The pH of WMS and NMS was decreased after APPJ treatment from 5.42 to 4.94, and 5.09 to 4.75, respectively. The water-binding capacity (WBC)  and swelling volume (SV)  of the starches were obviously increased by APPJ treatment. The surfaces of starch granules were wrecked, due to plasma etching. No changes in the crystalline types of both starches were observed. However, the relative crystallinities (RCs) of WMS and NMS were reduced from 46.7% to 42.0%, and 40.1% to 35.7%, respectively. Moreover, the short-range molecular orders of both starches were slightly reduced. In addition, APPJ treatment resulted in lower gelatinization temperature and enthalpies. Therefore, APPJ provides a mild and green approach to starch modification, showing great potential for applications in the food and non-food industry.In present study, a novel physical modification of waxy maize starch (WMS) and normal maize starch (NMS) was investigated by using an atmospheric pressure plasma jet (APPJ) treatment. The effect on the structure and physicochemical properties of both starches was demonstrated by treatment with a 5% starch suspension ( Starch is a natural carbohydrate, and a primary source of both stored energy and reduced carbon in most plant species. Starch is not only the main component of food, providing a vital energy for humans, but is also widely used in the non-food industries, such as paper, textiles, bioethanol, cosmetics, etc. ,2,3,4. SPlasma is considered to be the fourth state of matter in addition to gases, liquids, and solids , and conRecently, many scientists have employed plasma in various methods of modifying starches with changes in structure and properties ,20,24,25Waxy maize starch (WMS) was purchased from Hengrui starch Import & Export Co. Ltd. , with 12.1% moisture and 4.5% amylose. Normal maize starch (NMS) was purchased from Lihua starch Import & Export Co., Ltd.  with 10.9% moisture and 23.2% amylose. The reagents used here were of analytical grade .w/v). The slurry was thoroughly mixed by using a vortex mixer  before treatment.The WMS and NMS (5.0 g) were added to deionized water (100 mL) to prepare a slurry  with an APPJ treatment at 750 W of the input power supplied; the HF of power supply was 25 kHz, and H = 14 mm (distance from plasma jet probe to sample). The schematic diagram of APPJ  setup was shown in w/v) aqueous solutions according to Annapure’s method [The pH of untreated or treated WMS and NMS was estimated for 0.5% . Double-sided carbon tape was used to fix the starch samples onto the test metallic platform, and gold was sprayed for coating (120 s) using a sputter coater . An accelerating voltage of 25 kV was used for imaging . MicrogrXRD analysis was performed using a Bruker D8 ADVANCE X-ray diffractometer  operating at 40 kV and 30 mA. The starch samples (about 0.5 g) were placed in a circular test plate  and pressed. According to Wang’s method , the staw/w), and tablets approximately 1 mm thick were prepared. The absorbance spectra was computed between 4000 and 400 cm−1 with a resolution of 4 cm−1. The air was used as a blank, with a total of 64 scans for each sample at room temperature [−1 and normalized. The ratios of absorbance at 1047/1022 cm−1 were used to estimate the short-range ordered structure of starch [FTIR spectra of the starch samples was measured using a Vertex 70 FTIR spectrometer  equipped with a Deuterated triglycine sulfate (DTGS) detector. The dried KBr was fully ground and mixed with the dried samples at a percent of 1% . A minor amount of starch was placed in a test groove and pressed. Spectra was taken from in the range of 3200–100 cm testing . The fulThe gelatinization characteristics of the starch samples were characterized using a Q20 DSC . The baseline and furnace temperature were calibrated using indium, prior to testing. Nitrogen was used as purge gas at the rate of 15 mL/min. Starch samples (3.0 mg) were weighed directly in an aluminum pan, and distilled water was added to approximately 10.0 mg. The pan was hermetically sealed and allowed to equilibrate for 12 h at room temperature. The sample pans were heated from 30 °C up to 120 °C at the rate of 10 °C/min . A sealeP < 0.05) were conducted using the IBM SPSS Statistics 22.0 Software Program .Results were reported as the mean values and standard deviations of at least duplicate measurements three times. An analysis of variance (ANOVA) by Duncan’s test , peak temperature (Tp), conclusion temperature (Tc), and the enthalpy change (ΔH). The To and Tc of WMS were observed to start slightly prior after APPJ treatment. Similar results were by Zhang et al. and Chen [H of WMS was decreased from 16.78 to 14.81 by APPJ treatment (H has been reported to be influenced by the crystallinity structure of starch and granule stability [H of rice and tapioca starches by plasma treatment, respectively. Both the depolymerization and etching caused by plasma species might result in the decrease of ΔH [To, Tp, and ΔH were found (H of WMS was more obvious.The DSC thermograph of WMS and NMS by APPJ treatment was present in and Chen ,35. The and Chen . Furtherreatment . The ΔH tability . The dectability . Thirumdtability  and Wongtability  reportedse of ΔH ,29. Meanre found . In summIn summary, APPJ modification as an emerging eco-friendly technology changed the structure and physicochemical properties of WMS and NMS. Plasma etching caused by plasma species resulted in slight breakage of the surface of the starch granules, which further induced changes in the functional properties, such as increases of WBC and SV, and decreases in gelatinization temperature and enthalpy. As the treatment time increased, the RC of maize starches after APPJ modification was decreased, due to the depolymerization of starch by the active species of plasma. The FTIR and Raman results indicated that the short-range molecular order was slightly reduced. This study showed that APPJ could be employed as a novel physical method for starch modification. Thus, we expect that this study will motivate further research in the field of the green production of starch products.


