosphate and dipotassium hydrogenphosphate, citric acid, glycerol, disodium hydrogenphosphate, and EDTA were obtained from VWR . Acetonitrile (ACN), methanol (MeOH), ethylacetate were of HPLC grade and purchased from Fisher Chemicals.Phenacetin (PH), acetaminophen or paracetamol (PAR), tolbutamide (TB), 7-hydroxy-coumarin (OH-CM), dextrorphan-D3 (DXT-D3), coumarin (CM), chlorzoxazone (CZ), diethyldithiocarbamate (DDC), ketoconazole (KET), α-naphthoflavone (α-NFV), 8-methoxypsoralen (8-MPS), protease inhibitor , trifluoroacetic acid (TFA), dimethylsulfoxide (DMSO), triethylammonium bicarbonate (TEABC), dithiotreitol (DTT), methyl methane thiosulfonate (MMTS), calcium chloride, and phosphate buffered saline (PBS) were purchased from Sigma Aldrich . Midazolam (MDZ), 1-hydroxy-midazolam (OH-MDZ), 1-hydroxy-midazolam-D4 (OH-MDZ-D4), 4-hydroxy-tolbutamide (OH-TB), 6-hydroxy-chlorzoxazone (OH-CZ) were obtained from LGC standards . Dextromethorphan (DXM), dextrorphan tartrate (DXT), 7-hydroxycoumarin-D5 (OH-CM-D5), acetaminophen-D4 (PAR-D4), 4-hydroxytolbutamide-D9 (OH-TB-D9), quinidine (QND) and sulphaphenazole (SFZ) were purchased from Toronto Research Chemicals . NADPH was obtained from OYC Europe . Six-hydroxy-chlorzoxazone-13C6, OH-TB-D9, OH-CM-D5, PAR-D4 and DXT-D3 respectively).Stock solutions of each substrate and inhibitor were prepared in MeOH  or ACN ) and stored at −20 °C. Fresh working solutions were prepared by adding an appropriate amount of stock solution to HPLC-quality water. The stop reagent consisted of 55% ACN, 42% HPLC water, and 3% formic acid with internal standards .Microsomes were prepared from hepatic and intestinal tissues collected from sixteen different conventional pigs . Pigs of this age were selected as most human CYP450 enzymes reach adult activity at the age of 6 years, which corresponds to an age of 4–12 weeks in pigset al.55 and the intestinal microsomes were prepared using the mincing method described by Osselaere et al.56.Fresh samples were taken from the left liver lobe, mid-duodenum, mid-jejunum and mid-ileum, rinsed in PBS and immediately snap frozen in liquid nitrogen and stored at −80 °C. Hepatic microsomes were prepared according to Wilson g for 25 min at 4 °C. After centrifugation, the supernatant was transferred to ultracentrifugation tubes and centrifuged at 100,000 × g for 80 min at 4 °C. Samples were washed 4 times with 3 mL of buffer A solution and again centrifuged at 100,000 × g for 80 min at 4 °C. The microsomal pellets were suspended in 1.5 mL/g liver tissue of buffer A containing 30% glycerol, snap frozen and stored at −80 °C. All procedures were performed on ice.Briefly, liver tissues, stored for a maximum of 8 weeks, were thawed on ice in approximately 4 mL of a 0.25 M phosphate buffer (pH 7.25) containing 1.15% aqueous KCl (buffer A). Next, 4 grams of tissue was transferred to a petri dish and minced into small pieces, subsequently transferred to a Potter Elvehjem homogenizer and homogenized in 8 mL of buffer A. Glass tubes were rinsed twice with 4 mL of the same buffer solution and added to the homogenate. Samples were then centrifuged at 10,000 × 2PO4, 5.6 mM Na2HPO4 and one tablet of protease inhibitor per 50 mL of solution. After mincing, the tissues were transferred to a Potter Elvehjem homogenizer. Tissues were homogenized in 8 mL of homogenization buffer (50 mM phosphate buffer with 1 mM EDTA and 1 tablet of protease inhibitor per 50 mL solution). Glass tubes were rinsed twice with 4 mL of homogenization buffer. Samples were centrifuged at 18,000 × g for 20 min after which the supernatant was transferred and centrifuged at 100,000 × g for 67 min at 4 °C. Microsomal pellets were washed with a 125 mM phosphate buffer  and centrifuged for a second time at the conditions specified. Afterwards, microsomal pellets were suspended in 0.5 mL/gram tissue of resuspension buffer, snap frozen and stored at −80 °C. All procedures were performed on ice.Intestinal tissues were prepared according to an analogous procedure. Four gram of each segment was thawed on ice in 4 mL of thaw solution containing 1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate, 8 mM KH57, according to the manufacturer’s instructions.Hepatic and intestinal microsomal protein concentrations were determined by the Bradford assay2 and ACN to a final concentration of 1 mM and 5%, respectively. After evaporation in a speedvac, the samples were re-suspended in 0.1% formic acid. Four hundred ng sample was spiked with 50 fmol β-galactosidase  and 50 fmol Hi3 Escherichia coli  standards before injection. For the intestinal microsomes, samples of all sixteen pigs were pooled and measurements were performed in triplicate. Hepatic CYP450 quantity was determined for each pig.Microsomal proteins (20 µg) were reduced in 0.5 M TEABC and 1 mM DTT for 1 hour at 60 °C, followed by alkylation using 10 mM MMTS for 10 min at room temperature. Proteins were digested into peptides using trypsin  overnight at 37 °C with CaClm/z at 50–5,000) in resolution mode. Survey MS scans were acquired using a fixed scan time of 200 ms. Tandem mass spectra of up to eight precursor ions with charge state 2+ to 5+ were generated using CID in the trapping region with intensity threshold set at 3,000 cps, using a collision energy ramp from 6/9 V  up to 147/183 V . MS/MS scan time was set to 100 ms with an accumulated ion count ‘TIC stop parameter’ of 100,000 cps allowing a maximum accumulation time of 250 ms. Dynamic exclusion of fragmented precursor ions was set to 12 s. Ion mobility spectrometry wave velocity was ramped from 2,500 to 400 m/s. Wideband enhancement was used to obtain a near-100% duty cycle on singly-charged fragment ions. LockSpray of glufibrinopeptide-B (m/z 785.8427) was acquired at a scan frequency of 60 s.The peptides were separated using a nanoscale UPLC system  coupled to a HRMS Q-TOF Synapt G2-Si mass spectrometer . Peptides were first trapped in 0.1% formic acid on a 180 µm × 20 mm C18 Trap column. Separation was performed on a HSS C18 1.8 µm, 75 µm × 250 mm analytical column at a flow rate of 300 nL/min and a temperature of 45 °C. Mobile phase A and B were composed of 0.1% formic acid with 4% DMSO in UPLC-water and 80% ACN containing 0.1% formic acid, respectively. Peptides were separated with a linear gradient for 60 min at 1–40% solvent B and for 1 min at 40–85% solvent B. The HRMS instrument was operated in positive mode for High Definition-DDA, using a nano-ESI source, acquiring full scan MS and MS/MS spectra , supplemented with unreviewed CYP proteins and fragments of interest, the cRAP database , and sequences of spiked standard proteins. For relative quantification, the top three peptides were used and only proteins with at least one unique peptide were further considered. For absolute quantification, proteins were quantified using the top three peptides against Hi3 E. coli peptides, and only proteins with at least one unique peptide were further considered. Although it is hard to assume that the ionization efficiency is the same for each peptide and in each sample, this method has been proven reproducible (10% CV) and accurate (15% relative error) for the purpose of comparison58. Protein data was exported from Progenesis® for further statistical analysis. For the calculations of relative quantity of drug metabolizing CYP450, the mean quantity of each enzyme was calculated as the percentage of the mean total detected drug metabolizing enzyme.Data analysis of the raw files obtained from the Synapt G2-Si was performed in Progenesis® QI (Nonlinear Dynamics) version 2.3. Peptides with charge +1 were discarded. For relative quantification, data was normalized to all proteins. For absolute quantification, data was normalized to Hi3 Hepatic microsomes were incubated at 37 °C with TB, CZ, MDZ, CM, DXM, and PH at six different concentrations Table . The incg at 4 °C. Following centrifugation, the supernatant was transferred to 15 mL conical tubes containing 1 mL of a 100 mM phosphate buffer. Next, 7 mL of ethylacetate was added and samples were extracted for 15 min on an overhead shaker . The two phases were separated by centrifuging at 14,000 × g for 5 min. The organic phase was transferred and evaporated at 40 ± 5 °C under a gentle nitrogen stream. Samples were reconstituted in 200 µL of a 50/50 methanol/water solution and analyzed by LC-MS/MS as previously described59.After the specified incubation time, an aliquot of 200 µL was transferred to an Eppendorf tube containing 125 µL of ice cold stop reagent. Samples of the same animal, incubated with different probes, were pooled for each concentration and 125 µL of TFA was added. Subsequently, samples were centrifuged for 10 min at 16,200 × Intestinal microsomes were incubated as described above for hepatic microsomes. The final microsomal protein concentration was 0.75 mg/mL and incubation time was 10 min for all of the probe reactions. Each activity measurement  was performed in triplicate and data are presented as mean of these replicates.60. Km, Vmax and/or Hill coefficients were derived for each substrate and pig, in order to obtain the inter-individual variation.The activity data were fitted against the standard Michaelis-Menten equation . Considering the goal of