Balamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections. Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris—a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions—have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris. The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections. Balamuthia mandrillaris causes rare but life-threatening infections of the central nervous system (CNS), termed balamuthia or granulomatous amoebic encephalitis (GAE)  , 2. Onseis (GAE) , 3. Presis (GAE) , 3. Thesis (GAE) –6. Infecs (GAE) –, 8. Thusstimated .B. mandrillaris have not been performed, free-living B. mandrillaris amoebae have been isolated from water, soil, and dust across all continents   . The modhe brain . All of crobials –47 and trobials ––50. Suchobials ––.B. mandrillaris is almost always fatal, and no specific and highly successful treatment regimen is known  exhibit amoebicidal or amoebistatic activity  exhibits amoebicidal activity at low micromolar concentrations, well within the range of the estimated plasma concentrations achieved with recommended oral dosing  and screened 2,177 clinically approved compounds for reduction of trophozoite viability following 72 h of treatment at 20 µM. Compounds with high percent inhibition (∼40% or above) and B-score (∼5 or above) were nominated for secondary screening  values indicated that only two compounds, pentamidine isethionate and nitroxoline, demonstrated adequate potency against B. mandrillaris without high toxicity to human cells. Since the amoebistatic activity of pentamidine has previously been described  List of compounds meeting loosely applied criteria of a B-score of approximately 5 or greater and inhibition of approximately 40% or greater. The class and delivery method are annotated for each compound. Compounds that are classified as veterinary or are available only for external/topical administration are highlighted in yellow. Nitroxoline, the top hit compound based on B-score and percent inhibition, is highlighted in red. (C) Secondary screening of hit compounds to determine potency against B. mandrillaris trophozoites and toxicity to HFF-1 and H4 human cell lines. All IC50 values were approximated from 8-point dose-response curves. IC50 values highlighted in red are outside a desirable range for a hit compound, as they indicate either low potency against B. mandrillaris (IC50 > 30 µM) or high toxicity to human cells (IC50 < 1 µM). Nitroxoline and pentamidine isethionate were the only compounds that inhibited B. mandrillaris trophozoite viability at concentrations similar to or lower than concentrations that were toxic to human cells. Download Table S1, XLSX file, 0.3 MB.Tabulation of raw data from primary and secondary drug screens. (A) A primary screen of 2,177 clinically approved compounds measured the decrease in Copyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro anticancer activity, and free-radical metabolites are thought to be involved in cell death  is as a metal chelator that disrupts biofilm formation . Nitroxoll death . To testactivity . As demo10.1128/mBio.02051-18.5TABLE S250 values, and dose-response curve-fitting parameters for nitroxoline and all nitroxoline analogs tested for activity against B. mandrillaris. IC50, R2, and Hill slope values were calculated based on sigmoidal curves fitted to data from 8-point dose-response experiments performed in triplicate. Compounds 2, 4 through 12, and 14 were the most potent inhibitors of B. mandrillaris, most likely due to the presence of an 8-position hydroxyl group or bidentate ligand on the quinoline ring, each of which is predicted to be necessary for metal binding. Download Table S2, PDF file, 0.4 MB.Structures, ICCopyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. mandrillaris infections, we compared the in vitro performance of nitroxoline to that of pentamidine isethionate, miltefosine, and azithromycin, three drugs recommended by the CDC and commonly used in treatment of GAE (B. mandrillaris trophozoites and the toxicity to different human cell types using the following cell lines: HFF-1 (fibroblast), H4 , U87 , HEK-293T (kidney), and Hep-G2 (liver). Nitroxoline was the most potent inhibitor of B. mandrillaris trophozoites, with an IC50 of 2.84 µM compared to IC50 values of 9.14 µM, 63.23 µM, and 244.10 µM for pentamidine, miltefosine, and azithromycin, respectively  for all cell lines tested. The average Log10 selectivity index (CC50 for human cell toxicity/IC50 for B. mandrillaris inhibition) of nitroxoline across all cell lines was 0.832, compared to 0.049, −0.102, and −0.409 for pentamidine, miltefosine, and azithromycin, respectively  values calculated from dose-response experiments. IC50 and CC50 values were calculated from dose-response curves generated by measuring the viability of cultured cells 72 h after drug treatment. Cytotoxicity of compounds was measured in U87, H4, HEK-293T, HFF-1, and Hep-G2 cell lines. All experiments were performed in triplicate. SI values for each drug show the ratio of the CC50 value for a given cell line to the IC50 value for B. mandrillaris trophozoites. An SI value greater than 1 indicates that the drug inhibits B. mandrillaris trophozoites at lower concentrations than were seen with the human cell line used for comparison . Nitroxoline is the most potent inhibitor (lowest IC50) of both forms of B. mandrillaris and has the highest SI for all of the cell lines, with a mean SI of 6.79. Download Table S3, PDF file, 0.2 MB.Summary of ICCopyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. mandrillaris trophozoite viability, we also observed a general increase in the ratio of cysts to trophozoites correlated with increasing concentrations of some drugs. We investigated the propensity of nitroxoline, pentamidine isethionate, miltefosine, and azithromycin to induce encystment of B. mandrillaris by counting the number of cysts and trophozoites in culture samples following 72 h of treatment with different drug concentrations. We observed a dose-dependent reduction in the number of trophozoites in the population for all four drugs. Nitroxoline and pentamidine isethionate caused an increase in both the total number and the proportion of cysts in the population, while no substantial number of cysts was observed at any concentration of miltefosine or azithromycin  for 20 h before media were changed to remove drug or vehicle. Tissues were cultured for 4 days and then evaluated for damage by microscopic examination , uninfected primary brain tissue section 4 days after the start of culture. No significant movement of cells is observed, and a clearly defined edge marks the border of the tissue. Download Copyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02051-18.8MOVIE S2B. mandrillaris-infected primary brain tissue section 4 days after inoculation of amoebae (2 min duration). The movie is representative of two biological replicates. Highly motile B. mandrillaris trophozoites are observed throughout the field of view, and the tissue edges are not discernible due to loss of host cell density. Download Movie S2, AVI file, 4.7 MB.Representative movie showing the edge of an untreated (vehicle only), Copyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02051-18.9MOVIE S3B. mandrillaris-infected primary brain tissue section 4 days after inoculation of amoebae (10 min duration). The movie is representative of two biological replicates. Highly motile B. mandrillaris trophozoites are observed throughout the field of view, and the tissue edges are not discernible due to loss of host cell density. Download Movie S3, AVI file, 20.1 MB.Representative movie showing the edge of an untreated (vehicle only), Copyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02051-18.10MOVIE S4B. mandrillaris-infected primary brain tissue section 4 days after inoculation of amoebae. The tissue was treated with 35 µM nitroxoline simultaneously with inoculation of amoebae. Nonmotile B. mandrillaris cysts are observed individually primarily at the edge of the tissue and outside the tissue. The tissue edges are intact and do not show signs of host cell destruction by B. mandrillaris. Download Movie S4, AVI file, 3.3 MB.Representative movie showing edges of nitroxoline-treated, Copyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Balamuthia mandrillaris is almost always fatal  of up to approximately 30 µM (5.6 mg/liter) (–50 for B. mandrillaris trophozoites in vitro (B. mandrillaris frequently causes necrotizing vasculitis in the CNS with extensive blood-brain barrier (BBB) breakdown, which may affect the bioavailability of systemically administered drugs in the CNS of patients actively suffering from GAE  –65, whicin vitro . Althougin vitro . In addifrom GAE , 57. Furfrom GAE , 72. Whiin vitro results presented in this study strongly indicate that nitroxoline warrants further investigation as a potential treatment for B. mandrillaris infections. As there were many variables that were untested in our study that may affect the efficacy of nitroxoline in vivo, future work will ideally focus on providing in vivo validation of the amoebicidal activity of nitroxoline. In particular, in vivo studies will determine if efficacious concentrations of nitroxoline reach the relevant tissues as well as provide comparative evidence for amoebicidal activity with respect to currently recommended compounds. We note that nitroxoline is an approved compound in several countries, with an established safety profile , and 10 mM HEPES buffer. HFF-1 cells (ATCC SCRC-1041) were cultured in DMEM containing 15% (vol/vol) FBS, 2 mM l-glutamine, 100 U/ml penicillin/streptomycin, and 10 mM HEPES buffer (Gibco). All mammalian-cell incubation steps were carried out at 37°C with 5% CO2. All cell lines were tested for mycoplasma contamination using a Lonza Mycoalert mycoplasma detection kit (Lonza).Hep-G2 (ATCC HB-8065), U87 , H4 (ATCC HTB-148), and HEK-293T (ATCC CRL-3216) cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% (vol/vol) fetal bovine serum (FBS) (Gibco), 2 mM Balamuthia mandrillaris (ATCC PRA-291) was maintained axenically in 150-cm2 flasks (Corning) containing modified Cerva’s medium (axenic medium) with the following formulation: 20 g of Bacto Casitone (Difco), 68 ml of 10× Hanks’ balanced salt solution (Gibco), 10% fetal bovine serum, and 1× penicillin-streptomycin (200 IU/ml to 200 μg/ml) (B. mandrillaris resulted in 2 × 105 to 3 × 105 amoebae/ml in log phase. Routine subculturing was performed every 3 to 4 days by transferring 5 ml of amoebae into 15 ml of fresh axenic media.0 μg/ml) . Axenic B. mandrillaris was induced by galactose exposure . Amoebae were cultured in the induction medium until trophozoites were no longer observed (approximately 10 days). Galactose-induced cysts transition