We have read with great interest the paper published in Iranian Journal of Public Health in Sep 2016  were screened with these tests in Poland and 303 of them were revealed HIV positive  – that s


The Zika virus disease, also known as Zika fever is an arboviral disease that became epidemic in the Pacific Islands and had spread to 18 territories of the Americas in 2016. Zika virus disease has been linked to several health problems such as microcephaly and the Guillain–Barré syndrome, but to date, there has been no vaccine available for Zika. Problems related to the development of a vaccine include the vaccination target, which covers pregnant women and children, and the antibody dependent enhancement (ADE), which can be caused by non-neutralizing antibodies. The peptide vaccine was chosen as a focus of this study as a safer platform to develop the Zika vaccine. In this study, a collection of Zika proteomes was used to find the best candidates for T- and B-cell epitopes using the immunoinformatics approach. The most promising T-cell epitopes were mapped using the selected human leukocyte antigen (HLA) alleles, and further molecular docking and dynamics studies showed a good peptide-HLA interaction for the best major histocompatibility complex-II (MHC-II) epitope. The most promising B-cell epitopes include four linear peptides predicted to be cross-reactive with T-cells, and conformational epitopes from two proteins accessible by antibodies in their native biological assembly. It is believed that the use of immunoinformatics methods is a promising strategy against the Zika viral infection in designing an efficacious multiepitope vaccine. Zika Virus is an arbovirus of the genus Flavivirus known for causing Zika disease/fever in humans. Zika virus originates from Africa ,2, and iAlthough Zika is a potentially serious disease, there has been no medication or vaccine for Zika. Current treatments like rest, administration of fluids, and analgesics ,4 only dThe peptide vaccine is seen as a safer platform for vaccine development. By only using the parts of a protein that can elicit an immune response, unnecessary components that are potentially antigenic can be eliminated. In the case of Zika, the CD8+ T-cell activity, a target of this vaccine, has been shown to play a protective role against ADE in the dengue infection ,17. Immuhttp://www.ncbi.nlm.nih.gov/). The 39 American sequences were obtained from the BioProject accession: PRJNA344504 [Polyprotein sequences for Zika viruses were obtained from the NCBI database (NA344504 . http://www.rcsb.org/pdb/). In addition, some of these proteins would not be readily available and require modeling. For proteins to be modeled, a 3D structure of a template protein was downloaded instead. Template-based protein structures were decided based on their correlation to the target protein, sequence similarity, and model quality. Visualization of protein structures was done by the software DS Visualizer [Three-dimensional (3D) protein structures with ID codes were required for Zika proteins and HLA molecules  were obtained from the Protein Databank (PDB)  were employed by using the recommended method. The recommended method combines prediction results from several prediction methods into one consensus result when possible [The protein sequence in the FASTA format was used as input for the prediction tools. The prediction was done using NetCTLpan1.1  which uspossible . Predictpossible ,33, NetMpossible ,35, SMM possible  and Combpossible . http://tools.iedb.org/mhcii/) by using the recommended method. The IEDB recommended it combines the results from several prediction methods to obtain one consensus result when possible [The protein sequence in the FASTA format was used as input for the prediction tools. The prediction was done at IEDB  [The predicted T-cell epitopes were then subjected to a series of analyses. Analysis of the pMHC-I- T-cell receptor (TCR) binding was done for MHC-I epitopes using the IEDB Class I Immunogenicity Predictor (nicity/) , where nnicity/) . Followihttp://tools.iedb.org/population/) [The population coverage analysis was then done on conserved epitopes. A list of HLA alleles that would bind to each epitope was gathered. The program NetMHC 4.0 ,35 was ulation/) .Following the population coverage analysis, ten best epitopes for each HLA class were gathered. A cutoff of 80% averaged coverage was used, then the numbers of strong (SB) and weak binders (WB) were considered. To give higher weight on strong binders, WB was given half the value of an SB. Peptide antigenicity was checked by comparing each epitope to the complete sequence that ran through the EMBOSS antigenic  and lookhttp://www.iedb.org) [Prediction for linear epitopes was done using BepiPred-2.0  and LBtoedb.org) .To predict conformational epitopes, three-dimensional Zika protein structures were obtained either directly from PDB or through modeling. The prediction was done using DiscoTope 2.0  by uploaSimilar to the T-cell, consensus epitopes were passed through toxicity, conservancy, and antigenicity tests, then their similarity to human proteins was checked using BLASTp . In simiAll modeling was done using MODELLER 9.19 . For theModeling for proteins with minimal (1aa) difference from the target sequence was done by changing only the residue of interest while leaving the other residues rigid. MODELLER would then optimize the new residue. PDB structures of both the epitope (ligand) and MHC were used in docking. Epitope 3D structures were generated using PEP-FOLD3  by inputDocking preparation was done in AutoDock Tools . FirstlyA molecular dynamics simulation was performed to study the stability of docked peptide-MHC complexes. Docked epitopes were complexed with their HLA receptors and became input for MD simulation performed by GROMACS 2018 ,63. The force field AMBER99sb-ILDN  was usedMetsky et al.  uploadedThe HLA alleles used in this study are summarized in Two predictors were used in both finding MHC-I epitopes (HLA-A and B) and MHC-II (HLA-DR and DQ) to generate consensus epitope-HLA pairs. Two hundred and seventy seven epitope-HLA pairs were discovered for MHC-I, and 95 pairs for MHC-II. By ignoring HLA, there were 175 unique peptides for class I and 67 for MHC-II. To ensure the epitopes would work well as a vaccine component, the quality of these epitopes was then further evaluated by running them through immunogenicity, toxicity, conservation and population coverage tests. Ten of the best epitopes for MHC-I and II each were obtained. After further antigenicity and autoimmunity testing, nine MHC-I epitopes  and eighNine selected epitopes for MHC-I and eight selected epitopes for MHC-II were used for the molecular docking study. Each epitope was paired with an appropriate HLA molecule predicted to strongly bind the epitope. By looking at the NetMHC and NetMHCII results alongside the initial screening results, one HLA was selected to be paired with each epitope based on the binding quality, allele frequency in Southeast Asia, and prediction reliability (for NetMHCII). Some class II HLA structures were not available in PDB and had to be modeled. Structures for HLA-DQA1*05:01-DQB1*03:03, HLA-DQA1*02:01-DQB1*03:03 and HLA-DRA/DRB1*09:01 were generated using the homology modeling, while DRA/HLA-DRB1*15:02 that has only one residue difference from its reference structure was point mutated to its receptor. The PDB structures used to model these HLAs are listed in i) by using the formula:The AutoDock Vina uses its scoring function to calculate inter-atomic interactions and the difference of the Gibbs free energy (∆G) value, which depicts the binding energy between the protein and ligand ,82. ThisPeptide docked poses are displayed in The stability of HLAs and peptide epitopes were tested by 20 ns MD simulation. As a comparison, the same simulation was also done for the unbound form of the receptors. The RMSD analysis for WFHDIPLPW-HLA-C*04:01 a showed Root mean square fluctuation (RMSF) values depict the degree of movement of each residue. In both cases (WFHDIPLPW and WAIYAALTT), the overall RMSF appeared higher for the unbound structure, indicating a lower stability . The lasLastly, the radius of gyration (Rg) analysis depicts structure density by calculating the atomic root mean square deviation (RMSD) from a center of mass . A prote2 end, which was closed by Arg62 and Trp167 [The conformational analysis revealed several changes in the bound ligand WFHDIPLPW after 20 ns of MD simulation . The docd Trp167   that had passed immunogenicity (MHC-I only), toxicity and conservancy tests. Epitopes EWFHDIP (E), WRDRYKYHPDSPR (NS1), and DVPAPKE (NS3) were found to share epitopic residues with MHC-I, whereas epitopes LDPYWGDVKQD and WMDARVCSDHA from NS3 shared epitopic residues with MHC-II.Initially, a PDB search for tertiary Zika protein structures was performed. The only usable tertiary structures came from proteins C, E, prM and M, NS1, NS2B, NS3 protease, NS3 helicase and NS5. No tertiary structure that could be used as a template for proteins NS2A, NS4A and NS4B was found. To use the American consensus sequence, proteins prM, NS3 (protease and helicase domains) and NS5 were modeled using homology modeling and NS1 using point mutation H1R. The list of proteins, reference structures