this study, the concentrations of inhibitors were chosen to be 0.1 µM, 1 µM, 0.3 µM, 0.5 µM, 0.1 µM and 20 µM for α-NFV, 8-MPS, KET, SFZ, QND, and DDC respectively /Rate(control) and Rate/Rate(control). The incubations with inhibitors and two substrates were only performed on porcine hepatic microsomes, due to the limited availability and low activity of porcine intestinal microsomes.Second, two substrates were incubated simultaneously to investigate their influence on each other’s biotransformation rate. For the dual substrate incubation experiment, each possible combination of two substrates was investigated and incubated at concentrations around or below the KFirst, Eadie-Hofstee plots were examined for deviations from linearity. Biphasic plots are indicative of multiple enzyme involvement or of the presence of multiple binding sites within the enzyme. Furthermore, correlations between substrate-enzyme and substrate-substrate were analyzed to investigate changes in explained variability. In essence, at a fixed concentration, enzyme kinetics can be approximated by a linear function of enzyme concentration(s) (Eqs –d). Channd lowest concentration, Table m, is a constant over all individuals. This assumption is reasonable as no polymorphisms are known to date that affect the intrinsic clearance of substrate drugs44. Nevertheless, it should be noted that one polymorphism (1423 G- > A) in porcine CYP2E1 can lead to a decrease in protein expression61. However, it remains unclear if this polymorphism results in the alteration of the enzyme’s metabolic properties due to conflicting results63. Given this and a fixed concentration for each reaction, the Michaelis-Menten equation  can be ion  were calculated and used to assess enzyme involvement.Fourth, typical inhibitors for each of the reactions were included at previously reported Km, and Vmax values, were analyzed by an independent sample t-test. Normality was checked using the Shapiro Wilk test. If the Shapiro Wilk test statistic was significant, log transformation was used to calculate the t-statistic. Equality of variances was checked using the Levene’s test for equality of variances. The appropriate test statistic was used depending on the equality of variances.Stepwise linear regression, correlation analyses, and independent sample t-tests were performed with the SPSS® 24 software . Sex differences in hepatic protein abundances, K All procedures followed were in accordance to the ethical standards of the ethical committee of the Faculties of Veterinary Medicine and Bioscience Engineering of Ghent University .The entry information, which provides the associated accession number(s), of the detected and quantified enzymes, are shown in Supplementary Table m and Vmax  and for Vmax p = 0.192 (DXM)), which is reflected in the CYP450 quantities. Consequently, the parameters provided in Table Mean values for Michaelis-Menten parameters of hepatic CYP450 of 8 male and 8 female conventional pigs, 12 weeks of age are shown in Table 60. The Eadie-Hofstee plots for CZ, PH, and TB indicate deviation from standard Michaelis-Menten kinetics. Chlorzoxazone and PH are biphasic, while TB does not saturate in the range of TB concentrations tested. A Michaelis-Menten equation with two contributing enzymes was fit to the CZ and PH data, but the estimates obtained were inaccurate and not significant. However, a fit to a single enzyme kinetic equation resulted in an adequate fit  was observed Fig. , indicatmax was observed with duodenum > jejunum > ileum. No CM-hydroxylase activity could be detected in the intestine and only MDZ-hydroxylase and PH-O-deethylase activity were observed over all three segments  were 3 and 10 respectively. However, VResults of the incubation with substrate-inhibitor and dual substrates are presented in Tables i value for the human CYP1A2 enzyme (0.1 µM). However, when the concentration was increased to 1 µM, PH-O-deethylation (CYP1A2) activity decreased to 76% of the control value  or CYP2A . Notable is the apparent increase of MDZ-hydroxylation observed with all inhibitors, except KET.Eadie-Hofstee plots of hepatic and intestinal CYP450 activities are displayed in Figs 65. However, intestinal CYP450 activity is much lower in porcine microsomes, especially for CYP3A, which is 6–40 times lower67. In contrast to the Vmax of MDZ-hydroxylation, the Vmax for TB-hydroxylation (CYP2C) is similar to those observed in humans67, although direct comparison is difficult due to the different amounts of intestinal sample used for microsomes preparation. Hence no systematic comparison could be performed. In the current study, intestinal pieces of 4 grams were processed, while in previous studies67 mucosal scrapings of 1 foot sections or entire gut length were used. Likewise, the observed discrepancy for the CYP3A activity, can be attributed in part to the difference in intestinal microsomes preparation. Previously it has been shown that the elution of intestinal samples with EDTA resulted in the highest CYP450 activity when compared to the mucosal scraping method68. Furthermore, in the comparison between the EDTA elution method and the mincing method, it was shown that the latter resulted in higher enzymatic activities and more reproducible results69. Hence the mincing method was used in the current study.To the best of our knowledge, this is the first study to quantify and characterize porcine intestinal CYP450 enzymes. Duodenal relative quantitative results are remarkably similar between pigs and men, with CYP3A constituting 88% and CYP2C 12% of the detected CYP450 enzymes in pigs vs. 82% CYP3A and 16% CYP2C in humans . They investigated the CYP450 quantity in two adult conventional pigs, with a maximal difference of 5% in relative quantity for CYP2E1 compared to the current study. No evidence for sex differences was found, which corresponds with the recent results of Millecam et al. (2018)72. However, compared to humans, differences in relative quantities are observed, with CYP2C, CYP1A2, and CYP3A being less abundant  and CYP2A, CYP2D, and CYP2E being more abundant  15. However, it should be mentioned that due to the limited concentration range of TB, with no obvious observable saturation of the kinetics, it is recognized that the Km and Vmax estimates reported herein can be biased. Nevertheless, previous reported Vmax values in porcine hepatic microsomes range from 62–440 pmol/mg protein/min75 which are similar to the values reported here and within the range reported for humans. The affinity, on the other hand, is approximately 5 to 10 times lower76. It should be mentioned that the Km estimate of tolbutamide differs somewhat from previous experiments by Schelstraete et al. (2018)59. This can be attributed to the fact that for the Km estimation in the latter reference, the highest concentration level was included. In addition, the microsomes originated from different animals as described in this study.The 2.5 times lower quantity of porcine CYP2C enzymes seems to be reflected in the lower TB-hydroxylase activity compared to human19. Nevertheless, with respect to the selectivity, the CYP2C enzymes were most likely the major contributors to TB-hydroxylation, as they showed strong correlations with the activity. Furthermore, TB did not influence the biotransformation of other probes. However, KET could also inhibit the reaction moderately, a feature also seen in humans64. Therefore, in addition to the increasing correlations with MDZ-hydroxylation and CYP3A46, and biphasic intestinal metabolism, CYP3A enzymes probably contribute to a low affinity phase.In contrast to the similar biotransformation, SFZ did not inhibit TB-hydroxylation, indicating some qualitative differences with the human orthologuesmax of the CZ-hydroxylation reaction was similar between porcine and human microsomes 77. Remarkably the porcine and human CYP2E1 are almost equally abundant, representing 13% and 9% of drug-metabolizing CYP450 respectively70. However, the selectivity of chlorzoxazone for CYP2E1 has been questioned52. In humans, CYP1A2 and CYP3A also contribute to the reaction80, whereas in pigs recombinant CYP1A1, CYP2A19, and CYP2C33 are able to hydroxylate CZ47.The V47, CYP2A19 and CYP1A2 account for 72% of CZ-hydroxylation at a 5 µM concentration.At low substrate concentrations, CYP3A46 and PAR were selected from the regression analysis. Nonetheless, KET could only marginally inhibit CZ-hydroxylation (20%) and α-NFV did not inhibit the reaction. In contrast, 8-MPS inhibited the reaction to about 28%. Furthermore, CM, PH, and CZ affected each other’s biotransformation significantly, leading to the conclusion that mainly CYP2A19 and CYP1A2 are responsible for CZ-biotransformation. Indeed, when combining the CYP450 amounts in microsomes with data from recombinantly expressed CYP45047. Of note is that their cloned CYP3A differed 2 amino acids from the detected CYP3A29 in this study. Finally, at higher concentrations CYP2E1 was the variable explaining most of the variability in CZ-hydroxylation, suggesting its role in catalyzing the reaction.At higher substrate concentrations, CYP2E1, in addition to CYP2C and CYP3A enzymes, most likely metabolize CZ. Even though no marked influence was seen on