back to trophozoites after approximately 3 days of incubation in galactose-free axenic medium. Therefore, all assays with cysts were completed in the induction medium. To quantify amoebae for use in experiments, actively growing trophozoites or recently induced cysts were centrifuged at 3,000 rpm for 5 min, resuspended in axenic medium, and counted with a disposable hemocytometer . All incubation steps for B. mandrillaris growth were carried out at 37°C with 5% CO2.Following the findings of a previous study, encystment of exposure . B. mandB. mandrillaris amoebae were resuspended in axenic medium and distributed into opaque 384-well plates (Corning) at a density of 3,000 amoebae per well using a BioMek NX liquid handler. Negative-control wells were treated with vehicle only (0.2% DMSO in axenic medium), and positive-control wells simulating total destruction of amoebae were seeded with amoeba lysate generated by 3 consecutive freeze-thaw cycles. Following 72 h of incubation, 30 µl of CellTiter Glo reagent  was added to each well. The luminescence was measured with a Promega GloMax-Multi+ plate reader at 2, 4, and 8 h after CTG addition. The percent inhibition of B. mandrillaris was calculated based on the CTG luminescence measurements of treated wells relative to both positive and negative controls using the following equation: percent inhibition = 100 − 100*[(test well intensity − positive-control intensity)/(negative-control intensity − positive-control intensity)]. The B-score, representing a plate-based statistical approach for correcting row, column, and edge effects, was also calculated for each compound in the library  at the UCSF, was completed at 20 µM in 0.2% DMSO. All 2,177 drugs in the library were stored as 10 mM stocks dissolved in 100% DMSO (Sigma-Aldrich) at −20°C.  library . Raw lum library . Hit com library . CompounB. mandrillaris trophozoites, HFF-1 human fibroblast cells, and H4 human neuroglioma cells. Four 384-well plates were prepared prior to addition of drug as follows: one opaque plate (Corning) seeded with 3,000 HFF-1 cells per well in 60 µl complete media, one opaque plate seeded with 2,000 H4 cells per well in 60 µl complete media, one opaque plate containing 30 µl axenic media, and one clear plate (Corning) containing 30 µl axenic media. Opaque plates were used to measure the viability of trophozoites in the CTG assay, and clear plates were used for microscopic examination of cyst formation. HFF-1 and H4 plates were seeded 24 h prior to drug addition. The selected primary screen hits were added from 10 mM DMSO stocks into wells of all four test plates to reach final concentrations ranging from 0.06 µM to 30 µM . Negative-control wells received concentrations of DMSO corresponding to the amount of DMSO in each tested drug well (reaching a maximum of 0.3% DMSO). B. mandrillaris trophozoites were resuspended in axenic media and added to the plates containing drug dilutions in axenic media at a density of 3,000 amoebae per well in a final volume of 60 µl media. All plates were incubated for 72 h. Throughout the incubation period, the clear-bottom B. mandrillaris plate was monitored for large-scale changes to population encystment in response to drug treatment. After the 72-h incubation, 30 µl of CTG reagent was added to all wells of the opaque assay plates and luminescence was measured with a Promega GloMax-Multi+ plate reader at 2, 4, and 8 h after CTG addition. IC50 values for inhibition of B. mandrillaris viability were determined using the GraphPad Prism 4-parametric sigmoidal curve-fitting model, with the bottom and top constraints set to 0 and 1, respectively  in a final volume of 100 µl. After incubation for 72 h, 50 µl of CTG was added to all assay wells. Luminescence was measured using a Promega GloMax Multi+ luminometer 2, 4, and 8 h after CTG addition. IC50 values were determined using the GraphPad Prism 4-parametric sigmoidal curve-fitting model with bottom and top constrains of 0 and 1, respectively.Nineteen commercially available analogs of nitroxoline were selected for structure-activity relationship (SAR) experiments based on variances in functional groups that may play a role in the observed mechanism of action . Homogenous populations of B. mandrillaris cysts generated by galactose induction were seeded at 4,000 amoebae per well into opaque 96-well plates. HFF-1 (fibroblast), H4 , U87 , HEK-293T (kidney), and Hep-G2 (liver) cells were seeded at 3,000 cells per well in opaque 96-well plates 24 h prior to addition of drug. Stocks of nitroxoline  were dissolved in 100% DMSO at 10 mM. Stocks of azithromycin , pentamidine isethionate , and miltefosine  were dissolved in water at 10 mM. Drug stocks were serially diluted in water to generate 12-point dilution series, which were then used to treat assay wells containing B. mandrillaris or human cells at final concentrations ranging from 0.39 µM to 400 µM in 100-µl total well volumes. Control wells were treated with vehicle (DMSO or water) at concentrations corresponding to the final vehicle concentrations in each drug dilution series. After incubation for 72 h, 50 µl of CTG was added to all assay wells. Luminescence was measured with a Promega GloMax-Multi+ plate reader 2, 4, and 8 h after CTG addition. All dose-response experiments were performed with at least three independent biological replicates. IC50 values were determined as previously described. To quantify encystment under treated and untreated conditions, amoebae in individual assay wells were resuspended thoroughly and the number of cysts and number of trophozoites in 10-µl samples were counted by the use of a hemocytometer . Cysts and trophozoites are morphologically distinct, making them simple to distinguish with bright-field microscopy (example images are shown in 10.1128/mBio.02051-18.1FIG S1B. mandrillaris trophozoites and cysts. (A) Example images show B. mandrillaris trophozoites in log-phase growth. Trophozoites are pleomorphic and can be elongated or generally rounded, often with highly branched pseudopodia. (B) Example images show B. mandrillaris cysts induced by galactose exposure. Cysts are spherical and generally smaller in diameter than trophozoites and can have visibly distinct layers. Some cysts show signs of vacuolization that may be indicative of cell death. Download FIG S1, PDF file, 3.3 MB.Example bright-field images of Copyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02051-18.2FIG S2B. mandrillaris. (A and B) Heat maps of treatment effect (percent reduction of B. mandrillaris trophozoite viability relative to untreated control) across dose matrices for nitroxoline in combination with miltefosine (A) and pentamidine isethionate (B). The viability of B. mandrillaris trophozoite populations was measured following 72 h of drug treatment in three independent replicates. (C and D) Heat maps of excess effect over highest single agent (EOHSA) showing the additional effect of dose combinations beyond the effect of the most active single agent in the combination (EOHSA = Ecombo − EHSA) across dose matrices for nitroxoline in combination with miltefosine (C) and pentamidine isethionate (D). For both drug pairs, mild antagonism  was observed for a small number of combinations, whereas most combinations produced a modest additive effect . No strong antagonism or synergy was observed between nitroxoline and miltefosine or pentamidine isethionate. Download FIG S2, PDF file, 0.3 MB.Efficacy of nitroxoline in combination with miltefosine and pentamidine isethionate for inhibition of Copyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02051-18.3FIG S3B. mandrillaris trophozoites and removed after 20 h. Four days after exposure to B. mandrillaris, untreated tissues showed widespread damage and loss of cell density whereas nitroxoline-treated tissues remained intact and appeared similar to uninfected tissues. Large numbers of B. mandrillaris trophozoites can be seen at the edges of untreated tissues, while only clusters of cysts are observed in nitroxoline-treated tissues. (C) Images representative of two biological replicates of human brain tissue explants fixed and stained with DAPI 4 days after exposure to the indicated conditions (two images per condition). The number of host cell nuclei was dramatically reduced in untreated, B. mandrillaris-infected tissues compared to uninfected tissues, whereas B. mandrillaris-infected tissues treated with nitroxoline show no apparent loss of nuclei. Download FIG S3, PDF file, 0.5 MB.(A and B) Bright-field images representative of two biological replicates for human brain tissue explants before and after exposure to the indicated conditions. Nitroxoline or vehicle was added simultaneously with Copyright © 2018 Laurie et al.2018Laurie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. mandrillaris trophozoites were seeded into opaque 96-well plates (Corning) at a density of 4,000 amoebae per well and then treated with concentrations of nitroxoline that differed across the rows of each plate and with concentrations of either miltefosine or pentamidine isethionate that differed down the columns to generate 7-by-7 dose combination matrices. Final drug concentrations ranged from 0.5 to 10 µM, from 10 to 100 µM, and from 1 to 100 µM for nitroxoline, miltefosine, and pentamidine isethionate, respectively. After incubation with drug or vehicle was performed for 72 h, CTG was added to all assay wells and luminescence was measured with a Promega Glomax-Multi+ plate reader. The percent effect of each drug combination was calculated as the reduction in luminescence value relative to vehicle-only controls. The additive effect of drug combinations was assessed using the excess over highest single agent (EOHSA) model  single agent in the drug pair. Drug combination experiments were performed with three independent replicates.Experiments measuring the effect of drug combinations were carried out similarly to the dose-response experiments described above. A) model , 77. EOHB. mandrillaris were diluted to 2.5 × 105 amoebae in 10 ml media and were treated with 3.5, 7, 14, 28, 56, 84, and 112 μM nitroxoline, pentamidine isethionate, or miltefosine and incubated for 72 h. Following incubation, all remaining amoebae in each population (various mixtures of cysts and trophozoites) were pelleted at 3,000 rpm for 5 min and resuspended in drug-free HFF-1 medium. Each resuspended B. mandrillaris population was placed on a monolayer of HFF-1 cells that were seeded at 106 cells per flask in 10 ml at 24 h prior to inoculation. At that cell density, untreated amoebae typically consume 100% of HFF-1 monolayers within 24 h. Coculture flasks containing amoebae and HFF-1 cells were incubated until 100% of the HFF-1 monolayer was consumed as observed by daily microscopic inspection