and modeling methods are presented in Epitope prediction was first performed in its monomeric form, and then if possible as a biological assembly. The complete list of epitopes can be found in After acquiring pathogenic gene mutations, the Zika virus reached America and caused an outbreak in the naïve population ,6,7. To The specificity of each HLA’s epitope recognition  means thSome HLAs have been associated with protection or vulnerability to certain diseases; examples include Dengue type 2 protective HLAs HLA-DRB1*04 and HLA-DRB1*07 that have been reported to have lower frequencies in the patient population than the healthy population . IncludiScreening for T-cell epitopes was done for all 26 MHC-I and II HLA molecules. A peptide was said to be epitope for its respective HLA if it was predicted to bind strongly to the HLA molecule. Two predictors were used in both finding class I MHC epitopes (HLA-A and B) and class II (HLA-DR and DQ) to generate consensus epitope-HLA pairs.Consensus epitopes were then passed through several tests to ensure their quality as a vaccine component. For class I MHC, epitope immunogenicity was firstly evaluated. In this case, immunogenicity is defined as the ability of a peptide-MHC-I complex to bind with a T-cell receptor . PeptideWhen checking coverage for MHC-I, coverage numbers for Central America were very low , and thuEpitopes were then screened for the presence of antigenic residues. While this method is usually aimed towards B-cell epitope screening, T-cell epitopes may also come from digested B-cell epitopes . TherefoThe remaining peptides have passed various tests to ensure their quality as a vaccine component. The peptides are predicted to be able to strongly interact with MHC molecules, and be physicochemically and biologically suitable for use in humans to safely elicit the desired immune response. The coverage analysis over various geographic regions also means that these peptides would be suitable for use in a larger population.Mirza et al.  describeThe structure of HLA used will affect the epitope docking result. To obtain the desired docking conformation, holo structures of receptors were used, with the assumption that in real conditions, both the HLA and epitope will adopt conformations similar to the holo structure. Most bound MHC ligands also have a similar conformation ,90. TherFrom nine docked conformations returned by AutoDock Vina , one conIn the case of MHC-II, receptors from homology modeling had an overall lower quality when compared to their reference structures , and sinInter-protein interactions are dominated with non-covalent interactions . HydrogeMore detailed analysis of each interaction is required to obtain a more accurate interpretation. The complete list of hydrogen bonds for all epitopes is available in In the context of epitope searching, molecular dynamics (MD) simulation can be used to observe the stability of a peptide-MHC complex and evaluate the quality of the epitope ,97. OverWhen correctly bound to HLA, a typical nonameric peptide would have the residue in each position interact with the HLA molecule in a manner specific to that HLA  . The conThe discharge of residue nine from its pocket possible indicates an incompatibility with interacting HLA residues. Threonine itself is a polar amino acid, which goes against the accepted residues in the ninth position, Phe, Leu, Val, and Ile, all nonpolar . During All things considered, the docked epitope WAIYAALTT is sufficiently good for residues one to eight when docked to HLA*C:04:02. However, residue nine should not have been polar. Whether threonine in the ninth position makes this epitope, a bad epitope is yet to be determined. In this respect, however, the simulated structures from the result of each 20 ns of MD simulation analyses cannot be conclusive but open the way to further research developments.Several programs have been developed to predict B-cell epitopes from a protein sequence. Unfortunately, the prediction capabilities of these programs are still rather poor . The linInterestingly, the removal of pr sequence altered BepiPred-2.0 scoring for the M protein, giving rise to a new epitope previously undetected in prM. Care should be taken regarding the completeness of the sequence given for BepiPred-2.0.The elimination method used in this study had several shortcomings where longer epitopes tended to be eliminated in the conservancy test, and shorter epitopes tended to be eliminated in the autoimmunity test. Even so, mitigating these problems would require a variation of epitope lengths, such as trimming into several parts or inclusion of neighboring residues. While in vaccine/antibody design modifications may be required, each epitope was treated as one peptide for simplicity reasons.Despite no epitope having both MHC-I and II epitope residues, five epitopes that share epitopic residues with MHC have better potential as a good linear epitope due to cross-recognition by T-cells. Of particular interest are those recognized by CD4+ T-cells that help in B-cell activation . Among tIn conclusion, all 22 filtered consensus epitopes have the potential to become a linear epitope for B-cells. Among them, five epitopes, EWFHDIP (E), WRDRYKYHPDSPR (NS1), DVPAPKE (NS3), LDPYWGDVKQD (NS3) and WMDARVCSDHA (NS3) are the most potential candidates owing to the presence of T-cell epitope residues. Care should, however, be taken for DVPAPKE for its high similarity to the human protein.Despite conformational epitopes making up 90% of B-cell epitopes , predictThe search for E protein epitopes was done for both its monomeric and E-M heterodimer forms and reported in Most known Zika antibody-antigen complex structures come from the E protein , so it iThe NS1 protein was also checked in both its monomeric and dimeric form b. As witAside from the two proteins accessible extracellularly under natural conditions, the epitope search for other proteins, such as nonstructural proteins, is also interesting for its usefulness in laboratory testing. The epitope search for the C protein revealed several epitopes found only in its monomeric form, whereas no epitope was detected for its multimeric form . This stEpitope prediction using DiscoTope 2.0  uses spaFurther investigation on how the immunogenic peptide could induce T-cell responses by Paquin–Proulx et al. (2017). An interesting result showed that T-cell cross-reactivity could give a positive impact on the ZIKV infection in vitro. Paquin–Proulx et al. successfully demonstrated that the ZIKV CD4+ T-cell response induced by DENV vaccination could promote the appearance of cross-reactive antibodies mediating ADE. However, a better understanding of the interactions between the ZIKV and DENV immunity, such as induction by vaccination or by natural infection, will be necessary to provide safe and efficient vaccines . SeveralBy only using necessary peptides, the peptide vaccine is expected to minimize risks associated with unnecessary antigens. Furthermore, using in silico methods to screen for the best candidates can significantly reduce the time and materials spent in laboratories. In this study, the in silico search has been performed to find peptides that can potentially trigger the immune system, and be a component of the Zika vaccine. For the T-cell, the search method was adjusted for certain regions and their surrounding populations. Potential epitopes for both MHC-I and MHC-II have been screened for from the complete Zika protein sequence. Docking and molecular dynamics simulations have also been performed on the most promising epitopes. Regrettably, MD results were unsatisfactory for the best MHC-I epitope, WFHDIPLPW, which was probably caused by an improper starting docked structure. MD results for the best MHC-II epitope WAIYAALTT were quite promising apart from the harmful effect of one residue’s polarity.Coverage analysis does not account for HLA allele pairing, whereas for some HLA both chains are required to confer their specificity. Finding the frequencies of the allele pairs (chain A and B) is the ideal solution, but it is difficult to do. In this case, the epitope search using globally common alleles might give a better result. The linear and conformational epitopes of the B-cell have been successfully mapped. Several promising B-cell epitopes that have been verified by physicochemical modeling have been reported in this study. Most promising among them are EWFHDIP from the E protein, WRDRYKYHPDSPR from the NS1 protein, and DVPAPKE, LDPYWGDVKQD and WMDARVCSDHA from the NS3 protein, which are also predicted as T-cell epitopes. Of particular interest are LDPYWGDVKQD and WMDARVCSDHA since they would also be recognized by T helper cells which play a role in B-cell activation. Caution should be taken before using DVPAPKE for its similarity to a part of the human proteome.Conformational epitopes for the B-cell have also been searched for, for proteins C, E, prM, M, NS1, NS2B, NS3 (protease and helicase domains) and NS5. Particular attention was given to the proteins E and NS1 that can be found as an extracellular matrix under native conditions. These proteins are the primary antibody targets in the human body. This study also considered the change in epitope residues when a protein is in its monomeric or multimeric form.