CZ-hydroxylation by TB or MDZ, the associated parallel increase in correlation coefficient between CZ-hydroxylation and CYP3A, CYP2C enzymes at one hand, and TB and MDZ on the other hand, in combination with duodenal activity, suggests their involvement. However, Wiercinska and Squires expressed recombinant porcine CYP3A, but were unable to detect CZ-hydroxylase activity from this enzyme43. The CYP2A19 enzyme represents 31% of the hepatic CYP450 enzymes with a comparable Vmax value for humans, depending on the reference  and weak inhibition of the CYP1A2 associated inhibitor α-NFV (5%), the monophasic Eadie-Hofstee plot, and the absence of intestinal biotransformation, CYP2A19 is likely the sole important contributor to CM-hydroxylation.Coumarin is almost exclusively used as CYP2A6 probemax of the PH-deethylation is similar , although the amount of CYP1A2 is 4 times lower in pigs compared to humans suggesting higher efficiency of porcine CYP1A2 or involvement of additional enzymes. Indeed, as PH and CM could affect the other’s biotransformation rate, and 8-MPS inhibited PH-O-deethylation, CYP2A19 is likely involved. Hence, CYP2A19 seems to have broader substrate selectivity compared to human CYP2A6 as indicated by its capability of metabolizing PH and CZ. This apparent broader substrate selectivity of CYP2A19 is further supported by a report in which 1-ethoxyresorufin, another frequently used CYP1A2 substrate, was found to markedly affect coumarin-hydroxylation when co-incubated83. Finally, α-NFV was able to inhibit PH-O-deethylation (24%), indicating that CYP1A2 is involved in its biotransformation.In contrast, PH, a typical human CYP1A2 probe, is most likely metabolized in part by CYP2A19. This is supported by the fact the Vm value. Nevertheless, strong correlations (r = 0.80) were observed between PH-O-deethylation and MDZ-hydroxylation. In addition, CM- and CZ-hydroxylation explained most of the PH-O-deethylation variability. Although CZ is not very selective for CYP2E1, a parallel increase in correlation with this enzyme suggests CYP2E1 has low affinity for PH-deethylation, as observed in humans84.At higher concentrations, additional enzymes contribute to the PH-deethylation. The observation of a similar biphasic plot in hepatic and duodenal microsomes, suggests involvement of CYP2C and CYP3A enzymes, although in hepatic microsomes KET could not inhibit PH-O-deethylation at concentrations around the K48, paralleled by the approximately 3 times lower amount of CYP3A observed in the current study. Our results indicate that MDZ is largely selective for porcine CYP3A. Similar Eadie-Hofstee plots in hepatic and intestinal microsomes indicate the involvement of the same enzymes. Remarkably, MDZ reduced all reactions except CZ-hydroxylation, although this results possibly from nonselective binding of MDZ or a metabolite thereof, as supported by the observation of a sigmoidal profile, a characteristic observed for nonselective binding of other weak bases85. In addition, all inhibitors, except KET, enhanced MDZ-hydroxylation, which can be hypothesized to result from an increase in free concentration.The MDZ-hydroxylation rate is about 2 to 3 times lower compared to humans48. CYP3A22 had the highest quantity amongst CYP3A enzymes, which is reflected by the significant correlations with MDZ biotransformation at all concentrations. No significant correlation was found for CYP3A29, although it has a high intrinsic clearance and is the second highest quantified CYP3A enzyme48. A possible explanation is a polymorphism in the CYP3A29 sequence, supported by the existence of a CYP3A sequence in the Uniprot database, differing in only two amino acids . Recently, this sequence was recombinantly expressed. Unfortunately no CYP3A substrate was measured47.It has been shown that rCYP3A22 had the highest intrinsic MDZ clearance, followed by rCYP3A29 and rCYP3A4688, which reflects the 14 times higher quantity of CYP2D (28% vs. 2% in humans)90. However, porcine DXM-metabolism has been debated with CYP2B22 rather than CYP2D25 being responsible for DXM-O-demethylation90. Nevertheless, no CYP2B22 was observed in this study. Hence it is unlikely that CYP2B22 is the most important enzyme in DXM-O-demethylation.Dextromethorphan-O-demethylation was exceptionally high, being up to 7 times faster in pigs than in humans 89, the observed correlation in addition to the high biotransformation rate and linear Eadie-Hofstee plot, which is indicative for single enzyme involvement at the used concentrations, suppose that CYP2D25 is most likely responsible for DXM-biotransformation.Regression analysis selected CYP3A46, CYP2C33, OH-TB, and OH-MDZ as predictive variables. However, KET could not inhibit DXM-O-demethylation and only MDZ had a marked influence, on the DXM-biotransformation, although this probably resulted from nonselective binding. As a consequence, these enzymes are most likely not the major contributors to the DXM biotransformation. CYP2D25, however, also correlated significantly with the DXM-O-demethylation (r = 0.55–0.66). Although QND did not inhibit the reaction, in line with other reports91. This may be due to the higher amount and broader substrate-selectivity of CYP2A19. In addition, SFZ and QND showed no inhibition towards CYP2C and CYP2D25 respectively, indicating some differences in binding characteristics compared to human CYP450 orthologues. To conclude, the results presented herein support the use of the pig as an appropriate animal species for drug metabolism studies, based on the similarities in selectivity- and quantity- normalized activities, especially for the most important CYP3A subfamily. The current study provided an important step towards a consistent and systematic validation of the pig as a model for human drug metabolism.Tolbutamide, CM, MDZ, and DXM selectivity is similar between pigs and humans. This is an important feature for a potential preclinical animal species, as most of the currently available drugs are metabolized by CYP2C, CYP3A, and CYP2D. Moreover, being capable of producing the same metabolites at more or less equal rates is crucial in the safety and toxicological assessment of new drug candidates, as not only the parent molecule but also the metabolites can give rise to safety concerns. In contrast, CZ and PH are metabolized in part by CYP2A19. In humans, CZ is not metabolized by CYP2A6 and PH only to a limited extendSupplementary fileSupplementary Dataset 1


We also show that RAR-β signaling promotes the binding of the mitochondria to the anchoring protein, glucose-related protein 75, at the growing tip of neurite, thus allowing them to provide energy and metabolic roles required for neurite outgrowth.—Trigo, D., Goncalves, M. B., Corcoran, J. P. T. The regulation of mitochondrial dynamics in neurite outgrowth by retinoic acid receptor β signaling.Neuronal regeneration is a highly energy-demanding process that greatly relies on axonal mitochondrial transport to meet the enhanced metabolic requirements. Mature neurons typically fail to regenerate after injury, partly because of mitochondrial motility and energy deficits in injured axons. Retinoic acid receptor (RAR)-β signaling is involved in axonal and neurite regeneration. Here we investigate the effect of RAR-β signaling on mitochondrial trafficking during neurite outgrowth and find that it enhances their proliferation, speed, and movement toward the growing end of the neuron  Mitochondria are cellular organelles whose main functions are to generate ATP, buffer cytosolic calcium, and generate reactive oxygen species. Mitochondria also play an important role in the mechanism of axon extension, regeneration, and axon branching , 2. The This transport is regulated by various signaling pathways to efficiently respond to cellular needs and external factors. One of these pathways is hypoxia-inducible factor 1α (HIF-1α). Induced by decreased oxygen tension, the HIF-1α signaling pathway is traditionally associated with a first-level cell response to hypoxia, namely, by regulating mitochondrial recruitment, activity, and oxygen consumption , 6. Besi10via this signaling pathway.It has previously been shown, in refs. 13Here we show that in cultured cortical neurons, RAR-β, in parallel to promoting neurite outgrowth, promotes mitochondrial membrane depolarization in the soma and its anterograde transport and proliferation along the neurite by activating the hypoxia signaling pathway. We describe that HIF-1α is required for both retinoid-induced neurite elongation and mitochondrion regulation, and we additionally show that RAR-β activation promotes the accumulation of mitochondria in the growing neurite. This is accomplished by facilitating the interaction of mitochondria with the chaperone GRP75, possibly by mediating mitochondrion-ER interaction.d-lysine-coated 24-well cell culture plates, 75-cm2 flasks, or 35-mm glass-bottom culture dishes , depending on the experiment, at a density of 15 × 104 cells per ml. Cells were cultured in neurobasal medium  containing 2% B27 serum-free supplement, 2 mM l-glutamine, 1.5% glucose, penicillin (100 U/ml), and streptomycin (100 g/ml), incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cultures were 98% neurons, judged by