or until the predetermined endpoint of the experiment at 28 days post-B. mandrillaris inoculation was reached. The day on which complete clearance of the HFF-1 monolayer occurred was recorded for all conditions. All recrudescence assays were performed with three independent biological replicates. These methods were adapted from a minimum trophozoite amoebicidal concentration (MTAC) assay that was conducted with monolayers of MA104 monkey kidney cells  in 6-well culture plates and were cultured with media containing 66% Eagle’s basal medium, 25% Hanks balanced salt solution, 5% fetal bovine serum, 1% N-2 supplement, 1× penicillin-streptomycin, and glutamine in a 37°C incubator with 5% CO2. At 12 h after plating, slices were inoculated with 104B. mandrillaris trophozoites in 20 µl of complete media containing vehicle (DMSO) or 35 µM nitroxoline, which was added dropwise to the slice surface. Media below the cell culture insert was adjusted to matching vehicle or nitroxoline concentrations. At 20 h postinoculation, media below the cell culture insertion were replaced with fresh media containing no drug. At that time, amoebae on top of the insertion surrounding and within the tissue were left undisturbed. Bright-field and phase contrast images were captured during the live culture experiment at magnifications of ×4, ×10, and ×20 using an Evos FL Cell Imaging System. At 4 days postinoculation, slices were gently fixed in 3.7% paraformaldehyde (PFA) for 4 h at 4°C and then rinsed with phosphate-buffered saline (PBS), stained for 2 h at room temperature with DAPI  (0.3 µM)–PBS–1% Triton X-100, and then mounted with ProLong Gold antifade mountant (Thermo). Images of stained tissue were obtained with a Nikon Ti spinning disk confocal microscope at ×20 magnification. Confocal z-stacks were projected and adjusted in ImageJ  at the University of California, San Francisco. Primary brain tissue samples were sectioned perpendicularly to the ventricle to obtain slices 300 μm thick and ∼2.5 mmn ImageJ . Bright-


Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic debilitating disease with huge social-economic impact. It has been suggested that immune dysregulation, nitrooxidative stress, and metabolic impairment might contribute to disease pathogenesis. However, the etiology of ME/CFS remains largely unclear, and diagnostic/prognostic disease markers are lacking. Several long noncoding RNAs  have been reported to play roles in immunological diseases or in stress responses.In our study, we examined the expression signature of 10 very long lncRNAs  in the peripheral blood mononuclear cells of 44 ME/CFS patients.2O2 (oxidative stress) and poly (I:C)  increased the expression levels of NTT and MIAT.LncRNAs NTT, MIAT and EmX2OS levels were found to be significantly elevated in ME/CFS patients as compared with healthy controls. Furthermore, NTT and EmX2OS levels increased with disease severity. Stimulation of human monocytic cell line THP-1 and glioma cell line KALS1 with HOur study revealed a ME/CFS-associated very long lncRNA expression signature, which might reflect the regulatory response in ME/CFS patients to oxidative stress, chronic viral infection and hypoxemia. Further investigations need to be done to uncover the functions and potential diagnostic value of these lncRNAs in ME/CFS. Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic debilitating disease with huge social-economic burden. The prevalence rate is 1–3/1000 in Germany and in the US population, affecting more than 1 million Americans . AccordiLong non-coding RNAs are defined as RNAs that are more than 200 nucleotides long, which do not encode proteins. Through RNA–DNA, RNA–RNA, or RNA–protein interactions, lncRNA can affect different stages of gene regulation . LncRNAsGNAS1, encoding neuroendocrine secretory protein [EMX2 gene, and possibly regulates EMX2 [PSMD6, which encodes components of proteasome, involving antigen presentation by MHC class I and DNA damage repair [The ten lncRNAs are NTT 17 kb), NEAT1 23 kb), MALAT1 (7.5 kb), TUG1 (7.1 kb), MIAT (9.9 kb), His-1 RNA (8.4 kb), GNAS1-AS (8.9 kb), EMX2OS (7.3 kb), CR933609 (8.8 kb) and AK124742 (6 kb). NTT was first described in activated T cells, while NEAT1 has been reported to be involved in human lupus and in immune response to viral infections –13. MALA kb, NEAT protein , 20. Acctes EMX2 . Both EMtes EMX2 . For CR9 kb, MALAe repair , 26.2O2) and Toll like receptor 3 (TLR3) ligand poly (I:C)  on the levels of very large lncRNAs were also evaluated in human monocytic cell line THP-1 and glioma cell line KALS1.In this study, we aimed to investigate the expression signatures and potential diagnostic values of the ten very large lncRNAs (> 5 kb) in ME/CFS patients. Furthermore, the effects of oxidative stress . High Capacity cDNA Reverse Transcriptase Kit  was used to reverse-transcribe 2 μg RNA into cDNA using for real time PCR analyses. GAPDH expression was used as an endogenous control. All primers were designed and synthesized by Genomics BioSci & Tech, Taipei, Taiwan. The following primer sequences were used: 2. Cells were treated with different concentrations of peroxide (H2O2)  or poly (I:C)  for 6 h. RNAs were then extracted for further RT-PCR.Human monocytic cell line THP-1 and glioma cell line KALS1 were grown in LPS-free complete RPMI medium containing 10% fetal bovine serum, at 37 °C incubator with 5% COPrinciple component analyses were performed using Stata 13 software. Mann–Whitney U tests were used via GraphPad Prism 5 to analyze the difference of lncRNA expression levels between ME/CFS and controls. Receiver operating characteristic (ROC) curve analysis was performed using MedCalc v.14.lncRNA–CTGAPDH) of each individual subject were used to build a correlation matrix, and projected to principle component space by principle component analysis (PCA)  in peripheral blood mononuclear cells (PBMCs) of ME/CFS patients and healthy controls were evaluated by RT-PCR. The values of the ten ΔCT , and severe ME/CFS (with Bell score < 30) Fig. a–j. InteSince we have discovered that among the ten lncRNAs tested, only NTT, MIAT and EMX2OS levels were upregulated in ME/CFS, we plotted a new PCA plot using a correlation matrix of NTT, MIAT and EMX2OS ΔCT values Fig. a. In FigTo evaluate the diagnostic value of the combination of the expression of NTT, MIAT and EMX2OS, we first obtained the optimal cutoff ΔCT value of each of the three lncRNAs by using receiver operating characteristic (ROC) curves. The optimal cutoff ΔCT value for NTT, MIAT and EMX2OS were 9.49 (area under the curve (AUC) = 0.82, 95% CI 0.72–0.90); 6.82 ; 19.06 , respectively. Further ROC analysis was then performed and revealed that the criteria of increased expression  of any two of these three lncRNAs in diagnosing ME/CFS had a sensitivity of 67.4, and specificity of 86.7, AUC = 0.82, with 95% CI 0.71–0.90 , ZEB1 level was found to have a mild but significant elevation in ME/CFS PBMCs as compared with controls , but not in controls , but not in controls  or poly (I:C)  for 6 h. While NTT and MIAT were upregulated after stimulations, EMX2OS could not be detected in both cell lines. In THP-1 cells, NTT expression level increased to a mean of 1.47- and 3.06-fold after stimulation with 10 nM H2O2 and 100 μM poly (I:C), respectively , respectively , respectively; however, the level of IFNGR1  did not show an obvious change after both stimulations  treatment  stimulations, respectively  stimulations in KALS1 showed a similar pattern as those observed in THP-1  lncRNAs in immune regulation and disease processes are being discovered . Here we2O2 or poly (I:C) stimulations, an expression pattern similar to those found in ME/CFS patients’ PBMCs. However, we do not know yet whether this lncRNA profile found in our study is specific for ME/CFS or can be found in other diseases involving immune dysregulation or oxidative stress, such as autoimmune diseases and cancer. It has been reported that MIAT levels could be upregulated in high glucose conditions and in lung cancer, and NTT expressions might be found in processes involving T cell activation [Consistent with the hypothesis that disease pathology in ME/CFS could be driven by oxidative stress and viral infections, we found that NTT and MIAT levels in THP-1 and KALS1 cell lines could be increased after Htivation , 17, 29.ZEB1, a MIAT-regulated gene, with ME/CFS. Consistently, both THP-1 and KALS1 cell lines showed higher expression levels of MIAT and ZEB1 after stimulation of poly (I:C), a synthetic analog of double strand RNA representing active viral infection, a potential trigger of ME/CFS. Zinc finger E-box-binding protein (ZEB) 1 has been reported to be a transcription factor recruiting repressor complex to suppress IL-2 activation in T cells [ZEB1 might be associated with response to chronic inflammation in ME/CFS. In non-small-cell lung cancer cell line, knockdown of MIAT resulted in decreased ZEB1 expression, indicating cis-action of MIAT on regulating ZEB1 [IFNGR1, PBOV1, TNFAIP3, HIVEP2, BCLAF1 and MYB are located close to the chromosome position of NTT [IFNGR1 and PBOV1 expressions between ME/CFS and controls. However, a marked positive correlation between NTT and IFNGR1 levels were observed in ME/CFS, not in controls. This observation suggests that the NTT/IFNGR1 axis might play a subtle role in ME/CFS pathogenesis. Whether other downstream genes are affected by the upregulated NTT in ME/CFS and possibly play more important roles in disease pathobiology needs more experiments. Finally, according to lncrnadb, the lncRNA database, the expression level of EMX2OS under normal physiological condition is higher in brain, moderate in lymph nodes, and very low in leukocytes. Consistent with this, we could not detect EMX2OS in PBMCs of several individuals. However, most ME/CFS patients were found to have elevated EMX2OS in their PBMCs. The role of EMX2OS in PBMC is currently unclear, and the potential downstream gene EMX2 usually expressed in CNS could not be detected in all study subjects [MX2 upregulation was found in brain hypoxemia [The mechanisms of NTT, MIAT and EMX2OS in ME/CFS pathogenesis require further investigations. In our study, we detected an association of higher level of  T cells . Upreguling ZEB1 . Interesing ZEB1 , 31. Foring ZEB1 . Severaln of NTT . We founsubjects . Furtherypoxemia . ME/CFS ypoxemia .In addition to our study on using lncRNA signature as diagnostic marker for ME/CFS, profiles of blood mRNA expression and plasma metabolites have been suggested to show diagnostic values for ME/CFS , 7, 34. In conclusion, although the pathogenic mechanisms of very large lncRNAs in ME/CFS remains to be elucidated, we have first evidence that a lncRNA expression signature could be of diagnostic value in ME/CFS.