The present study applied an in situ CO2–NaHCO3 system to regulate photosynthetic biomass and starch production along with starch quality in a marine green microalga Tetraselmis subcordiformis under nitrogen-depletion (−N) and nitrogen-limitation (±N) conditions.Microalgal starch is regarded as a promising alternative to crop-based starch for biorefinery such as the production of biofuels and bio-based chemicals. The single or separate use of inorganic carbon source, e.g., CO2 (2%)–NaHCO3 (1 g L−1) system stabilized the pH at 7.7 in the −N cultivation, under which the optimal biomass and starch accumulation were achieved. The biomass and starch productivity under −N were improved by 2.1-fold and 1.7-fold, respectively, with 1 g L−1 NaHCO3 addition compared with the one without NaHCO3 addition. NaHCO3 addition alleviated the high-dCO2 inhibition caused by the single CO2 aeration, and provided sufficient effective carbon source HCO3− for the maintenance of adequate photosynthetic efficiency and increase in photoprotection to facilitate the biomass and starch production. The amylose content was also increased by 44% under this CO2–bicarbonate system compared to the single use of CO2. The highest starch productivity of 0.73 g L−1 day−1 under −N cultivation and highest starch concentration of 4.14 g L−1 under ±N cultivation were both achieved with the addition of 1 g L−1 NaHCO3. These levels were comparable to or exceeded the current achievements reported in studies. The addition of 5 g L−1 NaHCO3 under ±N cultivation led to a production of high-amylose starch , which could be used as a source of functional food.The CO2–NaHCO3 system significantly improved the biomass and starch production in T. subcordiformis. It could also regulate the starch quality with varied relative amylose content under different cultivation modes for diverse downstream applications that could promote the economic feasibility of microalgal starch-based biofuel production. Adoption of this system in T. subcordiformis would facilitate the CO2 mitigation couple with its starch-based biorefinery.The in situ COThe online version of this article (10.1186/s13068-019-1523-7) contains supplementary material, which is available to authorized users. The sta2–KI solution, absorbancies at 618 and 550 nm were measured. The Am/Ap was estimated from the ratio of the absorbancies by a graph in which the specific absorptions of the two compounds were introduced. Amylose content in total starch , amylose content in dry weight , amylose concentration  and amylose productivity  were calculated using the following equations:Amylose/amylopectin ratio (Am/Ap) was determined according to Hovenkamp-Hermelink et al.  with min2 and NaHCO3) and nitrogen source (KNO3) to produce 1 kg of biomass at laboratory production scale was calculated according to Nayak [V, L kg−1 biomass) was calculated as follows:The cost of carbon source (COto Nayak . Culture2 consumed for 1 L culture (−1) was calculated according to the following equation:2 concentration in the air (2%), F (vvm) was the aeration rate (0.4 vvm), −1) was the relative molecular mass of CO2 (44 g mol−1) and 24.5 was the molar volume of gas (L mol−1) at 25 °C (298.15 K). The cost of carbon source and nitrogen source for 1 kg of biomass  was calculated according to the following equation:2 (0.015 $ kg−1 [3 (0.2 $ kg−1 [3 (0.93 $ kg−1 [3 and KNO3 consumed for 1 L culture (kg L−1), respectively. The biomass value  based on starch quality  was assessed as follows:Psta was the price of starch ($ kg−1 starch). Since Psta is significantly affected by Am/Ap [Psta was proportional to the amylose percentage of total starch . The Psta was then estimated by extrapolation from the price of normal starch (0.29 $ kg−1 for Cam/ts of 27%TS) and high-amylose starch (6.35 $ kg−1 for Cam/ts of 60%TS) according to the Cam/ts obtained under different cultivation conditions (Table The amounts of CO3 $ kg−1 ), respecby Am/Ap , it was p < 0.05 were defined as statistically significant.Results are expressed as mean ± SD from three independent experiments. SPSS 16.0 software  was used to perform the statistical analysis. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) and Fisher’s LSD. Values of Additional file 1: Figure S1. The DIC species distribution of T. subcordiformis cultures. Figure S2. The total alkalinity (TA) of T. subcordiformis cultures. Figure S3. Cell morphology of T. subcordiformis cultures with different amounts of NaHCO3 addition under nitrogen depletion and nitrogen limitation.


Organelle genome studies of Fabaceae, an economically and ecologically important plant family, have been biased towards the plastid genome (plastome). Thus far, less than 15 mitochondrial genome (mitogenome) sequences of Fabaceae have been published, all but four of which belong to the subfamily Papilionoideae, limiting the understanding of size variation and content across the family. To address this, four mitogenomes were sequenced and assembled from three different subfamilies .Tamarindus, Libidibia, Haematoxylum, and Leucaena) in two subfamilies  occurred in relatively deep nodes, and was mainly caused by intercellular gene transfer and/or interspecific horizontal gene transfer (HGT). The second, more recent expansion occurred in the Papilionoideae as a result of duplication of native mitochondrial sequences. Family-wide gene content analysis revealed 11 gene losses, four  of which occurred in the ancestor of Fabaceae. Losses of the remaining seven genes  were restricted to specific lineages or occurred independently in different clades. Introns of three genes  showed extensive lineage-specific length variation due to large sequence insertions and deletions. Shared DNA analysis among Fabaceae mitogenomes demonstrated a substantial decay of intergenic spacers and provided further insight into HGT between the mimosoid clade of Caesalpinioideae and the holoparasitic Lophophytum .Phylogenetic analysis based on shared mitochondrial protein coding regions produced a fully resolved and well-supported phylogeny that was completely congruent with the plastome tree. Comparative analyses suggest that two kinds of mitogenome expansions have occurred in Fabaceae. Size expansion of four genera (This study represents the most exhaustive analysis of Fabaceae mitogenomes so far, and extends the understanding the dynamic variation in size and gene/intron content. The four newly sequenced mitogenomes reported here expands the phylogenetic coverage to four subfamilies. The family has experienced multiple mitogenome size fluctuations in both ancient and recent times. The causes of these size variations are distinct in different lineages. Fabaceae mitogenomes experienced extensive size fluctuation by recruitment of exogenous DNA and duplication of native mitochondrial DNA. Viscum scurruloideum at 66 kb  have been reported within species of various crop plants . Chang iosperm)  suggestecox2 gene from Leucaena and other legumes suggested a sequence insertion (ca. 2.9 kb) into the intron   using custom options . De novo assembly was conducted with low sensitivity in Geneious using approximately 30,000,000 plastome-filtered reads. All plastome-filtered reads were then mapped against assembled contigs. Putative nuclear genome contigs with low coverage (< 100X) were excluded. Among the remaining contigs, high coverage nuclear contigs  were excluded and mitochondrial contigs were selected by BLAST searches against reference Fabaceae mitogenome sequences at NCBI (https://www.ncbi.nlm.nih.gov/genome/organelle/) using BLASTN 2.8.0+ [Four species of Fabaceae, z et al. . Voucherz et al.  were usedica KJ4603, L. coTo complete and validate mitogenome assemblies, polymerase chain reactions (PCRs) were performed with primers designed in Primer3 . Primer Psilotum nudum: KX171638 and KX171639; Ginkgo biloba: KM672373; Liriodendron tulipifera: NC_021152) as BLAT reference sequences, each of which contained a set of 41 conserved, ancestral seed plant protein-coding genes [https://www.ncbi.nlm.nih.gov/genome/organelle/). The tRNAs were also checked by tRNAscan-SE v2.0 [In addition to the four completed mitogenomes, eight previously published mitogenomes were reannotated  were investigated using the CENSOR webserver  with defThe distribution of two kinds of repeats (tandem and dispersed) was examined in each mitogenome. Tandem repeats were analyzed using Tandem Repeats Finder version 4.09  with defhttps://www.ncbi.nlm.nih.gov/genome/organelle/; accessed on Oct. 16, 2018).To estimate size variation in mitogenomes of seed plants, 106 available complete mitogenomes from NCBI were evaluated (Cucurbita and Rosa) and two genera of Malphigiales  as outgroups  analyses were conducted on data from both genomes. In addition to 12 representative Fabaceae species, taxon sampling included two other genera of the nitrogen-fixing clade  were performed as described above.To test the phylogenetic position of Shared DNA among the 12 Fabaceae mitogenomes was estimated by pairwise comparisons using BLASTN with the same parameters as the repeat analyses. To avoid overestimation of shared DNA by including large repeats, all but one copy of repeats > 1 kb was manually deleted from all mitogenomes. Pair-wise Kimura 2-parameter (K2P)  genetic Lophophytum  as a query and 12 Fabaceae mitogenomes as a subject. A similar comparison was performed with Leucaena as a query and the 12 species (11 other Fabaceae species and Lophophytum) as the subject. The percentage of shared DNA between the comparisons was calculated by dividing the values for Lophophytum by the values for Leucaena.Shared DNA was also estimated to detect HGT using concatenated sequences of the multipartite mitogenomes (KU992322–KU992380 and KX792461) of the holoparasitic Additional file 1: Table S1. Assembly information of four completed Fabaceae mitogenomes. Table S2. Summary of repeat sequences in mitogenomes of Fabaceae. Table S3. Pairwise comparison of shared DNA (kb) among the mitochondrial genome of Fabaceae species. Table S4. Pairwise K2P distance among the mitochondrial genome of Fabaceae species. Table S5. Primer sequences and resulting amplification products from mitogenome finishing. Table S6. Accession numbers of mitogenomes used in comparative genomics and phylogenetic analyses. Table S7. Mitochondrial and plastid genes included in phylogeny reconstructions.Additional file 2: Figure S1. Read coverage of four complete Fabaceae mitogenomes. Figure S2. Factors contributing to mitogenome size in 12 representative Fabaceae. Figure S3. Gene content of the 12 Fabaceae mitogenomes. Figure S4. Maximum likelihood phylogenies for rpl2 and rps19. Figure S5. Variation in the cox2 gene among Fabaceae mitogenomes. Figure S6. Schematic relationships of shared mitochondrial DNA between Fabaceae and holoparasitic Lophophytum .