βIII-tubulin staining. Unless mentioned otherwise, mouse primary cortical cultures were treated with 100 nM CD2019  or vehicle  for 72 h. CD2019 is a RAR-β agonist capable of inducing axonal outgrowth in central primary cultures with a 5-fold and 12-fold selectivities for RAR-β over RAR-α and RAR-γ, respectively , an (aryloxyacetylamino)benzoic acid analog that was determined by a reporter assay to inhibit HIF-1α protein accumulation and its target gene expression under hypoxic conditions, without altering HIF-1β levels  for 30 min prior to fixation. Alternatively, cells were loaded with 20 nM tetramethylrhodamine, methyl ester  for 45 min, prior to being placed in an incubator attached to a confocal microscope. TMRM is a cell-permeant fluorescent dye, sequestered by active mitochondria.Multichannel fluorescence images were captured using a Zeiss LSM 700 laser-scanning confocal microscope  with a ×63 oil-immersion Aprochromat objective with an image size of 512 × 512 pixels, with a pinhole aperture of 1 Airy unit. Settings for gain, contrast, and brightness were optimized initially and held constant throughout each study so that all sections were digitized under the same conditions.z stacks of the whole imaged neuron were taken  as previously described in ref. https://fiji.sc/) of ImageJ software  was used to measure pixel signal intensity and colocalization studies.For colocalization studies, For neurite outgrowth assays, treated primary cortical neuron cultures were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 4 min, and stained with Alexa Fluor 488 phalloidin, or alternatively with the neuron-specific anti–βIII-tubulin. These were then imaged by confocal microscopy, and ImageJ-Fiji software was used to measure the length of the longest neurite in each neuron.GRP75 small interfering RNA (siRNA)  and Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) in OptiMem (2% v/v) were prepared and allowed to stand for 5 min. They were then mixed and incubated at room temperature for 20 min before addition to cortical neurons plated in glass bottom dishes or 6-well plates, in a total volume of 1 ml, or in 24-well plates, in a total volume of 250 μl, with a final siRNA concentration of 10 nM. Cells were incubated for 7 h before adding an equal volume of supplemented neurobasal medium and incubated for additional 24 h, following which the medium was replaced with fresh supplemented neurobasal medium. Cells were incubated for an additional period of 24 h.Proteins were separated by SDS-PAGE on 10% (w/v) polyacrylamide gels and then transferred to a 0.45-μm pore size nitrocellulose membrane  using a Trans-Blot SD Semi-Dry Transfer Cell . Nitrocellulose membranes were incubated in blocking solution consisting of 5% (w/v) skimmed milk powder in PBS-T  for 1 h at room temperature, followed by incubation with the appropriate primary antibody diluted in blocking solution overnight at 4°C. Membranes were washed 3 times in PBS-T and incubated with species-specific secondary antibodies in blocking solution for 2 h at room temperature in the dark, after which the membranes were washed as above. Protein levels were corrected for loading differences by normalizing against β-actin levels. Proteins were detected by scanning at 700 and 800 nm using the Odyssey Detection System .r value)  , obtainet test. Comparisons were made between appropriate groups, with α = 0.05.Statistical studies were performed using Prism 5 for Mac OS X and Prism 6 for Windows . Unless stated otherwise, data were analyzed using nondirectional, 2-tailed, 2-sample, equal variance Student’s P = 0.0018) . This increase was more significant when the number of mitochondria in the neurite terminal was tallied individually , indicating that this increase in mitotracker red signal results from an elevated mitochondrial presence in the growing neurite following RAR-β activation  results in neurite elongation  C showed tivation D–F.This population increment could result either from increased recruitment and transport toward the neurite or from greater mitochondrial proliferation in this region.P = 0.019) and retrograde directions (P = 0.048). However, even though the speed of moving mitochondria appears to also be increased independently of direction of transport, RAR-β activation increases the percentage of mitochondria moving anterogradely from 8.9 ± 3.5% to 15.2 ± 3.7% (P = 0.009), not altering the percentage of mitochondria moving toward the soma (P = 0.43)  revealed that acute activation of RAR-β  increases the speed of movement of healthy mitochondria, both in anterograde ( = 0.43) G–I.P = 0.99 for anterograde-moving, P = 0.54 for retrograde-moving, and P = 0.74 for immobile mitochondria), analysis of mitochondrial size distribution showed a significant increase in smaller mitochondria, particularly <1 µm, from 20.6 ± 1.0% to 29.3 ± 5.0% (P = 0.0026). Moreover, neuronal activation of RAR-β affected mitochondrial dynamics, substantiated by the increase in number of mitochondrial fission events, from 0.0077 ± 0.006 per micrometer in vehicle-treated neurons to 0.0204 ± 0.012 per micrometer in agonist-treated neurons (P = 0.035)   and prev= 0.035) , 23.P = 0.13 for anterograde-moving, P = 0.16 for retrograde-moving, and P = 0.29 for immobile mitochondria). This decrease in mitochondrial membrane potential was confirmed to be a statistically significant 20% decrease in TMRM integrated density signal when all the mitochondria in the neuron were taken into account (P = 0.012), suggesting that this decrease in membrane potential is more focused on the cell body. This was indeed confirmed when quantification of mean TMRM signal discriminated between cell body, where a 22% decrease in mean TMRM signal intensity was observed (P = 0.019), and the neurite, with mean TMRM signal intensity remaining unaltered (P = 0.819)  suggests a decrease in mitochondrial membrane potential in the neurite (= 0.819) F–K.P = 0.036) and 33% increase at 72 h (P = 0.049) , which inhibits HIF-1α accumulation in a concentration and time-dependent manner and significantly suppresses transcriptional activity of HIF-1α target genes, obtaining complete inhibition at similar concentrations (P = 0.018), in a manner not rescuable by CD2019, which had a similar decrease in HIF-1α levels to 24% of control (P = 0.0002) (P < 0.005).These observations, together with the data showing mitochondrial recruitment to the neurite terminal, suggests that RAR-β promotes mitochondrial recruitment toward the growing neurite, where energy production is promoted trations . Neurons 0.0002) C, D. In P = 0.48) but prevents the increase in speed of moving mitochondria observed with acute activation of RAR-β, decreasing the mean speed of moving mitochondria to that of vehicle-treated neurons . While not altering mitochondrial presence in the neurite or the percentage of mitochondria moving in either direction, when compared with control, the increase in anterograde-moving mitochondria observed with activation of RAR-β was prevented with HIF-1α inhibition, not altering the percentage of retrograde-moving mitochondria. Additionally, the decrease in TMRM signal intensity observed following activation of RAR-β is also absent when HIF-1α is inhibited , and colocalization between immunostained GRP75 and mitotracker red increased from 0.35 ± 0.03 in vehicle-treated neurons to 0.47 ± 0.04   or vehicle for 72 h shows that in the presence of the RAR-β agonist there is an increase in colocalization of GRP75 and the mitochondrion dye mitotracker red, suggesting that not only is GRP75 up-regulated in RAR-β activated neurites but its binding to mitochondria is also elevated, established by colocalization analysis. Indeed, RAR-β activation caused a 40% increase in GRP75 signal coincident with mitotracker red (P < 0.05). Live imaging of GRP75-silenced neurons additionally revealed that acute treatment with RAR-β agonist (30 min prior to imaging) causes a significant increase of 102% in the number of mitochondria moving in the retrograde direction (P = 0.039)  E–H. Havir = 0.6 between ER and mitochondria. The levels of coincident signal between ER and mitotracker red in the whole neuron were unaltered following RAR-β activation, but when this analysis was restricted to the distal 20 μm2 of the neurite, an increase of 40% (P = 0.025) was observed in agonist-treated neurons  following activation of RAR-β. These results indicate that GRP75 is required to maintain recruited mitochondria at the neurite terminal.We further show that GRP75 is required for neurite outgrowth, because its silencing with siRNA ablates the effect of neurite elongation. Interestingly, silencing of GRP75 also resulted in a drastic increase in mitochondrial retrograde transport  or functional reasons , and GRP75 has long been described to determine ER-mitochondrial coupling, mediating the interaction of the mitochondrial voltage-dependent anion channel with the reticular inositol 1,4,5-triphosphate receptor (Indeed, RAR-β activation is accompanied by increased association between the ER and mitochondria in the distal end of the neurite, where mitochondrion-ER interaction is exacerbated. This interaction could be increased because of structural purposes (receptor .Thereforreceptor .