Gloriosa superba seeds using the Super Critical Fluid (CO2) Extraction (SCFE) technology. The seeds were purified upto 99.82% using column chromatography. Colchicine affinity was further investigated for anticancer activity in six human cancer cell lines, i.e., A549, MCF-7, MDA-MB231, PANC-1, HCT116, and SiHa. Purified colchicine showed the least cell cytotoxicity and antiproliferation and caused no G2/M arrest at clinically acceptable concentrations. Mitotic arrest was observed in only A549 and MDA-MB231 cell lines at 60 nM concentration. Our finding indicated the possible use of colchicine at a clinically acceptable dose and provided insight into the science behind microtubule destabilization. However, more studies need to be conducted beforethese findings could be established.Colchicine was extracted from  Gloriosa is used in the treatment of various clinical conditions like inflammation, edema, rheumatism, skin infections, arthritis, alopecia, and many others  and for the negative ion mode [(M + H) + m/z 554.2620] to ensure mass accuracy during the MS analysis. The Lock–Spray scan time was set at 0.25 s with aninterval of 30 s.Analysis was performed on a Waters Xevo G2-XS QTof equipped with Acquity UPLC-I Class and Unifi software. Separation was carried out using Acquity UPLC HSS-T3 column . The column was maintained at 50 °C throughout the analysis, and sample temperature was kept at 10°C. The elution was carried out at a flow rate of 0.4 mL/min using gradient elution 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). Solvent gradient program was 95%–85% of the mobile phase A during 0–4 min, 85%–80% A during 4–15 min, 80%–75% A during 15–20 min, 75% A during 20–28 min, 75%–60% A during 28–31 min, 60%–10% A during 31–32 min, 10% A during 32–34 min, 10%–95% A during 34–35 min, followed by 95% A during 35–37 min. A total of 1µl of the test solution was injected for the screening and the chromatograph was recorded for 30 min.The LC–MS QTof instrument was equipped with an ESI ion source operating in a positive and negative ion mode. A mass range of 50–1000 Da was set with a 0.2 s scan time. The main working parameters for mass spectrometry were set as follows, ionization type—ESI, mode—MSve mode) A, capillve mode) B, source6 and grown overnight at 37 °C in a humidified atmosphere of 5% CO2. The six human cancer cell lines, lung cancer (A549), breast cancer (MCF-7 and MDA-MB231), pancreatic carcinoma (PANC-1), colon carcinoma (HCT116), and cervical cancer (SiHa) were obtained from National Centre for Cell Science (NCCS) Pune, India. Cell lines were cultured in a DMEM medium supplemented with 10%FBS and 1X antibiotic. The cultured cells were seeded in 24-well plate at a density of 0.05 × 104 cells/mL). After 24 h incubation, the cells were treated with different concentrations of colchicine and Gloriosa extract  0–2560 nM for 48 h. Cells were washed with PBS (twice), fixed with 10% formalin for 30 min and washed with H2O. The cells were then stained with 0.5% (w/v) crystal violet (25% (v/v) methanol) for 25 min. After washing with water until no color was eluted, the cells were dried overnight. Crystal violet [Cell lines were seeded in 96-well plates  and then imaged at 0, 24, 48, and 72 h post-scratch. Three independent areas per treatment group were recorded, averaged for each time point, and plotted against time to calculate the rate of migration. The cell-free areas were measured using Carl Zeiss AxioVision SE64 Rel. 4.9.1 software (Germany). Cells were seeded at a density of 0.05 × 102. The cells treated with colchicine were harvested and fixed in ice cold 70% ethanolin ice, for 1 h. Then, the cells were incubated for 30 min at 37 °C in a PBS solution containing 1 mg/mL RNaseA and 1 µg/mL propidium iodide (PI) solution. The cell cycle analysis was performed using Amnis Image Stream flow cytometer  and the data were analyzed based on 8000 events recorded using IDEAS image Stream Analysis Software.The cells were grown in 24-well plate for 24 h to study the effect of colchicine on cell cycle and were then treated with colchicine of different concentrations . The cells were then incubated overnight at 37 °C in a humidified atmosphere of 5% COp ≤ 0.05 was used as the criterion for statistical significance.Experimental values are expressed as mean ± standard deviation of at least two experiments, in triplicates. The level of w/w), quantified by HPLC.Colchicine  was isoGloriosa seeds using Super Critical Fluid Extractor (SCFE) , on the basis of the colchicine content, i.e., 27% colchicine content (w/w) (This was the first time colchicine was extracted from r (SCFE) . The extnt (w/w) . 1H NMR and 13C NMR spectroscopy, using JEOL 400 MHz instrument (USA).The isolation methodology involved column chromatographic separation in which the charcoal column separated most of the impurities, whereas the assay of 99.82%with a final yield of 85.2% was achieved when passed through the alumina columnand the final product was analyzed by HPLC . StructuScreening of the phytochemical composition of the Gloriosa seeds was analyzed using UPLC–MS QTof , where 32 compounds were identified in the positive mode of ionization  and 10 oEffect on cell viability was evaluated using crystal violet staining assay  against Cell migration was studied using in vitro scratch assay in six human cancer cell lines over a period of 72 h . In the The effect of colchicine on cell cycle progression was studied in six human cancer cell lines . The resThe untreated PANC-1 and MCF-7 cells showed a normal distribution in the cell cycle phase , while thetreated cells with 20–60 nM showed increasing4C  and decreasing 2C  content in both cell lines. Similarly, the untreated HCT116 cells distributed 64% in the G0/G1 and 19% in the G2/M phase within creasing 4C and decreasing 2C content at both concentrations (40 nM and 60 nM). In MDA-MB231, the control showed a normal cell cycle distribution; 69% of cells in the G0/G1 and 19% in the G2/M phase , whereasGloriosa superba seeds. Super Critical Fluid (CO2) extractor (SCFE) was used to obtain the crude seed extract (2 with 3 g water as co-solvent) could be admitted as the best method to extract colchicine from the seeds of Gloriosa  by disrupting the formation of the mitotic spindle required for mitosis . It was In conclusion, to avoid side effects of impurities present in colchicine, we developed methods of extraction and purification that produced a refined colchicine with high levels of activity, demonstrating its possible medicinal use. The present findings support that purified colchicine can be used in lung cancer, breast cancer, colon cancer, pancreatic cancer, and cervical cancer, because it was merely toxic at low concentrations and caused no mitotic arrest. Further pharmacological and clinical studies are required to establish these findings.


Forming microspheres or microbeads from nanofibrous materials has recently attracted research interest for their applications in various fields, because these structures greatly impact cellular behaviors and functions. However, conventional methods of preparing microspheres or microbeads have limitations, such as limited variety of material. Here, we propose a new fabrication process for forming a nanofibrous microsphere composed of bacterial cellulose (BC), which is synthesized through fermentation by specific bacteria. The process uses a hydrogel fiber containing spherical cavities. The bacteria encapsulated into the cavities produce BC, resulting in the formation of BC microspheres. Because of its simplicity, robustness, and cost-effectiveness, this process is promising for applications, such as in biochemical engineering and cell delivery systems. A sphere with a diameter in micrometer range is known as a microsphere, and is widely used in several fields. In biochemical engineering, microspheres provide a large surface area for anchorage-dependent cells. Because of these features, microspheres are referred to as microcarriers, and enable efficient cell culture in large bulk media ,4,5,6,7.Komagataeibacter xylinus. This bacteria produces cellulosic nanofibers with a diameter of 40–60 nm [Tg is 300 °C [The natural class of nanofibrous materials known as bacterial cellulose (BC) is produced by specific bacteria such as 40–60 nm  at a rat40–60 nm . Additio40–60 nm , exceedi40–60 nm , and glas 300 °C . These vs 300 °C ,19. Biods 300 °C  or 2,2,6s 300 °C . Additios 300 °C . Adhesios 300 °C ,24,25 tos 300 °C , chondros 300 °C , osteobls 300 °C , and cars 300 °C .We previously proposed a new process for forming microspheres from BC . This siKomagataeibacter xylinus (NBRC 13693) was used to produce BC. All chemicals for cultivating K. xylinus including glucose, mannitol, MgSO4·7H2O, ethanol, polypeptone, and yeast extract, were purchased from Wako Pure Chemical Industries . Deionized water was prepared using a Millipore system . SU-8 (SU-8 10 and 3050) and polydimethylsiloxane (PDMS) (Sylpot 184 W/C) for fabricating PDMS master molds were obtained from MicroChem Corp.  and Dow Corning Toray Co., Ltd. , respectively. Photomasks for photolithography were manufactured by Tokyo Process Service Co., Ltd. .K. xylinus for the continuous phase and corn oil with span 80 by syringe pumps (AS ONE), droplets were continuously generated at the tip of the glass needle. The droplets were collected in a cold corn oil bath. The mixture of gelatin and bacterial suspension was prepared by mixing the gelatin solution  and bacterial suspension . For the continuous phase of the emulsion, corn oil containing span 80 (3 wt %) was used. The composition of the culture medium for K. xylinus was 5 g polypeptone, 5 g yeast extract, 100 g glucose, 5 g mannitol, 1 g MgSO4·7H2O, 5 mL ethanol, and 1 L deionized water.To prepare monodisperse gelatin microspheres, a co-flow microfluidic device was used . The micK. xylinus and 9 mL corn oil containing span 80 with a magnetic stirrer at 750 rpm for 3 min, an emulsion was prepared. In this process, a conical centrifuge tube and magnetic rotor  were used.The composition of the liquid was the same as that used in the microfluidic method. By stirring a mixture of 1 mL gelatin solution containing g for 1 min. Subsequently, the gelatin microspheres were transferred into 1.5 wt % Na-alginate solution after removing the corn oil. By extruding the solution into 150 mM CaCCl2 solution through a syringe needle, a Ca-alginate hydrogel fiber was formed. The fiber was cultured in the culture medium for K. xylinus for 2 days at 30 °C. Inside the cavities, which are created by gelatin microspheres, BC microspheres were produced. The BC microspheres were collected by dissolving the Ca-alginate with 0.5 M ethylenediamine tetraacetic acid (EDTA) solution. The collected BC microspheres were rinsed by autoclaving them with 1 M NaOH for removal of the bacteria.The prepared emulsion was poured into cold water (~2 °C) in a plastic tube, followed by centrifugation at 420cq, or inner diameter of the glass needle wd, the diameter of the droplet d is precisely controllable d˜=a+qc for gelatin microsphere preparation. The plots show the same tendency as Here, we demonstrated a new process to form BC microspheres by cultivating BC-producing bacteria inside a Ca-alginate hydrogel fiber having spherical cavities created by using gelatin microsphere as a sacrificial architecture. By using an emulsification method to prepare the gelatin microsphere, the minimum diameter of the BC microsphere which the proposed process can form was estimated around 10 μm. Additionally, BC microspheres with high monodispersity were successfully produced by employing a co-flow microfluidic device. Surprisingly, the diameter of the BC was smaller than the one of the cavity in 15%, approximately. These results would be beneficial to the application of the BC microsphere production using the process.