To the Editor: In the article “What Are the Odds You Will Read This Article?” published in the Croatian Medical Journal (CMJ will not misunderstand the article and assume that there is a controversy about the causal link between estrogen and uterine cancer ( Journal , Hrabač  Journal . Estroge Journal . Therefoe cancer ,3. The ae cancer .For non-expert audiences a presentation of odds ratios in regard to non-collapsibility and its interpretation that is sometimes similar to risk ratios would have been a much better choice because that is what scientists really care about most of the time. A few added references may help readers to understand the odds ratios in practice -6.There exist innumerable examples of questionable exposure-disease associations that could have been selected to illustrate points about measures of association in epidemiological studies. A statistical sketch based on wrong premises and not on veritable published medical knowledge might mislead the readers rather than explain concepts about association between two binary variables. Finally, to appreciate the association and causation in epidemiological studies we recommend to readers the historical publications by Bradford Hill, for example “The Environment and Disease: Association or Causation?” .


Journal of Foot and Ankle Research (JFAR). We would like to thank our predecessors Professor Hylton Menz, Professor Alan Borthwick and Mike Potter for all the hard work over the last 10 years. During this time they have served the Podiatric Community with dedication, commitment, and diligence while bringing to the journal a strong vision and a wealth of knowledge that has tremendously improved the visibility and impact of the journal. Our three predecessors have been recognised for their work as Emeritus Editors for the journal. The new editorial team will continue to seek their expert guidance for taking JFAR to further heights of recognition and readership.It is a great honour and privilege to be appointed for the new editorial team of the Professor Bowen and I have had the privilege of serving as Deputy Editor and Associate Editor, respectively for JFAR. Therefore, we understand the nuances and importance of peer review in the manuscript handling process. We believe that scientific values must be upheld in manuscripts and that the editorial team should work efficiently. The editorial team will be strengthened by new Deputy Editors  and Associate Editors , as well as members of the editorial board and advisors from all over the globe.We believe that the most critical components of any scientific journal’s success are the submission of high-quality manuscripts, the dedication of members of its Editorial Board, and excellence of those reviewing the manuscripts. These days, when publication in major journals can take several months after a paper is accepted, the open-access online format of JFAR provides a platform for fast publication without undue delay.Looking back over its last 10 years, the journal has published on a wide diversity of research. Perhaps the most rewarding aspect of the journal has been the opportunity provided to first-time authors to have their work published. There is a bias in academic publishing towards those experienced academics with established reputations, and it is vital that early career scholars are given the prospect of publishing their work. Any academic journal is by definition a collaborative activity, involving the energy and goodwill (and often the patience!) of authors, reviewers, editorial team and production staff. Another lesson we learn from innovation is that it is a continuing process, and change is inevitable and to be welcomed.As so many of the articles in this journal argue, innovation is a long-term concern, and assessing its achievements requires a long-term perspective. Our vision would be initially to continue with the current format but in the future consider broadening the scope of the journal by adapting ‘new emerging themes’ through proactively seeking and promoting thematic issues. This future vision will need to be endorsed by Australian Podiatric Association, College of Podiatry (UK) and ably supported by our allies in the Canadian Federation of Podiatric Medicine. Finally, we would like to thank at this very early stage all those involved in building the journal over the years, and especially all the authors and referees for all their hard work in enhancing scholarship. The journal is now well established. It has a first-rate publisher in BMC; with excellent distribution, and is included in ISI Web of Science. The number and quality of the submissions continues to improve from its already strong base.Professor Catherine Bowen and Professor Keith RomeEditors-in-Chief


ClinicalTrials.gov, PubMed, a geodatabase and geographic maps to enable geospatial study search and real-time rendering of study locations or research networks on a map. Thus, disease-specific clinical studies or whole research networks can be shown in a geographic proximity. Moreover, a semantic layer enables multilingual disease input and autosuggestion of medical terms based on the Unified Medical Language System. The portal is accessible on https://studyportal.uni-muenster.de. This paper presents details on implementation of the novel search platform, its search evaluation and future work.StudyPortal was implemented as the first multilingual search platform for geographic visualization of clinical trials and scientific articles. The platform queries information from  ClinicalTrials.gov provide powerful search platforms to study the current clinical research landscape [Clinicaltrials.gov [ClinicalTrials.gov, a user can select a number of search fields and will gain access to a list of registered trials that meet the search criteria. Each listed trial provides information, as for example, the study design, its therapeutic area or medical condition, sponsors, principal investigators and more importantly for the scope of this work: the study site locations. By linking these locations to geographic coordinates and further processing, we unlock two use cases for clinical research and patient care, which are highly relevant but yet unexploited:International trial databases as andscape –3. The iandscape , 5. A laandscape . Howeverials.gov . Sensitiials.gov . Using tFirst, health care providers and their patients suffering from cancer or chronic diseases could access an overview of suitable clinical trials with potentially new suitable treatment options, close to the patient’s place of residence or patient’s preferred location.Second, clinical researchers could generate a map-based overview of clinical research networks that have conducted similar research and therefore could synergistically share expertise. This is particularly useful when new research networks are being formed or extended and suitable research partners need to be identified.Currently, both of the presented use cases are only realizable by several tedious manual searches. In addition, more advanced location queries, as for instance: “Find the nearest clinical studies within a given distance of 200 km from a specific location” are not executable, since geospatial relations  of studies are not available in clinical trial databases.Clinicaltrials.gov as trial registry, PubMed as medical literature database and a geodatabase in order to render a geographic map of relevant trials or research networks in real-time. As a patient-oriented feature, the search function should support multilingual entry and autosuggestions of diseases to find matching trials. This way, medical terms by laypeople and/or non-English speaking users are mapped to medical concepts, e.g. heart attack or ataque al corazón (Spanish) or Herzinfarkt (German) are mapped to the same concept “myocardial infarction”.The objective of this work is to implement a novel research platform that processes As previous work, key requirements of both use