Detailed x-ray reciprocal space mapping (RSM) measurements have been taken in order to study the strain, along with cross-section scanning electron microscope (SEM) images and x-ray diffraction (XRD) curve measurements. It has been found that it is critical to achieve a crack-free GaN HEMT epi-wafer with high crystal quality by obtaining a high quality AlN buffer, and then tuning the proper thickness and aluminium composition of the two transitional AlxGa1−xN layers. Finally, HEMTs with high performance that are fabricated on the epi-wafer have been demonstrated to confirm the success of our strain engineering and above analysis.We have designed and then grown a simple structure for high electron mobility transistors (HEMTs) on silicon, where as usual two transitional layers of Al However, there is a large lattice mismatch and a large thermal expansion coefficient (TEC) difference between GaN and silicon, typically causing extensive cracks in the post-growth cooling down process [In the last decade, III-nitride devices have been widely used in many applications, such as general illumination , radio-f process ,6,7,8,9,xGa1−xN-based interlayers [xGa1−xN strain-release layers [xGa1−xN method is very popular due to its easiness to achieve and analyze the strains in each layer. In detail, for the graded AlxGa1−xN buffer layers, a large number (>4) of AlxGa1−xN layers [Until now, a number of approaches have been proposed, such as AlN/GaN super-lattice layers ,11, AlxGerlayers ,12,13,14erlayers ,16 and se layers ,18,19,20N layers  and diffN layers . HoweverxGa1−xN layers and then a GaN/Al0.2Ga0.8N heterostructure. This simple structure allows us to analyze the strain more clearly for the graded AlxGa1−xN transitional layers with different compositions and thicknesses. By measuring X-ray reciprocal space mapping (RSM), the strain components in our AlxGa1−xN layers can be obtained and then feed back into epi-wafer growth. Finally, the HEMTs with high performance have been demonstrated, verifying the quality of our GaN HEMT epi-wafers.In this paper, we design a very simple HEMT epi-structure with one AlN buffer layer, only two graded transitional Al2 ambiance to remove any contaminants and native oxides. Subsequently, the temperature is decreased to 1000 °C and a trimethylaluminium (TMA) pre-flow is conducted without any NH3 flowing for 40 s. A thin low-temperature AlN (LT-AlN) layer is then grown, followed by a high temperature AlN (HT-AlN) layer grown at 1297 °C. The thickness of the AlN layer is 260 nm in total. Next, two layers of Al0.35Ga0.65N and Al0.17Ga0.83N have been further grown as strain-compensation transitional layers. After finishing the growth of the two AlxGa1−xN transitional layers, a 1.2 µm (0001) GaN layer was then grown, followed by a final 25 nm Al0.2Ga0.8N barrier layer. During the cooling down procedure, N2 and NH3 are used as cooling gases in order to eliminate any micro cracks generated on the final AlxGa1−xN layer which has been accepted as a result of H2 enhanced surface etching [ etching .0.35Ga0.65N layer, the Al0.17Ga0.83N layer and the GaN layer are 258 nm, 180 nm, 290 nm and 1.18 µm, respectively.The whole wafer has been examined across two inches by optical microscopy, confirming that it is crack-free except for the edge region of the wafer, as shown in xGa1−xN layer, thus filtered by the AlxGa1−xN layers. However, there are still a few cracks penetrating the GaN layer which extend to the surface, as shown in By comparing The crystal quality has been further characterized by X-ray diffraction  measurements as shown in According to Reference , the scrThus, we can getWith our growth method, the edge dislocation density is almost 7.3 times the screw dislocation density, thus edge dislocation dominates all the dislocations. In future, we will focus on reducing the edge dislocation to further optimize the GaN buffer layers.xGa1−xN and AlN layers in RSM maps. The solid white line shows the fully relaxed AlxGa1−xN layers grown AlN. In x = 0, making it difficult to investigate the in-plane strain distribution of AlxGa1−xN transitional layers. We rotated certain angles to the  is the coordinates in the map, corresponding to the latticed constants ;  is the Bragg reflection direction; ; z axis and the extended line interpolated from the two points described above; 0 and a0 are the relaxed parameters from Vegard’s law; Ci,j(x) are the elastic constants.Based on the RSM measurements, the compressive in-plane strain components accumulated in each AlGaN = 3.189 Å, cGaN = 5.185 Å, aAlN = 3.112 Å, cAlN = 4.982 Å are used. For the elastic constants, C13 = 103 GPa, C33 = 405 GPa for GaN [13 = 108 GPa, C33 = 373 GPa for AlN [xGa1−xN layer, then use these initial For the GaN and AlN lattice constant, a for GaN  and C13  for AlN  are usedxGa1−xN growth, compressive strain accumulated. Finally, during the GaN growth, compressive strain increases at first then decreases due to the dislocation relaxation. Given that the GaN thicknesses and dislocation densities are almost the same for the wafer centre and edge, an equal amount of compressive strain accumulated in GaN layers and was consumed by dislocations during the growth procedure for wafer centre and edge. However, cracks form only at the wafer edge after cooling down, so we can conclude that not enough initial compressive strain accumulated during the AlxGa1−xN transitional layers for the wafer edge leads to the cracks formed at the edge region when cooling down to room temperature.From 2 V−1 S−1, which is less than, but still comparable to the reported 2150 cm2 V−1 S−1 in Reference [To evaluate the whole HEMT epi quality, Hall measurements have also been conducted. The Ti/Al/Ti/Au (20/150/50/80 nm) alloys have been deposited as pads and thermally annealed in order to form Ohmic contacts. The Hall result shows that a carrier mobility of the two-dimensional electron gas (2DEG) is as high as 1600 cmeference . The hig2 ambient atmosphere for 30 s in order to form Ohmic contacts for the source and drain of the HEMT. Finally, a Ni/Au (50/150 nm) alloy is deposited in order to form a Schottky gate for the HEMT.To further verify the epi quality, GaN HEMT devices are also fabricated on the HEMT epi-wafer. The fabrication process is described as below: A 300 nm depth mesa is etched down to define the active region for the HEMT by inductive coupled plasma (ICP) etching, then the metal stack of Ti/Al/Ti/Au (20/150/50/80 nm) is deposited and then annealed under a 850 °C Ngs = 1 V and Vds = 8.8 V and specific-on resistance of 0.427 Ω·mm2, comparable to the reported value of 688 mA/mm at Vds = 9 V in Reference [eference , considexGa1−xN transitional layers. The compressive strain of GaN on the Al0.35Ga0.65N and Al0.17Ga0.83N layers on the AlN buffer is good enough to compensate the tensile strain between GaN and silicon. As a result of a high crystal quality AlN buffer layer, high quality GaN HEMTs on silicon with a mobility of 1600 cm2 V−1 S−1 and a current density of 290 mA/mm for 120 µm-gate HEMTs have been achieved.To conclude, we have achieved a crack-free GaN HEMT epi-wafer grown on silicon by properly tuning two Al


These studies have revealed sulfide, pH, and temperature are the main constraints on phototrophy. However, the taxonomic and physiological diversity of anoxygenic phototrophs suggests that, within these constraints, specific geochemical parameters determine the distribution and activity of individual anoxygenic phototrophic taxa. Here, we report the recovery of sequences affiliated with characterized anoxygenic phototrophs in sites that range in pH from 2 to 9 and in temperature from 31°C to 71°C. Transcript abundance indicates anoxygenic phototrophs are active across this temperature and pH range. Our data suggest sulfide is not a key determinant of anoxygenic phototrophic taxa and underscore a role for photoheterotrophy in terrestrial geothermal ecosystems. These data provide the framework for high-resolution sequencing and  Proteobacteria are more abundant at lower pH and lower temperature, while phototrophic Chloroflexi are prevalent in circumneutral to alkaline springs. In contrast to previous studies, our data suggest sulfide is not a key determinant of anoxygenic phototrophic taxa. Finally, our data underscore a role for photoheterotrophy (or photomixotrophy) across geochemical gradients in terrestrial geothermal ecosystems.Extant anoxygenic phototrophs are taxonomically, physiologically, and metabolically diverse and include examples from all seven bacterial phyla with characterized phototrophic members. pH, temperature, and sulfide are known to constrain