As expected, MI reduced cardiac contractility and relaxation with no changes in perfusion. The impaired cardiac hemodynamics were associated with increased reactive oxygen species (ROS) levels (Sham: 19.3±0.5 vs MI: 23.8±0.3 µM), NADPH oxidase (NOX) activity (Sham: 42.2±1.3 vs MI: 60.5±1.5 nmol·h−1·mg−1) and monocarboxylate transporter 1 (mct1) mRNA levels (Sham: 1.0±0.06 vs MI: 1.7±0.2 a.u.), but no changes in superoxide dismutase (SOD), catalase, NADH oxidase (NADox), and xanthine oxidase activities. Lactate perfusion in MI hearts had no additional effect on ROS levels, NADox, and NOX activity, however, it partially reduced mct1 mRNA expression (MI-Lactate 1.3±0.08 a.u.). Interestingly, lactate significantly decreased SOD (MI-Lactate: 54.5±4.2 µmol·mg−1·min−1) and catalase (MI: 1.1±0.1 nmol·mg−1·min−1) activities in MI. Collectively, our data suggest that under pathological stress, lactate lacks its ability to modulate the expression of cardiac LOC-related genes and the perfused pressure in hearts submitted to chronic MI. Together, these data contribute to elucidate the mechanisms involved in the pathogenesis of heart failure induced by MI.Lactate modulates the expression of lactate oxidation complex (LOC)-related genes and cardiac blood flow under physiological conditions, but its modulatory role remains to be elucidated regarding pathological cardiac stress. The present study evaluated the effect of lactate on LOC-related genes expression and hemodynamics of hearts submitted to myocardial infarction (MI). Four weeks after MI or sham operation, isolated hearts of male Wistar rats were perfused for 60 min with Na Lactate is a metabolic intermediary compound that links energy metabolism to different organs and tissues . The hea2 utilization , both metabolic shifts of cardiac metabolism toward carbohydrate utilization and cardiac increase of lactate transport are adaptations that improve cardiac function, especially after myocardial ischemia . Many stlization ,9. Takinlization , we can Considering the high indexes of mortality and the incidence of HF worldwide, especially from ischemic etiology ,11, it iad libitum. Experiments were conducted according to the procedures stated in the Guide for the Care and Use of Laboratory Animals  and approved by the Ethics and Research Committee of the Universidade de São Paulo (#2011/55).Male Wistar rats weighing 250–300 g were used in the present study. Rats were placed in acrylic boxes lined with wood chips and access to water and standard chow ip) and xylazine  and after thoracotomy, the ADCA was occluded preventing left ventricle  blood supply. Some animals underwent the same procedures except the coronary artery ligation and were included in the sham-operated group. The day after surgery, echocardiographic analysis was conducted, and only infarcted animals that had lack of motility of the left ventricle anterior wall in M mode were included in the study. The animals were then assigned to three experimental groups: sham-operated perfused with Krebs-Henseleit (KH) solution (Sham), MI perfused with KH solution (MI), and MI perfused with KH solution plus sodium lactate (20 mM) (MI-lactate).MI was obtained by ligation of the anterior descending coronary artery (ADCA). Animals were anesthetized with ketamine . During the perfusion, the solutions were filtrated , bubbled with a carbogenic mixture (95% CO2 and 5% O2) and maintained at 37°C and pH±7.4. Subsequently, the left ventricle was accessed through the mitral valve, and a needle puncture was used to perforate the heart apex to avoid liquid accumulation. After that, a soft latex distensible balloon was placed into the left ventricle and inflated until diastolic pressure attained ±10 mmHg. Both aortic and balloon cannulas were connected to pressure transducers and signals were amplified, digitalized, and stored for further analysis of the developed pressure (DP), heart rate (HR), maximum positive and negative dP/dt (+dP/dtmax and −dP/dtmax) and perfusion pressure (PP) .After four weeks of the MI or sham surgery, the animals were euthanized and their hearts removed and placed on a Langendorff apparatus to test the effect of lactate perfusion as previously described by our group . BrieflyAfter the placement of the latex balloon into left ventricle, the hearts were equilibrated for 40 min beating spontaneously. KH or KHL were perfused for 60 min as previously described by Gabriel-Costa et al. . After 6g for 20 min at 4°C. The supernatant was used to measure lactate concentration. To determine lactate levels, the samples were first incubated with 0.2 M of glycine-semicarbazide buffer and 0.02 M of NAD+, and the absorbance was measured at a wavelength of 340 nm (R1). Then, 2 mg/mL of LDH, obtained from rabbit muscle, was added in the preparation and incubated for 60 min at 40°C. After that, a second reading was collected at the same wavelength (R2). R1 and R2 were used to calculate the net absorbance with and without LDH, with the following formula A = (R2 – 0.9R1) – (B2 – 0.9B1), where A is the net absorbance, B1 is the blank absorbance without LDH, and B2 is the blank absorbance with LDH. The concentration of lactate in mM was inferred by using the values of the net absorbance in a standard curve obtained between absorbance and increasing concentrations of lactate.The lactate levels in the cardiac tissue and perfusate  were evaluated based on the technique of Rosenberg and Rush . Brieflyg for 15 min at 4°C. The supernatant obtained was centrifuged for 30 min at 4°C at 100,000 g and the pellet resuspended in 1 mL of the same buffer. Another ultracentrifugation was obtained at the same conditions, and finally, the supernatant was resuspended in 1 mL of a phosphate buffer of composition: 50.0 mM sodium phosphate, 1.0 mM EGTA, 2.0 mM MgCl2, 250.0 mM sucrose, and 5 μg/mL aprotinin and 34.8 μg/mL PMSF, pH 7.2. The EPM was obtained by centrifugation of homogenized left ventricle tissue at 3,000 g for 15 min at 4°C and another subsequent centrifugation to obtain the pellet of the preparation (NADox and nicotinamide adenine dinucleotide phosphate oxidase (NOX) activities were measured in enriched microsomal membrane (EMM) and enriched plasma membrane (EPM) of the perfused hearts, respectively. The EMM was obtained by homogenization of left ventricle tissue with phosphate buffer containing: 50.0 mM sodium phosphate, 0.5 mM dithiothreitol, 1.0 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N0,N0-tetraacetic acid, 250.0 mM sucrose, pH 7.2, and 5 μg/mL aprotinin, 34.8 μg/mL phenylmethanesulfonylfluoride (PMSF). The homogenate was centrifuged at 3,000 paration .2O2 was used to transform the values to nmol or µmol H2O2·min-1·mg-1. Since the preparations contained SOD, the results were referred as total H2O2 (Both NADox and NOX were measured under similar conditions. The samples were incubated in a medium containing phosphate buffer 150 mM), pH 7.2, Amplex Red (50 μM), superoxide dismutase (SOD) (100 U/mL), and horseradish-peroxidase (0.5 U/mL). The reactions occurred with or without the corresponding substrates NADH or NADPH (0.1 mM), respectively. To calculate the specific activity of both enzymes, the slope of the curve with the addition of NADH/NADPH was subtracted from the slope of the curves with addition of water for each sample. The fluorescence was detected at a wavelength of 563 nm to excitation and 587 to emission. A standard curve with known concentrations of H0 mM, pH 2O2 and fluorescence transforming the values in μM of H2O2.ROS generation was measured in EMM after reperfusion since NADox is present in microsomal fractions. The conditions used in this assay were similar to those employed in enzymatic measurements. The samples were incubated only with 50 μM of Amplex Red, 100 U/mL of SOD, and 0.5 U/mL of horseradish-peroxidase (HRP) for 35 min at 30°C. After that, fluorescence levels at the end of the curve (Plato) were used to infer the ROS generation using a formula obtained in a standard curve with known concentrations of Hg for 15 min at 4°C and absorbance was read at 293 nm. Subsequently, the same procedure was conducted after the incubation of the samples for 20 min at 37°C. XO activity was obtained by subtracting the second from the first absorbance values. Calculation of XO activity was based on molar extinction coefficient of uric acid.Xanthine oxidase (XO) activity was obtained as previously described by Veskoukis et al. . Samples2PO4, 50.0 mM K2HPO4, pH 7.8, followed by centrifugation at 10,000 g for 20 min at 4°C. The rate of cytochrome C reduction inhibition was measured in the absence or in the presence of the sample in a reaction medium containing: 1.18 mM xanthine, 19.0 mM cytochrome C, and XO, diluted in sodium phosphate buffer  for 5 min. SOD activity was calculated subtracting the rate of cytochrome C oxidase reduction inhibition in the presence and absence of the samples , 4 (mct4), ldh, peroxisome proliferator receptor coactivator type 1 alpha (pgc1-α), and cytochrome oxidase IV (coxiv) and cyclophilin were used as a reference genes. All primers were synthesized by Fermentas and their sequences are shown in -ΔΔCt. Control group levels were arbitrarily set to 1.Total RNA was isolated from left ventricle samples using Trizol . The RNA concentration and purity (260:280 nm ratios) were assessed in a spectrophotometer  and integrity was observed in an agarose gel  electrophoresis. The cDNA was synthesized using Revertaid First Strand cDNA synthesis kit . The genes analyzed were: t-test or one-way analysis of variance and Tukey's post hoc, when necessary. The results were considered significantly different when P<0.05.Data are reported as means±SE. The means were compared with non-paired vs 1.8±0.1 mM, respectively) at 60 min, but it increased significantly after lactate perfusion (MI-lactate group: 22.3±0.8 mM). These values were kept constant throughout the experiment . Cardiac lactate basal levels were not altered by MI . However, lactate perfusion induced a significant increase in lactate levels when MI-lactate hearts were compared to both Sham and MI hearts (4.7±0.8 mM). We also tested whether lactate administration would acidify the isolated heart perfusate along the 60 min of lactate perfusion at 20 mM . The increased cardiac ROS levels in both MI groups were associated to a high NOX activity in MI groups , while NADox . The reduced SOD and catalase activities in lactate-perfused MI hearts were not accompanied by changes in nrf-2 mRNA levels, a redox-sensitive transcription factor that regulates SOD and catalase expression  in cardiac cells with no further increment after lactate perfusion. In fact, the enhancement of ROS concentration was associated to NOX but not NADox activity. These data are in agreement with an established consensus, which states that MI induces both sympathetic nervous and renin-angiotensin systems hyperactivity that recognizably up-regulate components of NOX complex and ROS levels in cardiac muscle  and lusitropic (−dP/dt) function, which were not changed by lactate perfusion in isolated MI hearts. Interestingly, lactate perfusion in MI hearts did not reduce cardiac perfusion pressure as we previously observed in isolated healthy hearts . We belimct1 mRNA expression and antioxidant enzymatic defense. Although increasing evidence suggests that switching metabolic fuel usage toward glycolytic fuel (e.g. lactate) oxidation increases cardiac function (Taken together, our data provide evidence that MI blunted the ability of lactate in modulating LOC-related gene expression and cardiac perfusion in isolated hearts. The practical implications of our findings are that lactate did not rescue cardiac function and hemodynamics in ischemic HF. Instead, it contributed to decrease function , our dat