cases were identified as a result of semi-structured interviews with two patient support groups (inflammatory bowel disease and rheumatoid arthritis) and two senior physicians at the local university hospital of Münster in Germany. Four key requirements were identified, which are currently not available on existing international trial registries to the best of our knowledge. KF 1: Generation and visualization of research networks as graphs on a geographic map. Each node of the graph represents a study site of a conducted or conducting trial with facility details on the map. An edge between two nodes represents a collaboration of two sites in one conducted trial. KF 2: Multilingual entry of medical conditions with support of autosuggestion to determine the actual disease concept. Each autosuggested concept is linked with an official description by the Medical Subject Headings (MeSH). KF 3: The search can be filtered for a specific perimeter to find suitable studies within a preferred proximity. KF 4: Further nodes and edges should be shown for any suitable publication found on PubMED (MEDLINE database) in order to boost sensitivity of the research networks and to go beyond clinical trials. That is, if an article is tagged with a MeSH term that is semantically equivalent to the entered medical condition, the affiliations of the lead authors (defined as the first two and last two authors) will be analyzed with the geodatabase and the corresponding locations of the affiliations will be added to the research network. Duplicates will be removed by crosschecking Clinical trial (NCT-IDs) and publication identifiers (PMIDs). Figure ClinicalTrials.gov and PubMed, as both of them use MeSH terms. Trial information is frequently imported from a relational database by ClinicalTrials.gov into StudyPortal’s PostgreSQL database. Imported data contains information on trials, facilities and sponsors including facilities’ locations and ZIP codes, city names and country names. The facilities are mapped against a geospatial location by using the geonames.org database [ClinicalTrials.gov pointing to the specific city ; 2) a city of a specific country mentioned in the affiliation text. 3) a city with the highest population (if multiple cities with the same name would be matching and no country information was available).StudyPortal is a Java-based web service implemented as a REST API using Leaflet Library  as frontdatabase  and thendatabase . PubMed database . Articlenot visualized correctly. To efficiently assess completeness and correctness of visualization, a specific visualization-test platform is accessible via the sub-URL https://studyportal.uni-muenster.de/researcher-network. Here, NCT-IDs of trials and PMIDs of scientific articles can be entered directly and the corresponding research network will be visualized immediately without requiring further user input but using the same core databases and the aforementioned integration procedures. Details of evaluated studies and article are provided in the supplement [The evaluation on information completeness and visualization correctness for this platform is based on manual cross-checking on other external well-established sources: The WHO International Clinical Trials Registry Platform (ICTRP)  for clinpplement .https://studyportal.uni-muenster.de. Import routines to retrieve data from the three core databases ClinicalTrials.gov, MEDLINE and Geonames.org are running on a monthly basis. Figure ClinicalTrials.gov for detailed study description. Using a navigation view, the user can select a specific study and visualize the corresponding subnetwork as illustrated in Fig. The platform is accessible on While the search radius can be freely selected, the system can directly visualize global study networks: Fig. ClinicalTrials.gov or MEDLINE. Full tables on evaluation with details on the misses are provided in the supplement [Expert-based cross-comparison showed that 14 out of 15 (93%) articles and 11 out of 15 (73%) trials were retrievable on StudyPortal. Among of those, all of them (100%) were visualized correctly according to their geographic position. All five misses  were caused by information gaps in The integration of geodatabases, the largest international trial registry, and the largest biomedical literature database was unexploited so far. StudyPortal links these well-maintained but disconnected sources to generate an unprecedented view of studies and research networks on geographic maps. Though all aforementioned key features are implemented and running, the early implementation state cannot encompass further relevant functionalities, which are shortly discussed. Future work will enable extensive filtering of study recruitment status, study phases, interventions, study design and a set of PubMed advanced search filters. These data are already available in structured format and will be planned for next major software release. Moreover, approaches to analyze for hot spot research networks using graph theory-based indicators as centrality measures enables visualization of network evolution over time are subject to current implementation plans as well.ClinicalTrials.gov database is not a dedicated international trial database as the WHO study registry. For instance, three of our 15 test trials were Japanese trials that were not listed on ClinicalTrials.gov. Moreover, many PubMed articles might not be found by our system since these articles were only e-published but not added to the MEDLINE exports of PubMed.Our results indicate that scientific articles could be found and visualized correctly more often (93% vs 73%) than clinical trials. This observation was not surprising as almost all of the tested Web of Science articles were addressing research-intense disease entities and our implementation utilized PubMed, which is one of the largest and most used biomedical databases . The lowClinicalTrials.gov [ClinicalTrials.gov.Incomplete study registration is a major limitation of the integrated data sources . The Stuials.gov . These iials.gov . As prelStudyPortal is the first platform to enable a geospatial overview of biomedical literature and clinical trials. For the majority of tested studies, the presented platform enables an accurate visualization of the study landscape.


Concerns have been raised that the transplants performed in the local context at the time of procedures reported in this article  may haveDetails as to the donor sources and methods of obtaining informed consent from donors were not reported in , and wheThe authors stated that no vulnerable populations were involved in their research and they provided a letter from the Ethics Committee of Human Organ Transplantation at First Affiliated Hospital, Zhejiang University which confirmed that the organ donations involved in the article took place 2001–2012, and that these organ donations received ethics approval and conformed to local regulations. However, the authors did not provide donor consent forms in response to the journal’s request, and the document provided by the Ethics Committee of Human Organ Transplantation did not specifically support the authors’ claim about vulnerable populations or clarify whether organs had been procured from prisoners.In addition, the Methods section of  did not The authors did not respond to inquiries about the availability of underlying data supporting this study.PLOS ONE Editors retract this article.Owing to the lack of documentation to demonstrate this study had prospective ethical approval, insufficient reporting, unresolved concerns around the source of transplanted organs and whether they included organs from prisoners, and in compliance with international ethical standards for organ/tissue donation and transplantation, the The corresponding author notified the journal that all authors disagree with the retraction. XY confirmed their disagreement, the other authors either could not be reached or did not respond directly.