phototrophs, but how these factors dictate the distribution and activity of specific taxa of anoxygenic phototrophs has not been reported. Here, we hypothesized that within the known limits of pH, temperature, and sulfide, the distribution, abundance, and activity of specific anoxygenic phototrophic taxa would vary due to key differences in the physiology of these organisms. To test this hypothesis, we examined the distribution, abundance, and potential activity of anoxygenic phototrophs in filaments, microbial mats, and sediments across geochemical gradients in geothermal features of Yellowstone National Park, which ranged in pH from 2.2 to 9.4 and in temperature from 31.5°C to 71.0°C. Indeed, our data indicate putative aerobic anoxygenic phototrophs within the IMPORTANCE There is a long and rich history of literature on phototrophs in terrestrial geothermal springs. These studies have revealed sulfide, pH, and temperature are the main constraints on phototrophy. However, the taxonomic and physiological diversity of anoxygenic phototrophs suggests that, within these constraints, specific geochemical parameters determine the distribution and activity of individual anoxygenic phototrophic taxa. Here, we report the recovery of sequences affiliated with characterized anoxygenic phototrophs in sites that range in pH from 2 to 9 and in temperature from 31°C to 71°C. Transcript abundance indicates anoxygenic phototrophs are active across this temperature and pH range. Our data suggest sulfide is not a key determinant of anoxygenic phototrophic taxa and underscore a role for photoheterotrophy in terrestrial geothermal ecosystems. These data provide the framework for high-resolution sequencing and in situ activity approaches to characterize the physiology of specific anoxygenic phototrophic taxa across a broad range of temperatures and pH. Cyanobacteria are abundant in alkaline hot springs  across a diurnal cycle chlorophylls (BChls or Chls) to capture solar energy and convert it into chemical energy . Anoxyged AsO33− . Anoxyged AsO33− . Phototrxotrophs –14. Someotrophs ––17. Two bacteria , 15–17. al cycle .There is a long and rich history of literature on phototrophs in terrestrial geothermal springs where reduced substrates, e.g., hydrogen, hydrogen sulfide, and sometimes ferrous iron, to support anoxygenic phototrophs, are typically abundant. Sequences affiliated with anoxygenic phototrophs have been recovered from terrestrial hot springs in Yellowstone National Park (YNP) , 19–28. 19–bchY and chlL/bchL). bchY encodes a component of chlorophyllide (Chlide) oxidoreductase, which reduces the B-ring of Chlide a  , revealing fine-scale interactions in phototrophic mats and ecological diversification in cyanobacteria  , 42, 43.hlL/bchL , 44–46, Chlide a –49.Here, we examined the distribution, abundance, and potential activity of putative anoxygenic phototrophs across geochemical gradients in geothermal features of YNP, ranging in pH from 2.2 to 9.4 and in temperature from 31.5°C to 71.0°C. Within this framework, we hypothesized that biotic and abiotic factors, including pH, temperature, and sulfide, would select for specific anoxygenic phototrophic taxa due to key differences in the physiology of these organisms.in situ microcosms to inform the range of temperature, pH, and sulfide concentration where anoxygenic phototrophs are abundant and active. We used 16S rRNA gene amplicon sequencing and stable isotopes of C and N to identify specific taxa of putative anoxygenic phototrophs and their potential metabolism within this temperature and pH range. From these data, we assessed biotic and abiotic factors that affect the distribution of specific anoxygenic phototrophic taxa.Terrestrial geothermal springs provide an exceptional opportunity to study diverse microbes, including phototrophs, and their potential metabolisms across geochemical gradients  and microbial mats/biofilms (G to L) sampled in this study. Download Copyright © 2019 Hamilton et al.2019Hamilton et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00498-19.8TABLE S113C values, dissolved organic carbon (DOC), and DOC δ13C values are also provided. Na, K, Ca, and Mg were determined via inductively coupled plasma optical emission spectroscopy. Sample type is indicated: fil, filament or mat, microbial mat/biofilm. Des., sample designator; Sulf, sulfide; nd, not determined; bdl, below detection limit. Detection limits: sulfide, 5 μg/liter S2−; Fe2+, 20 μg/liter. Library names for the 16S rRNA amplicon libraries included in the present study are provided. All sequence data, including raw reads with quality scores and mapping data for this study, are available from the NCBI Sequence Read Archive (SRA) database under with the BioProject number PRJNA513338. Download Table S1, XLSX file, 0.02 MB.GPS coordinates, pH, conductivity, temperature, aqueous geochemistry, and stable isotope analyses. Total carbon and nitrogen and stable isotope analysis results for biomass and dissolved inorganic carbon (DIC), DIC δCopyright © 2019 Hamilton et al.2019Hamilton et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the bchY, a proxy for anoxygenic phototrophs.(i) Distribution of  Anoxygenic phototrophs occur throughout temperature and pH ranges similar to those of oxygenic phototrophs, but anoxygenic phototrophs are less prevalent at low pH. In this study and both previous and unpublished work . This dbchY transcripts by qRT-PCR.(ii) Abundance of  We observed a range in bchY transcript abundance from 3.2 × 103 to 1.5 × 108 copies per gram of dry mass (gdm) (bchY transcripts were more abundant at higher pH (pH >6) , except for Sylvan Springs Area 4 (SSA4) and Geyser Creek Area 2 (GCA2)  (bchY transcript abundance and pH as well as temperature (P > 0.05)  and in samples with up to 19 μM sulfide (bchY transcripts were observed in Rabbit Creek Area 1 (RCA1) and RCA2  and One Hundred Springs Plain 2 (OHSP2) . Despite the low abundance of bchY transcripts, these conditions (in terms of pH and temperature) are suitable for photosynthesis based on the distribution of chlL/bchL and bchY  . In gene (pH >6) . Most sa2 (GCA2) . At 71.0to 73°C) . Based o > 0.05) . In cont sulfide . The lowand bchY  and the hemistry . An incrportance . In circportance , 56, 57.(iii) Anoxygenic photosynthesis activity. We employed in situ microcosms (in the presence of NaH13CO3) to assess assimilation of inorganic carbon (autotrophy) in a subset of samples (n = 17) across the range of pH and temperature of our sample sites. Light-dependent inorganic carbon assimilation (photoautotrophy and photosynthesis) was significantly higher than that of dark assimilation (chemoautotrophy) in all sites except Sentinel Meadows 1 (SM1)  , suggest 1 (SM1) . Assumin 1 (SM1) . We obse 1 (SM1) , indicat 1 (SM1) . These s 1 (SM1) . Similar10.1128/mSystems.00498-19.9TABLE S2P values for t tests for each comparison of C uptake rates. P values in boldface are below 0.05. All carbon isotope values are given as absolute values. All samples have 13C-labeled bicarbonate added. Light, labeled bicarbonate added; DCMU, DCMU added; Dark, aluminum foil wrapped. St. dev., standard deviation of assays performed in triplicate. Treat., amendment. Site and area designations are provided in Table S2, XLSX file, 0.01 MB.Rates of carbon assimilation and Copyright © 2019 Hamilton et al.2019Hamilton et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (iv) Diversity of anoxygenic phototrophs. Alpha diversity patterns for the overall microbial community and the putative phototroph fraction of the community are similar: the acidic sites had slightly higher alpha diversity values than alkaline sites independent of the temperature when either the full data set  Alpha diversity metrics for the complete data set. (B) Alpha diversity statistics for sequences affiliated with characterized phototrophs. Physicochemical data and sample designations provided in Copyright © 2019 Hamilton et al.2019Hamilton et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00498-19.4FIG S4FIG S4, PDF file, 0.3 MB.16S rRNA gene sequences binned at the phylum (A), class (B), and genus (C) levels recovered from samples across a range of geochemical spaces in YNP. Site and area designations are provided in Copyright © 2019 Hamilton et al.2019Hamilton et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Gemmatimonadetes and Firmicutes were rare. OTUs affiliated with characterized phototrophs within the Proteobacteria were more prevalent (but not abundant) at low pH (<6.4) and lower temperature (<58°C) (Chlorobi (in the phylum Bacteriodetes) were more abundant in sites with pH >6.4 and temperature of >58°C  were recovered from sites across the range of temperatures and pH  . Sequencof >58°C . We obsene sites . SequencSM sites . We onlyeratures . Sequencs and pH . There aChloroflexus and Roseiflexus were recovered from circumneutral to alkaline sites  or anoxic (C. aggregans) conditions    71). In. InChlorEctothiorhodospira, a genus of anoxygenic purple phototrophs within the Gammaproteobacteria, were present in SSA1, SSA2, and SSA3  from SSA1 and SSA3  and is the most acidophilic anaerobic anoxygenic phototrophic purple bacterium characterized to date  and temperature (B) groups are plotted over boxplots displaying the median and quartiles of each group (OTUs with relative abundance of <1% were omitted to avoid overplotting). Download FIG S5, PDF file, 0.1 MB.Relative abundance of OTUs affiliated with Copyright © 2019 Hamilton et al.2019Hamilton et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Proteobacteria, sequences affiliated with known phototrophic Proteobacteria were rare in sites above pH 6.4 and sites with temperatures of >58.0°C  only in sites where light-dependent photoassimilation was also observed. This observation is consistent with other studies of circumneutral geothermal systems, including high-spatial-resolution characterization of mats in the Lower Geyser Basin , but hasbacteria . These sn waters . Sequencn waters . These sChloroflexi and Oxyphotobacteria were the most abundant putative phototrophs. Cooccurrence of Chloroflexi and Cyanobacteria has been reported in microbial mats between a 50°C to 65°C temperature range in terrestrial hot springs in Thailand , which is consistent with other hot spring studies  and White Creek Area (WCA) had δ13C values that ranged from −20.00 to −17.87‰ and −22.75 to −20.55‰, respectively. The three samples from SM had δ13C values of −19.07, −16.19, and −14.64‰. Two samples from the Mud Volcano Area (MVA), Greater Obsidian Pool Area 1 (GOPA1) and GOPA2, had the lightest biomass δ13C values, at −25.37 and −25.87‰, respectively. These large ranges in biomass δ13C values could be attributed to fractionation from DIC sources varying in δ13C values, with measured δ13C values of DIC ranging from −3.74 to 4.30‰. Thus, calculating fractionation factors is valuable for removing this effect.We did not observe any general trends between biomass δ15N values close to the value of atmospheric air (0‰ ± 2‰) indicate most of the N incorporated in the biomass is sourced from nitrogen fixation, while more positive values are associated with limited fixed N from allochthonous sources and/or volatilization of NH3 and more negative values with subsurface boiling concentrating isotopically light NH3  by calculating the difference between the biofilm δ13C and DIC δ13C. Δ13C values ranged from −27.01 to −9.96‰   + OH−).We examined carbon isotope fractionation (Δo −9.96‰ . In siteynthesis  or with ynthesis . In geneand GCA) . Abioticarbonate , 84, in rocesses . At pH 710.1128/mSystems.00498-19.6FIG S62−). Download FIG S6, PDF file, 0.2 MB.Carbon isotopic fractionations in biomass mass collected for the present study. Values are plotted against pH (A) and temperature (B) and with increasing pH (C), increasing temperature (D), and increasing sulfide concentration (E). n.d., not detected; b.d.l., below detection limit  of organic carbon in the deeper mat layers could become an increasingly important source of DIC for anoxygenic phototrophs. Thus, a thick mat generated by oxygenic phototrophs would create increasing niche space for anaerobic heterotrophs and anoxygenic photoautotrophs, and if this is the case, then we would expect to see a cooccurrence of oxygenic phototrophs and anoxygenic phototrophs. A systematic study of these biotic and abiotic factors is necessary to determine the cause for variation in the signal and the positive relationship observed between rates of anoxygenic photosynthesis and increasing Δ13C. Regardless, δ13C and Δ13C signals reported in this study are consistent with those preserved in the rock record , it is harder for DIC to diffuse into the mat. Thus, the release of CObchY genes and recover bchY transcripts and 16S rRNA gene sequences affiliated with phototrophic Cyanobacteria and anoxygenic phototrophs from sites below pH 6  values between rarefied abundances of the top 50 most abundant OTUs classified at the class level and environmental variables. Copyright © 2019 Hamilton et al.2019Hamilton et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Cyanobacteria and Chlorobi  (4(T) tend to be low (<100 μM), due in part to the equilibration of aqueous NH4+(aq) with NH3(g) (pK 7.6 at 90°C) and the subsequent volatilization of NH3(g)  , despitef NH3(g) , 82. As uires Mo , 91.P < 0.01) related to the composition of microbial communities in our samples  indicated that, in addition to pH and temperature, potassium and conductivity were statistically significant environmental factors  with either temperature or pH . Sulfide3–from YNP ), Chlora sulfide ), and “C sulfide ).2S could be an electron donor. Chloroflexus can grow photoautotrophically with sulfide or hydrogen as an electron donor for carbon fixation via the 3HPB cycle, and Chlorobi can use sulfide as an electron donor for carbon fixation via the rTCA pathway. In general, circumneutral and alkaline sites exhibited higher rates of oxygenic and anoxygenic photosynthesis in higher temperature sites. Above a threshold of ∼1 μg C/g biomass C/h, there was an apparent correlation of increasing fractionation with increasing rates of anoxygenic photosynthesis  and the rTCA cycle (in autotrophic Chlorobi) typically generate a much smaller fractionation; however, these values can vary by temperature  and the Geyser Creek Area  in the Gibbon Geyser Basin (GGB); Sentinel Meadows , Boulder Geyser , and the White Creek Area  in the Lower Geyser Basin (LGB); the Rabbit Creek Area  in the Midway Geyser Basin (MGB); the Greater Obsidian Pool Area  in the Mud Volcano Area (MVA); and the One Hundred Springs Plain  area of Norris Geyser Basin (NGB). GPS coordinates of sample sites are provided in Samples were collected from geothermal features in Yellowstone National Park in October of 2015, June of 2016, and July of 2017. Geothermal features were targeted to capture a range of temperatures and pH as well as other geochemical parameters, including sulfide (see n = 3 for each site) and C and N content and δ13C and δ15N determination (n = 1 for each site) were collected using flame-sterilized spatulas or forceps. Samples were placed in sterile 2.0-ml vials and immediately frozen on dry ice and stored at –80°C until nucleic acid extraction or processing for C, N, δ13C, and δ15N analysis.Samples for nucleic acid extraction . Water samples were filtered through 0.2-μm polyethersulfone syringe filters  and analyzed for cation concentration , anion concentration (Cl− and SO42−), trace element concentration , dissolved inorganic carbon (DIC) concentration and δ13C value, and dissolved organic carbon concentration and δ13C value, as described previously , and conductivity was measured with a YSI 30 conductivity meter and probe . Sulfide, Feeviously –103. FieFor ion chromatography analysis of anions, water was filtered into 15-ml centrifuge tubes (presoaked in 18.2 MΩ/cm deionized [DI] water) and stored at 4°C until analysis. Major anions were determined using a Dionex ICS2500 ion chromatography (IC) system  by the Earth and Environmental Systems Institute (EESI) , the STAR Lab at the Ohio State University, or the Analytical Geochemistry Laboratory in the Department of Earth Sciences at the University of Minnesota as described previously.3  followed by triple rinsing with 18.2 MΩ/cm DI water) and immediately acidified with 400 μl of concentrated OmniTrace Ultra concentrated nitric acid . Samples were stored at 4°C until analysis. Analysis of cations was conducted via a Thermo X-Series II quadrupole collision cell technology-enabled inductively coupled plasma mass spectrometer  by the EESI at the Pennsylvania State University, the STAR Lab at the Ohio State University, or the Analytical Geochemistry Laboratory in the Department of Earth Sciences at the University of Minnesota.Samples for cation analysis were filtered into acid-washed 15-ml centrifuge tubes  . Raw delta values were converted to final values using laboratory standards  calibrated against standards NBS-19 and L-SVEC.Four-milliliter samples for DIC concentration analysis were filtered into Labco Exetainers  preflushed with helium. Samples were stored cap-side down at 4°C until analysis. DIC concentrations and δ13C-labeled DIC, triple rinsed with 18.2 MΩ/cm deionized water, and then