DHCR7 rs12785878, CYP2R1 rs10741657, GC rs4588) have been associated with serum vitamin D. We studied the association of these variants with serum vitamin D in 2163 patients with T2D from the “Sapienza University Mortality and Morbidity Event Rate (SUMMER) study in diabetes”. Measurements of serum vitamin D were centralised. Genotypes were obtained by Eco™ Real-Time PCR. Data were adjusted for gender, age, BMI, HbA1c, T2D therapy and sampling season.Mortality rate is increased in type 2 diabetes (T2D). Low vitamin D levels are associated with increased mortality risk in T2D. In the general population, genetic variants affecting vitamin D metabolism (p = 1 x 10–4) and GC rs4588 (p = 1 x 10–6) but not CYP2R1 rs10741657 (p = 0.31) were significantly associated with vitamin D levels.DHCR7 rs12785878 (p = 1.1 x 10–11) and insufficiency (<30 ng/ml) .One unit of a weighted genotype risk score (GRS) was strongly associated with vitamin D levels (GC rs4588, but not CYP2R1 rs10741657, are significantly associated with vitamin D levels. When the 3 variants were considered together as GRS, a strong association with vitamin D levels and vitamin D insufficiency was observed, thus providing robust evidence that genes involved in vitamin D metabolism modulate serum vitamin D in T2D.In conclusion, DHCR7 rs12785878 and  The rate of mortality of patients with T2D doubles that of non-diabetic individuals of similar age , thus maThe biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 plays a central role in a wide variety of metabolic pathways. Vitamin D insufficiency, affecting as many as 50% healthy adults in developed countries, has been linked to autoimmune , infectiAlthough serum levels of vitamin D are to some extent under the control of modifiable determinants such as dietary intake and synthesis in the skin, classical twin studies showed that Vitamin D levels are 50–80% heritable , 24, thuGenes affecting vitamin D metabolism are candidates for the control of serum vitamin D levels.In this respect, three large GWA studies of serum 25-hydroxyvitamin D reported that variants at three loci reached genome-wide significance above all the others . They weDHCR7, which encodes the enzyme 7-dehydrocholesterol reductase, thereby affecting Vitamin D synthesis  (Table The rs12785878 SNP at p = 2.1 × 10–5). Similarly, no changes were observed in the above-mentioned association when insulin treatment vs. oral anti-hyperglycaemic drugs was taken into account as a covariate (adjusted-p = 1 × 10–4), in order to address a possible confounding effect of diabetes treatment.The observed association was still significant in a multivariable model comprising adjustments for gender, age, BMI, HbA1c and sampling season , respectively].Though not reaching a formal statistical significance, a tendency toward an association was observed between the CYP2R1 rs10741657 SNP and mean (95% CIs) vitamin D levels  (Table p = 1 × 10–6).The GC rs4588 SNP was significantly associated with means (95% CIs) vitamin D levels both in unadjusted analysis [means: 24.0 (95%CI = 23.4–24.6), 22.3 (95%CI = 21.6–22.9), 20.9 (95%CI = 19.6–22.1) ng/ml in GG, GT and TT individuals, respectively, p = 1.6 × 10–10, adjusted-p = 1.1 × 10–11). Similar results were observed when the unw-GRS was used (beta (SE) = −0.049 (0.008); p = 2.2 × 10–9, adjusted-p = 1.3 × 10–9); proportions of individuals carrying the different number of risk alleles are shown in Table p = 1.6 × 10–9) across w-GRS quartiles .Aggregating information from multiple SNPs, each with small effects, into a single genetic risk score (GRS) has become a useful tool for examining the cumulative predictive ability of genetic variation at known loci on different disease outcomes and related phenotypes . To inveThe present study, carried out in a large population of patients of European ancestry with T2D, is the first testing the association between variability in three genes involved in vitamin D metabolism and serum vitamin D, a biomarker of mortality risk in diabetic patients .DHCR7 and GC, respectively, while only a non-significant trend was observed for rs10741657 at CYP2R1. More importantly, when the three SNPs were considered in a combined fashion as indicated by a genetic risk score, a very strong association with both serum vitamin D and hypovitaminosis was observed. Hence, we do have a genetic marker of vitamin D levels, which can be used, for example by a Mendelian randomization approach [We found strong evidence of association between low vitamin D levels and SNPs rs12785878 and rs4588, harboured by approach , to addrapproach  in T2D iDHCR7, CYP2R1 and GC are key loci involved in the hereditary control of vitamin D levels [p-values of those reaching genome-wide significance, and have been replicated in a second large study [Our results are consistent with previous studies conducted in the general population, reporting that D levels –27, 33. DHCR7, CYP2R1 and GC variability is independent of hyperglycaemia or other intrinsic features of diabetes status. Of note, we observed a mean difference between genotypes of 4–5 ng/dl in vitamin D levels. Although it is reasonable to believe that this difference between genotypes in vitamin D level may not be clinically relevant, it is conceivable that in diabetic subjects, who are already affected by low vitamin D levels, a further genetically induced decrease may have in the long term detrimental consequences. Only longitudinal studies may answer this question.Given the known role of T2D itself in lowering vitamin D levels, our present finding clearly indicates that the effect of Strengths of our study are the large, homogeneous and clinically well-characterized cohort of patients with T2D derived from the SUMMER Study in Diabetes . Also, vWe acknowledge that no information on vitamin D supplementation, which might have influenced vitamin D levels in our patients, was available. Epidemiological data from the ARNO Diabetes Italian registry  show thaIn conclusion, this study demonstrates a strong independent association between variants in genes involved in the metabolism of vitamin D and its circulating levels in patients with T2D, establishing for the first time the role of these genetic factors in the regulation of vitamin D levels also in the clinical setting of type 2 diabetes. Based on the present findings, further prospective studies may now be designed aimed at addressing the intrinsic nature of the association between low serum vitamin D and increased mortality rate in T2D .ClinicalTrials.gov, NCT02311244; URL: https://clinicaltrials.gov/ct2/show/NCT02311244?termZSUMMER&rankZ5).The first 2163 consecutive study subjects of the “Sapienza University Mortality and Morbidity Event Rate (SUMMER) study in diabetes” cohort were studied. This is an observational, prospective, collaborative study aimed at unravelling new molecular predictors of mortality and vascular morbidity in patients with T2D  . The diagnosis of hypertension was based on the presence of elevated systolic (>140 mmHg) and/or diastolic (>90 mmHg) blood pressure, and/or the current use of antihypertensive medications.Briefly, consecutive patients with T2D of age >18 years and European ancestry were recruited from the outpatient clinics of 10 Italian centres. All patients have undergone a structured interview in order to collect information on family history of diabetes and cardiovascular disease and on current treatments. All subjects have had a complete work-up including clinical examination, anthropometric measurements and laboratory tests. BMI was calculated as body weight (kg)/height (mStudy populations underwent fasting blood sampling to assess glycosylated hemoglobin (HbA1c), total cholesterol, HDL-cholesterol, triglycerides, white blood cell count, uric acid, serum creatinine and urinary albumin/creatinine ratio (ACR). Low-density lipoprotein (LDL) cholesterol value was obtained using Friedwald formula. In addition, measurements of serum 25-hydroxyvitamin D levels  were carried out in all study patients. In order to avoid the introduction of a bias linked to the different inclination of the sunrays during the different seasons of the year, the sampling period was taken into account for the statistical analyses .DHCR7 rs12785878 T>G, CYP2R1 rs10741657 G>A, GC rs4588 G>T.The following SNPs have been studied in DNAs from all 2163 individuals used for the present study: DHCR7 rs12785878 T>G, C_2958430_10 for CYP2R1 rs10741657 G>A and C_8278879_10 for GC rs4588 G>T. The assay was carried out on an Eco™ Real-Time PCR System by Illumina  in a total volume of 10 μl. After an initial polymerase activation step at 95° C for 3 min, amplification was performed using 45 cycles of denaturation (95° C for 15 s), annealing and extension (60° C for 1 min).Genotyping of SNPs was assayed using the TaqMan assays (Applied Biosystems) C_32063037_10 for 2 test. Univariate and multivariate linear regression analysis was used to assess the effect of each single SNP (assuming an additive genetic model of inheritance) on continuous outcome. Multivariable analyses were performed using linear regression models adjusting for gender, age, BMI, HbA1c and sampling season as binary variable . Results were reported as beta values along with their 95% confidence intervals (CIs). A P-value < 0.05 was considered as statistically significant. All analyses were performed using SPSS version 20.0 .Patients’ baseline characteristics were reported as mean ± SD and percentages for continuous and categorical variables, respectively. Log transformation was used for 25-OH D levels, that was non-normally distributed. Differences between continuous variables across genotype classes were evaluated by ANOVA models. Categorical variables distribution was compared between groups by χn = 2163), we had at least 96% power with a type I error of 5% and 80% power with a type I error of 1% to detect the same effect size of each SNP described by Wang et al. [In our study the vitamin D was distributed with standard deviation equal to 10.4. In the whole sample  was created by summing the number of the risk alleles associated with lower vitamin D levels of the 3 SNPs, carried by each subject. A weighted genotype risk score (w-GRS) was created by summing the weighted effect size on serum vitamin D of the 3 SNPs carried by each subject.The study was conducted in accordance with the Declaration of Helsinki. The study protocol has been approved by the coordinating centre’s Ethic Committee and, thereafter, by the Ethics Committee of each centre outside the Umberto I “Sapienza” University Hospital, in Rome. Written consent was obtained from all subjects before the study.


Polygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses. Research has attempted to identify biosynthesis genes and metabolic pathways in this species, and quantitative real-time PCR (RT-qPCR) has commonly been used to detect gene expression because of its speed, sensitivity, and specificity. However, no P. cuspidatum reference genes have been identified, which hinders gene expression studies. Here, we aimed to identify suitable reference genes for accurate and reliable normalization of P. cuspidatum RT-qPCR data.ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, and eIF6A) and three novel , were analyzed in different tissues  without treatment and in leaves under abiotic stresses  and hormone stimuli . Expression stability in 65 samples was calculated using the △CT method, geNorm, NormFinder, BestKeeper, and RefFinder. Two reference genes (NDUFA13 and EF-1γ) were sufficient to normalize gene expression across all sample sets. They were also the two most stable genes for abiotic stresses and different tissues, whereas NDUFA13 and SKD1 were the top two choices for hormone stimuli. Considering individual experimental sets, GAPDH was the top-ranked gene under ABA, ETH, and GA3 treatments, while 60SrRNA showed good stability under MeJA and cold treatments. ACT, UBC, and TUB were suitable genes for drought, UV, and ABA treatments, respectively. TUA was not suitable because of its considerable variation in expression under different conditions. The expression patterns of PcPAL, PcSTS, and PcMYB4 under UV and SA treatments and in different tissues normalized by stable and unstable reference genes demonstrated the suitability of the optimal reference genes.Twelve candidate reference genes, including nine common (NDUFA13 and EF-1γ as reference genes to normalize P. cuspidatum expression data. To our knowledge, this is the first systematic study of reference genes in P. cuspidatum which could help advance molecular biology research in P. cuspidatum and allied species.We propose  Polygonum cuspidatum is a perennial herb of the Polygonaceae family that is a well-known traditional Chinese medicine. Its first medicinal application records date back over 1800 years in Mingyi Bielu [yi Bielu . More reyi Bielu .P. cuspidatum to date, such as anthraquinones, flavonoids, stilbenes, organic acids, coumarins, catechins, and lignans [P. cuspidatum than in other plants [P. cuspidatum also show significant health-promoting effects in cells or animal models [P. cuspidatum responses to abiotic stresses and hormone stimuli as well as in various tissues needs to be thoroughly explored to better understand the production of these active ingredients.Numerous active compounds have been isolated and identified from  lignans . The conr plants , and sevr plants . Other cl models , 7. Intel models , 9. HoweMYB4 in Arabidopsis responds to UV and SA stresses to suppress phenylpropanoid pathway gene expression [Stilbenes and flavonoids, derivatives of the phenylpropanoid pathway, actively participate in the regulation of resistance to stresses, including pathogens, ultraviolet (UV) radiation, low/high temperature, drought, heavy metals, methyl jasmonate (MeJA), and ethylene (ETH) , 9. Phenpression , 13.The analysis of gene expression patterns can provide insights into complex metabolic processes. Quantitative real-time PCR (RT-qPCR) is commonly used to detect gene expression in different species because of its simplicity, speed, sensitivity, specificity, and high throughput . HoweverGentiana macrophylla [Achyranthes bidentata [Euscaphis konishii [P. cuspidatum, PcPKS1 [PcPKS2 [PcCHS1 [PcSTS [PcMYB1 [PcWRKY33 [P. cuspidatum reference genes has become a major hurdle for gene expression studies in this species. Because of a lack of sequence and expression information, we performed transcriptome sequencing of P. cuspidatum vegetative tissues  and leaves under different treatments (UV and MeJA) in our laboratory (unpublished data), providing a wealth of resources for our current selection of suitable reference genes.In this context, the △CT method , geNorm rophylla , Achyranidentata , and Euskonishii . In P. c, PcPKS1 , PcPKS2  [PcPKS2 , PcCHS1  [PcCHS1 , PcSTS [1 [PcSTS , PcMYB1  [PcMYB1 , and PcWP. cuspidatum. Twelve genes  were screened out from the P. cuspidatum transcriptome, and their expression levels were detected by RT-qPCR across all samples. These included three tissues , 32 abiotic-treated samples , and 30 hormone-treated samples . Expression stability was calculated using △CT, geNorm, NormFinder, BestKeeper, and RefFinder. Additionally, the expression of three target genes  under UV and SA treatments and in different tissues was normalized separately by the most and least stable genes to demonstrate the suitability of the recommended reference genes. Our results provide some useful resources for the future quantification of gene expression in P. cuspidatum and allied species.The present study was undertaken to characterize candidate reference genes for RT-qPCR data normalization in ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, and eIF6A) and three novel reference genes  were identified from the P. cuspidatum transcriptome as candidate reference genes. The full-length cDNAs of these 12 genes, which were used to design specific qPCR primers, were submitted to NCBI GenBank  agarose gel electrophoresis, which revealed a single band of expected size for each primer pair  varied from 86% (TUB) to 103% (SKD1) with correlation coefficients (R2) ranging from 0.994 to 0.999  were evaluated in 65 samples collected from different tissues without treatments and leaves under abiotic and hormone stimuli using CT values. Raw CT values of the 12 genes across all samples were shown in Additional file GAPDH) to 33.46 (SKD1) across all samples. Because the gene expression level is negatively correlated with the CT value, ACT was the highest expressed gene with the lowest mean CT value (21.60), whereas SKD1 was the least abundant gene with the highest mean CT value (27.42) among the 12 genes. The expression variation among the 65 samples for each gene ranged from 4.12 (eIF6A) to 8.779 (SKD1). No gene showed an unchanged expression level under all conditions, so it was necessary to identify reference genes under specific experimental conditions in P. cuspidatum.The expression levels of the 12 candidate reference genes  (3) “Different tissues” , and (4) “All” . More specifically, the stability of the 12 candidate reference genes was analyzed by the △CT method, geNorm, NormFinder, BestKeeper, and RefFinder.In our study, each reference gene was evaluated in 11 experimental sets which were analyzed individually at first. To obtain a more comprehensive analysis, the sets were then divided into four different groups: (1) “Abiotic stress”  (2) “Hormone stimuli”  followed by NDUFA13 (0.35), indicating that these were the three most stable genes in geNorm analysis. NDUFA13 and EF-1γ were the two top-ranking genes in NormFinder and △CT analysis, while eIF6A and 60SrRNA were top in BestKeeper analysis. According to the calculations performed by RefFinder, EF-1γ and NDUFA13 were the most appropriate reference genes under abiotic stress conditions. The comprehensive ranking order for every specific abiotic stress was not entirely consistent. EF-1γ, NDUFA13, and 60SrRNA were also identified as the three most stable reference genes under heat treatment, but EF-1γ and NDUFA13 fell outside the list of the four most stable genes, though 60SrRNA ranked top under cold conditions. For salt treatment, YLS8 was the top-ranked gene, followed by EF-1γ, TUB, and NDUFA13. For UV treatment, UBC had the highest stability, while EF-1γ, NDUFA13, and 60SrRNA also exhibited good stability. NDUFA13 was the optimal reference gene under drought treatment, followed by EF-1γ and 60SrRNA with low CVs , while NDUFA13 and EF-1γ ranked second and fourth, respectively. NDUFA13 and EF-1γ were recommended in different tissues of P. cuspidatum by RefFinder , Hormone stimuli (SA), and in different tissues . The two most stable reference genes  and one unstable gene (TUA) were selected for normalizing qPCR data.To ensure the accuracy and reliability of our results, the relative expression patterns of PcPAL was significantly induced at all analyzed stress times. However, the expression levels at 16 h and 32 h with TUA normalization were much higher than those with either UBC, EF-1γ, or their geometric mean. The expression of PcMYB4 was inhibited by UV treatment, initially reduced after reaching the lowest level at 8 h, then increased afterwards. However, the expression levels of PcMYB4 at 16 h and 32 h were overestimated with TUA normalization. Similar misjudgments occurred in the analysis of PcSTS expression data. Some divergences in the results were also observed under SA treatment . TUA normalization similarly led to erroneous interpretation of the relative expression patterns of PcMYB4. In different tissues , composed of the four subunits EF-1α, EF-1β, EF-1δ, and EF-1γ, plays a central role in protein biosynthesis [EF-1α, encoding a G-protein, was used for the normalization of qPCR data in some medicinal plants such as G. macrophylla [A. bidentata [EF-1γ had previously been evaluated in P. ginseng [Nilaparvata lugens [EF-1γ was one of the two most suitable reference genes with stable expression levels under various experimental conditions.Eukaryotic translation initiation factor and ribosomal RNA have been reported as reference genes in many studies, such as e indica , and eIF ginseng . In our ynthesis . EF-1α, rophylla  and A. bidentata . The maiidentata . The exp