Given the gap between patients in need of a renal transplantation (RTx) and organs available, transplantation centers increasingly accept organs of suboptimal quality, e.g. from donors with acute kidney injury (AKI).To determine the outcome of kidney transplants from deceased donors with AKI (defined as ≥ AKIN stage 1), all 107 patients who received a RTx from donors with AKI between August 2004 and July 2014 at our center were compared to their respective consecutively transplanted patients receiving kidneys from donors without AKI. 5-year patient and graft survival, frequencies of delayed graft function (DGF), acute rejections and glomerular filtration rate  were assessed.Patient survival was similar in both groups, whereas death-censored and overall graft survival were decreased in AKI kidney recipients. AKI kidney recipients showed higher frequencies of DGF and had a reduced eGFR at 7 days, three months and one and three years after RTx. However, mortality was noticeably lower compared to waiting list candidates. Rejection-free survival was similar between groups.In our cohort, both short-term and long-term renal function was inferior in recipients of AKI kidneys, while patient survival was similar. Our data indicates that recipients of donor AKI kidneys should be carefully selected and additional factors impairing short- and long-term outcome should be minimized to prevent further deterioration of graft function. Donorber 2016 . Thus, cber 2016 .Lately, attention was drawn to deceased donors who experienced acute kidney injury  prior to donation . Causes In the context of RTx, contradictory data on the outcomes of kidneys with AKI have been published. There is a series of small studies yielding that despite higher rates of delayed graft function (DGF) in grafts from AKI donors, there is no significant difference in graft function and one-year survival after transplantation between kidneys with and without donor AKI ,10. In cThere is only limited retrospective data on the outcome of kidneys with donor AKI in Germany –14. All We herein provide long-term data on the outcome of kidneys with donor AKI from a large German Transplant center reflecting the characteristics of the German transplantation system.Prior to analysis, data of all patients were anonymized and de-identified. The local ethics committee  approved the study. Methods in this study were carried out in accordance with the current transplantation guidelines and the Declarations of Istanbul and Helsinki. Written informed consent was given by all participants at the time of transplantation for recording their clinical data.We retrospectively analyzed all 107 kidney transplant recipients who received a renal allograft from donors with AKI between August 2004 and July 2014 at the University Hospital Münster. We compared them with their respective 107 patients transplanted immediately afterwards and receiving kidneys from donors without AKI. This matching strategy was applied to avoid bias due to changing therapeutic strategies and guidelines over the relatively long study period of 10 years. We used initial creatinine level and ultrasonography findings for decision making whether to accept or discard an organ as these findings are easily available and do not extend the cold ischemia times, unlike biopsy. Machine perfusion was not performed in any of the cases. Recipients were selected according to Eurotransplant allocation criteria. Patients with combined transplants or missing follow-up data were excluded from analysis.A group of all 703 patients who were on the waiting list at our center from July 2006 up to January 2015 was assessed for survival analysis.Data were collected from the patients’ files and the Eurotransplant network information system (ENIS).AKI was defined according to the AKIN classification in congruence with the largest analysis currently available . CreatinMain outcome measures were patient survival, overall graft survival and death-censored graft survival. Patient survival was defined as time from RTx or day of listing, respectively, to death (from any cause) or last contact for patients alive. Patients on the waiting list were removed from the follow-up on the day they received a kidney transplantation which was considered as a competing event.Overall graft survival was defined as time from RTx to death (from any cause), graft failure or last contact whatever occurred first. Graft failure was defined as reinitiation of dialysis treatment. Death-censored graft survival was defined as time from RTx to graft failure where death without prior graft failure was regarded as censored. Further outcome parameters were incidence of delayed graft function , serum creatinine and eGFR at 7 days, three months and one and three years after transplantation as well as frequency of biopsy-proven acute rejections (BPAR) and rejection-free survival within the first year after RTx.Whole blood was analyzed for creatinine  and renal function was determined by calculating the eGFR using the CKD-EPI equation.st and 3rd quartiles. Absolute and relative frequencies are given for categorical variables. Groups were compared using Student’s t-test for normally distributed data, Mann–Whitney U test for skewed distributed continuous variables and Fisher's exact test for categorical variables.Statistical analysis was performed using IBM SPSS Statistics 25 for Windows . Normally distributed continuous variables are shown as mean ± standard deviation (SD) and not normally distributed continuous variables as median and 1Univariable and multivariable binary logistic regression analysis were performed to estimate the probability of DGF. A stepwise forward variable selection procedure  was performed including the following variables: AKIN stage, recipient age and sex, BMI, time on dialysis, prior kidney transplantation ≥ 1, number of HLA mismatches, current ≥ PRA 20%, induction therapy, cold ischemia time, donor age. All selected variables were then included in a final multivariable model. Results are presented as odds ratios (OR) with 95% confidence intervals. Rejection-free survival was estimated by Kaplan-Meier method and groups were compared by log-rank test .Univariable and multivariable linear regressions were performed to determine independent factors influencing allograft function (eGFR) at indicated time points. Variables included were AKIN stage, recipient age and sex, recipient BMI, time on dialysis, prior kidney transplantation, number of HLA mismatches, current ≥ PRA 20%, cold ischemia time, induction therapy, donor age. Results are presented as regression coefficients (β) with 95% confidence intervals.Survival analyses were based on a maximum follow-up of 5 years after RTx. Patient survival as well as death-censored and overall allograft survival (death was regarded as event) were estimated using Kaplan-Meier method , and groNo adjustment for multiple testing was performed and all analyses are regarded as explorative.Exploratory p-values ≤0.05 were considered as statistically noticeable.During the study period 796 RTx from deceased donors including 111 organs with AKI from 86 corresponding donors were performed at our center. Four patients were excluded due to combined RTx or missing follow-up data. Except for a higher age in recipients of AKI kidneys, the groups were similar with respect to all analyzed baseline characteristics .Donor characteristics are shown in Estimated 5-year patient survival was similar between recipients of AKI and non-AKI kidneys , whereas patients remaining on the waiting list showed a noticeably lower 5-year patient survival (41.2%) compared to recipients of non-AKI (log-rank p<0.001) and AKI kidneys (log-rank p = 0.011) . 5-year Recipients of donors with AKIN stages 1 and 2 had higher frequencies of DGF (n = 31 (34.4%) and n = 4 (30.8%), respectively) when compared to non-AKI recipients (n = 20 (18.7%)). While DGF frequency did not differ between patients with AKIN stage 1 and 2 kidneys, DGF occurred much more frequent in kidneys with AKIN stage 3 with three out of four patients experiencing a DGF. Due to the small numbers of events in some AKI stages, we aggregated these data and compared donors with any AKI and without AKI.DGF occurred more frequently in AKI kidney recipients than in non-AKI kidney recipients in both univariable  and multAllograft function was investigated at 7 days, three months and one and three years post transplantation. Patients who received an organ with AKI showed a decreased eGFR at all investigated time points after transplantation . Donor ARejection-free survival within one year after transplantation was similar between recipients of grafts with and without AKI .The gap between organ supply and demand in kidney transplantation has led to different strategies to reasonably enlarge the donor pool.Some of the approaches aim at an expansion of the living donor pool: While ABO-incompatible transplantation is established in the clinical routine of many transplantation centers, HLA-incompatible transplantation is expected to become increasingly important following data from a large study showing a clear survival benefit in these patients ,18. UntiAt present, over 70% of all kidney transplantations in Germany utilize kidneys from a deceased donor . IdeallySo far, long-term data on the outcome of AKI kidneys is limited. Defined standard criteria to estimate the quality of kidneys with AKI are still missing. To our knowledge, we herein present the largest 5-year follow-up of patients with AKI kidneys from a large German Transplant center reflecting the characteristics of the German transplantation system. The aim of our study was to assist on-call clinicians in their decision-making to accept or decline kidneys form donors with AKI.Our and other German data suggest that AKI kidneys are currently used with caution: Most transplanted AKI kidneys were staged as AKIN 1, only a few AKIN 3 kidneys were regarded as suitable for surgery.Though recent studies on the outcome of kidneys with donor AKI did not show any difference in patient survival, results regarding transplant survival are heterogeneous. Several