dried (60°C for 3 days). Natural abundance samples were not treated with acid. Samples were ground/homogenized with a cleaned mortar and pestle , weighed, and placed into tin boats, sealed, and submitted to the UC Davis Stable Isotope Facility for analyses.C and N stable isotope signals of biomass were determined via a Costech Instruments elemental analyzer (EA) periphery connected to a Thermo Scientific Delta V Advantage IR-MS at the UC Davis Stable Isotope Facility. Microcosm samples were thawed, and biomass was rinsed with 1 M HCl to remove any extra −1] × 1,000, expressed in delta notation (δ) and reported as per mil (‰). Carbon isotopic values are reported as isotopic ratios of 13C to 12C from the equation [(13C/12C of sample)/(13C/12C of standard)−1] × 1,000 and expressed in delta notation (δ13C). δ13C values are reported using the Vienna Pee Dee Belemnite (VPDB) standard. The difference between DIC δ13C values and biomass δ13C values is expressed as Δ13C and represents the fractionation of 13C from the source inorganic carbon (DIC δ13C) and biomass δ13C values. Nitrogen isotopic values are reported as isotopic ratios of 15N to 14N from [(15N/14N sample)/(15N/14N standard)−1] × 1,000, expressed in delta notation (δ15N). The standard for reporting nitrogen isotopic values is atmospheric N2.All isotopic data are reported as isotope ratios, relative to standards of known value, using the equation  . Assays were stopped by flash freezing vials on dry ice. Incubation duration times were recorded in the field. All vials were stored at –80°C until processed (described above). Reported values of 13C-labeled DIC uptake (carbon fixation rates) reflect the difference in uptake between the biomass in the assays that received NaH13CO3 and the natural abundance biomass samples. Using the organic carbon content, the uptake rate was calculated from the total micrograms of C taken up divided by the grams of organic C per gram of sediment, and that was divided by the number of hours of incubation . Carbon assimilation rates are provided in 13C uptake rates, one-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons between treatments was conducted using Tukey’s honest significant difference (HSD) within the R software package . Mean rates with P values of <0.05 were considered significantly different (Microcosms were initiated by addition of NaHifferent . Our micifferent .https://jgi.doe.gov/user-programs/pmo-overview/protocols-sample-preparation-information). Briefly, RNA was incubated at 37°C in the presence of DNase I and DNase I reaction buffer  for 30 min, followed by a cleanup step using the RNeasy MinElute cleanup kit , modified according to the JGI protocol. Concentrations of DNA and RNA were determined using a Qubit 3.0 fluorometer , and equal volumes of each extraction were pooled prior to sequencing or qRT-PCR analyses. DNA was extracted from the filter used for the field blank water sample as a negative DNA extraction control. No DNA was detected in any of the filter controls, and sequencing failed to generate amplicons (see below for amplicon sequencing details). RNA extracts were screened for the presence of contaminating genomic DNA by performing a PCR using ∼1 ng of RNA as the template and bacterial 16S rRNA gene primers 1100F and 1492R as described previously  according to the manufacturer’s instructions. Total RNA was extracted from triplicate ∼250-mg samples using a RNeasy power biofilm kit , which includes a DNase treatment, according to the manufacturer’s instructions. A second DNase treatment was performed according to the Joint Genome Institute (JGI) Sequencing Technology DNase treatment protocol (eviously .remove.rare” function within mothur.Amplicons were sequenced using MiSeq Illumina 2× 300-bp chemistry at the University of Minnesota Genomics Center (UMGC). We targeted the V4 hypervariable region of bacterial and archaeal 16S small subunit rRNA gene using the modified primers 515F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3′). Amplicon libraries were created by UMGC by following their improved protocol for library preparation, which enables detection of taxonomic groups that often go undetected with existing methods . Each saChloroflexi, Cyanobacteria, and Gemmatimonadetes; class Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Chlorobia; and family Heliobacteria. We recognize that not all representatives from these groups are known phototrophs and have provided higher taxonomic resolution in the supplemental information.Following removal of chimeras and singletons, sequences were binned into operational taxonomic units (OTUs) based on a sequence similarity of 97.0% for archaea and bacteria. OTUs assigned to mitochondria or chloroplast were removed and not included in the rest of the analyses. OTUs with unassigned taxonomy by mothur were further refined using BLASTN and GreenGenes databases using BLCA and assigned taxonomy when possible . FollowiRank abundance plots were generated in R, version 3.4.3 , using tmetaMDS” and “envfit,” respectively . Linear regression models between relative abundance of OTUs and pH or temperature were built using the “stat_fit” method in ggplot2. Spearman correlation R values were plotted using ComplexHeatmap  were calculated and visualized in Cytoscape , a Spearman rank correlation test was conducted using the base R package. 13C assimilation rates were plotted across pH, temperature, and sulfide space, and Spearman rank correlation lines were drawn and plotted with correlation coefficients (R) and P values using the ggpubr package. The association between bchY transcript abundance and environmental parameters  were calculated and plotted using the same methods.All statistical analyses were carried out in R, version 3.4.3 . Rarefacectively   indicates the lowest sample size for the site SSA2. Site and area designations are provided in Copyright © 2019 Hamilton et al.2019Hamilton et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the bchY was assayed by amplifying a 500-bp fragment of bchY using bchY_fwd (5′-CCNCARACNATGTGYCCNGCNTTYGG-3′ and bchY_rev (5′-GGRTCNRCNGGRAANATYTCNCC-3′) as described previously (chlL/bchL was assayed by amplifying a 390-bp fragment of chlL/bchL using chlLbchL-54F (5′-CARATYGGHTGYGAYCCNAARCAYGA-3′) and chlL*bchL-167R (5′-AAYGRYTTYGAYDSBWTHTTYGC-3′), as described previously   , 43. bchhlL/bchL , 44, 45.bchY transcripts was assayed by amplifying a 500-bp fragment of bchY using bchY_fwd and bchY_rev as described previously  database under the BioProject number


EGFR mutational status as a model for genetic profile. Clonogenic assays of EGFR mutant (n = 6) and wild-type (n = 9) non-small cell lung carcinoma (NSCLC) cell lines were performed independently by two oncologists. Clonogenic survival parameters SF2, SF4, SF6, SF8, mean inactivation dose (MID), D10, D50, α, and β were obtained using the linear quadratic model. The differences in the clonogenic survival parameters between the EGFR mutant and wild-type cell lines were assessed using the Mann–Whitney U test. As a result, for both datasets, the p values for SF2, SF4, D50, α, and α/β were below 0.05, and those for SF2 were lowest. These data indicate that a genetic profile of NSCLC cell lines might be predictive for their radiation response; i.e., EGFR mutant cell lines might be more sensitive to low dose- and low fraction sized-irradiation.In the era of precision medicine, radiotherapy strategies should be determined based on genetic profiles that predict tumor radiosensitivity. Accordingly, pre-clinical research aimed at discovering clinically applicable genetic profiles is needed. However, how a given genetic profile affects cancer cell radiosensitivity is unclear. To address this issue, we performed a pilot in vitro study by utilizing  Radiation therapy is one of the most important treatments for cancer. In the clinical practice of definitive radiation therapy, the doses required for clinical tumor control vary widely even for tumors arising in the same anatomical sites. For example, an integrated analysis of 50 single-institution studies performed by Okunieff et al. revealed that the dose required for 50% clinical tumor control ranged from 21.4 to 90.3 Gy for breast cancer, 50.4 to 83.4 Gy for supraglottic cancer, and 24.3 to 64.4 Gy for cervical cancer .EGFR mutant and wild-type cell lines by non-parametric two-sided Mann–Whitney U test. Correlation of the survival data between datasets was examined by Spearman’s rank order test. Differences were considered statistically significant at p < 0.05. Analyses were performed using Prism8 .Clonogenic survival parameters were compared between