ginseng  and Nilaa lugens . In our SKD1, YLS8, and NDUFA13 have also been selected for gene normalization by the application of large amounts of omics data. SKD1, encoding a protein that contributes to vacuolar trafficking and maintenance of the large central vacuole of plant cells [SKD1 was preferable under “Hormone stimuli” conditions, but had to be discarded under “Abiotic stress” and “Different tissues” conditions because of its low expression and high variation. YLS8 encodes a protein involved in mitosis, and performed well under ABA, MeJA, and salt conditions, but poorly in other individual sets and the three combination groups. These results were not identical to those obtained previously [NDUFA13 was remarkably stable in the individual sets (except in the ABA group) as well as in the combination groups, suggesting it is an almost ideal reference gene. NDUFA13, encoded by the nuclear genome, is an accessory subunit of the mitochondria respiratory chain complex I, which transfers electrons from nicotinamide adenine dinucleotide to ubiquinone [NDUFA13 showed stable expression under various conditions in this study. NDUFA13 was previously reported as a reference gene in Apostichopus japonicus [NDUFA13 as a reference gene in plants.Novel reference genes such as nt cells , was firnt cells . In thiseviously , 47. Notiquinone . The facaponicus , but no PAL, STS, and MYB4 are three important regulating genes in the phenylpropanoid pathway. When using the stable reference genes, the relative expression patterns of PcPAL and PcMYB4 were in agreement with previous reports; for example, MYB4 was down-regulated by exposure to UV-B light in Arabidopsis thaliana [Brassica rapa [PAL was induced by exposure to UV and SA in A. thaliana [Juglans regia [STS is found in only a few plants, and not in Arabidopsis or tobacco. Research into STS expression is currently concentrated in grapes, where it was shown to be strongly induced by UV-C [STS in response to SA in P. cuspidatum, with rapidly decreasing expression lasting for 12 h. These differences may reflect variations in UV wavelengths or species. Moreover, our results are consistent with P. cuspidatum transcriptome data. Evidently, there were obvious underestimates or overestimates in our results following normalization by the least stable gene, TUA. Our results demonstrate the importance of using a stable reference gene for normalization to obtain accurate results.thaliana  and Brasica rapa , and PALthaliana  and Juglns regia . To our  by UV-C  and SA [ by UV-C . We obseBased on the above analysis, we suggest that no single gene should be used for normalization in all species, tissues, or treatments. Therefore, suitable reference genes for given species and conditions should be explored.P. cuspidatum and under different treatment conditions. NDUFA13 and EF-1γ were identified as the best two reference genes for normalizing RT-qPCR gene expression data. Their reliability and effectiveness were verified by PcPAL, PcSTS, and PcMYB4. To our knowledge, the current work is the first systematic analysis of suitable reference genes that will facilitate further research into the molecular biology of P. cuspidatum and other closely related species.We evaluated the expression stability of 12 candidate reference genes in different tissues of P. cuspidatum were collected from the medicinal plant garden of the Institute of Botany, Chinese Academy of Sciences . Seeds were surface-sterilized and sown on Murashige and Skoog (MS) agar medium in a growth chamber at 24 °C with a 16 h/8 h light/dark cycle. After 1 month, similar seedlings were submitted to different treatments. For hormone treatment, the seedlings were uniformly sprayed with 0.5 mM MeJA, 1 mM SA, 0.1 mM ABA, 0.5 mM GA3 or 0.5 g/L ethrel. For cold and heat treatments, the seedlings were respectively kept in 4 °C and 42 °C illumination boxes. For UV irradiation, the seedlings were placed under a UV-B transilluminator for 20 min. For salt treatment, the seedlings were transplanted to MS agar medium containing 100 mM NaCl. Leaf samples were collected at 0, 2, 4, 8, 16, and 32 h after UV and MeJA treatments, and 0, 2, 4, 8, 12, and 24 h for the other treatments. For drought treatment, plants grown for 2 months in plastic pots containing a soil/vermiculite mixture (1:1) under the same conditions were kept in dry soil for 1 week, then rehydrated and sampled daily . Tissue-specific samples  were collected from plants grown for 2 months in the soil. All 65 samples, including three tissue-specific samples , 32 stress-treated samples , and 30 hormone-treated samples , were separately collected in three biological repeats. All samples were immediately frozen in liquid nitrogen and stored at − 70 °C before RNA extraction.Seeds of 260/280 < 2.2 and OD260/230 > 2.0 were used for subsequent cDNA synthesis. Total RNA (3 μg) was reverse-transcribed into cDNA using the Hifair™ II 1st Strand cDNA Synthesis SuperMix  with oligo (dT) primers according to the manufacturer’s instructions. cDNA was diluted at 1:20 with the EASY dilution solution , then stored at − 20 °C until required as template for qPCR.Total RNA was isolated from samples using the Plant Total RNA Purification Kit  following the manufacturer’s instructions. The RNA integrity was checked on a 1% (m/v) agarose gel. The quantity and quality of the total RNA samples were assessed by recording absorbances at 260/280 nm and 260/230 nm with the NanoDrop 2000 spectrophotometer . Only RNA samples 1.8 < ODCV) [CV calculation formula was: CV = standard deviation of reads per kilobase of transcript per million mapped reads (RPKM)/average RPKM. We downloaded the sequences of nine classic reference genes  identified in other species, and carried out BLASTn queries against the P. cuspidatum transcriptome. Then, the highest ortholog sequences were checked against the Arabidopsis genome database and recorded in Table SKD1, YLS8, and NDUFA13, were selected based on their low CV and appropriate RPKM values  . In thisR2) and amplification efficiency (E) for all primer pairs were evaluated by standard curves using fourfold dilutions of the pooled cDNA .Specific primers were designed using Primer 5 software and synthesized by GENEWIZ Company . Gene characteristics and primer details are shown in Table All qPCR was carried out in 96-well plates using QuantStudio™ Real-Time PCR Software  with the Hieff™ qPCR SYBR® Green Master Mix . Each 10 μL reaction included: 5.0 μL of 2× Hieff™ qPCR SYBR® Green Master Mix, 0.2 μL of each primer (10 μM), 1.0 μL of diluted (1:20) cDNA template, and 3.6 μL of RNase-free water. The cycle program for product amplification was: 95 °C for 5 min (hot-start activation) followed by 40 cycles of 95 °C for 10 s (denaturation), 58 °C for 20 s , and 72 °C for 20 s (extension). The melting curve was generated after 40 cycles to test the specificity of each primer pair across the temperature range of 60–95 °C at a heating rate of 0.05 °C/s. Three technical replicates were used for each sample.E (%) = (10 − 1/slope − 1) × 100 [The CT values represent the expression level of each candidate reference gene. The amplification efficiency was calculated by: 1) × 100 . The sta1) × 100 , geNorm 1) × 100 , NormFin1) × 100 , BestKee1) × 100 , and Ref1) × 100 .SD) based on the relative expression of all pairwise combinations of candidate reference genes. The gene with the lowest SD was identified as the most stable reference gene [M-value) for each gene. Genes with the lowest M values have the most stable expression. geNorm determines the pairwise variations (V) of one specific gene with all others. A cut-off value of Vn/n + 1 < 0.15 means that further addition of reference genes no longer makes any significant contribution to the normalization. For instance, V2/3 < 0.15 means that two reference genes are sufficient for data normalization [SV) for each gene, which takes intergroup and intragroup relationships into consideration. A lower SV indicates a higher stability [r), standard deviation (SD), and coefficient of variance (CV). A more stable gene exhibits a lower SD ± CV value [The △CT method was employed to rank the genes by calculating the average standard deviation  under UV and SA treatments and in different tissues. The homologous P. cuspidatum genes AtMYB4 and AtPAL1 were cloned and named PcMYB4 and PcPAL, respectively (Table PcSTS (EU647245.1) was isolated in previous studies from our laboratory [−△△CT method.To validate the selected reference genes, qPCR was performed to analyze target gene expression levels , TUA (2), TUB (3), GAPDH (4), EF-1γ (5), NDUFA13 (6), UBQ (7), UBC (8), 60SrRNA (9), SKD1 (10), YLS8 (11), eIF6A (12), PcMYB4 (13), PcPAL (14), PcSTS (15). Figure S2. Melting curves of 12 candidate reference genes and 3 target genes in Polygonum cuspidatum.Figure S3. Standard curves of 12 candidate reference genes and 3 target genes in Polygonum cuspidatum.Additional file 2: Raw CT values of the 12 candidate reference genes across all samples of Polygonum cuspidatum.Additional file 3: Table S1. Gene expression stability ranked by △CT, BestKeeper, NormFinder, geNorm and RefFinder under individual condition. Table S2. RPKM values of 3 target genes in Polygonum cuspidatum transcriptome. Table S3. RPKM values of 3 novel genes covering 7 transcriptomes data of Polygonum cuspidatum.Additional file 4. cDNA sequences of 12 candidate reference genes and 3 target genes. Coding sequence (CDS) were marked green.Additional file 5. Genomic DNA sequences of 12 candidate reference genes and 3 target genes. The exons were shown in green shading, qPCR primers were marked yellow.