small studies including the German ones report similar graft and patient survival rates after transplantation despite consistently higher rates of DGF in recipients of donor AKI kidneys –14,19–22We herein separately assessed patient survival, death-censored and overall graft survival. In univariable Cox-regression, the recipient age was not associated with death-censored graft survival. Moreover, multivariable analysis for eGFR did not reveal a significant influence of recipient age on kidney function at any of the investigated time points post transplantation, which strongly suggests that the different outcomes are not driven by a difference in the mean age between the groups. As patient survival was similar between the groups, it makes it even more unlikely that the difference in recipient age might have contributed to the different outcomes, suggesting a negligible impact of potential age-related factors as disease burden, functional status or comorbidities in our cohort. The similar rejection-free survival rates as well as comparable frequencies of the respective allocation system between the groups also makes it unlikely that an age-related higher susceptibility to acute rejections or allocation of lower quality organs to elderly patients might have contributed to the difference in allograft function and survival in the present study.In congruence to the literature, we observed higher rates of DGF in donor kidneys with AKI. DGF is usually recognized to limit function as well as the prognosis of graft and recipients ,29. Inte2 at three months, one and three years after transplantation. Furthermore, in donor AKI recipients we observed a noticeable influence of DGF on short- and long-term kidney function. One theoretical reason for this finding could be a higher rate of expanded criteria donors (ECDs) in our AKI group, which have been shown to be associated with a worse outcome compared to standard criteria donors [Many studies observed that a higher percentage of DGF in the AKI groups did not lead to a clinically relevant decrease in 6-month or 12-month eGFR ,30. Thisa donors , especiaa donors . As we fFurther covariables that were independently associated with DGF and lower eGFR were prior RTx and number of HLA mismatches and recipient BMI and donor age, respectively. This is in accordance with previous studies reporting higher rates of DGF and impaired graft function in presensitized patients ,32. BaseThere is comprehensive literature confirming the influence of early adverse events on long-term allograft outcome. However, it has recently been suggested by Gaston et al. in a prospective multicenter cohort study that late graft failure is not necessarily dependent on pretransplant or early events initiating an inalterable sequence of fibrosis and atrophy, designated as the non-specific diagnosis of chronic allograft nephropathy. It rather represents the real-time impact of new onset graft dysfunction beyond 90 days, regardless of cause, that occurs over the life of an allograft . In the Our study has some limitations. Data was analyzed retrospectively and all inferential statistics have to be interpreted exploratory. Due to the sample size and the small number of events (patient and graft death), the inclusion of all clinically relevant variables in a multivariable model was not possible to reduce further potential bias. No sensitivity analysis or validation of the selected multivariable models was performed. Therefore, the applied statistical models are not valid to prove associations between potential predictors and the corresponding outcomes. Consequently, prospective studies are needed to confirm our findings.Although we have found a reduced graft survival of AKI kidneys, we would like to highlight a few things: We could observe that patient survival between AKI and non-AKI recipients did not differ and was noticeably better compared to patients remaining on dialysis. Therefore, organs with AKI should not be simply discarded. However, as DGF rates are high and prognosis is limited the recipient should be carefully selected and additional covariables that can contribute to DGF or inferior graft function in the short- as well as long-term course  should be considered.S1 FigKaplan-Meier curves for patient (A), death-censored (B) and overall graft survival (C) in recipients of AKI kidney by DGF status. Survival curves and 5-year rates of AKI kidney recipients with DGF (red lines) and without DGF (blue lines) were estimated by Kaplan–Meier method and compared by log-rank test. Band plots represent 95% confidence intervals (log transformation) for pointwise Kaplan-Meier survival estimates. Hazard ratios (HR), 95% confidence limits (CI) and Wald p-values are from univariable Cox-regression. Patients with DGF showed a noticeable reduced patient and overall graft survival . Univariable Cox-regression revealed a noticeable association between DGF and patient and overall graft survival.(DOCX)Click here for additional data file.S1 TableBesides donor AKI, donor age was noticeably associated with death-censored and overall graft survival.HR = Hazard ratio, CI = Confidence interval, ref = reference. P-values are from the Wald tests.(DOCX)Click here for additional data file.S2 TableAdditionally, a multivariable model with stepwise forward variable selection procedure was performed. All variables with p ≤ 0.05 (Likelihood ratio test) were included in the final multivariable model .R = Odds ratio, CI = Confidence interval, ref = reference, P-values are from the Wald tests.(DOCX)Click here for additional data file.S3 TableRecipients of kidneys from donors with AKI show a noticeably decreased eGFR at all times. P-values are from Mann-Whitney U tests.(DOCX)Click here for additional data file.S4 TablePatients with DGF show a noticeably decreased eGFR at all times. P-values are from Mann-Whitney U tests.(DOCX)Click here for additional data file.S5 TablePatients with DGF and no DGF show a comparable eGFR at all time points. P-values are from Mann-Whitney U tests.(DOCX)Click here for additional data file.


The lintnerization followed by autoclaving treatment significantly increased the solubility and water holding capacity, reduced the swelling power, and disrupted the crystalline structure of the starch granules. The native rice starch with autoclave treatment exhibited the highest swelling power among the samples, while the acid hydrolyzed starch was followed by autoclave treatment showing the lowest swelling power (1 g/g) at 90 °C. Fourier transform infrared analysis revealed the modified structures and bonding of the starch materials with the shifting of C=O stretch. However, the antioxidant properties and pasting properties were observed to decrease with the lintnerization, autoclaving, and freeze-thawing treatment of the native starch. The highly resistant starch content and low glycemic index value of the autoclaved RD 31 starch indicates the potential of the resistant starch’s application for the formulation of functional foods targeting the diabetic population.The objective of this study was to assess the effects of lintnerization, autoclaving, lintnerization followed by autoclaving, and freeze thawing treatments on the production of resistant starch from Pathumthani 80 (RD 31) rice. The produced resistant starch was further evaluated for some important physicochemical properties including pasting properties, swelling behavior, digestibility, water holding capacity, and functional properties including glycemic index and antioxidant properties. The lintnerization treatment and autoclaving significantly ( With the increase in consumer awareness toward healthier foods, there is a growing interest in functional foods with a low glycemic index. Glycemic index (GI) is defined as the increment in the blood glucose area following the test food and is expressed as the percentage of the corresponding area following a carbohydrate equivalent load of a reference product . The glyBased on physical and chemical attributes, resistant starch can be classified into five groups as RS 1 , RS 2 (raw starch granule), RS 3 (retrograded starch), RS 4 , and RS 5 (Amylose-lipid complex) . The degree of resistant starch formation is dependent on the nature of native starch and the treatments involved. Lintnerization or acid hydrolysis of α-1,4 and α-1,6 glycosides linkages from amylose and amylopectin develop resistance starch type 3 (lintnerized starch). Acid hydrolysis increases the crystalline content of starch, which, in turn, is resistant to enzymatic hydrolysis . SeveralRice starch is a major source of nutrients and carbohydrates. Normally, rice starch can convert into glucose, which is the main energy source for metabolic function. During long-term overeating, carbohydrates potentially lead to some health problems such as type 2 diabetes, obesity, and colon diseases. Therefore, the development of starch resistant to enzymatic digestion from the native starch is recently gaining interest. The consumption of resistant starch is indicative of lowering the glycemic index. The starch digestion rate, the blood glucose level, and insulin responses of the most rice-based products are determined based on the amylose concentration. Amylose with its tightly packed structure exhibits high resistance to digestion compared to amylopectin . The resThe high amylose content of RD 31 is an important factor for determining its cooking and eating quality, hard texture, and its suitability for the development of resistant starch. The development of functional foods with the fortification of resistant starch has gained interest by food developers and nutritionists mainly due to fiber-fortification and the potential physiological benefits including the lowering of glycemic index . To the Phatumthani 80 (RD 31), which is a type of a paddy rice, was provided by the Pathumthani Rice Research Center, Ministry of Agriculture and Cooperatives (MoAC), Thailand. The resistant starch test kit was bought from Megazyme International Ltd., Ireland. All the other chemical reagents used were of an analytical grade and bought from Sigma-Aldrich, Switzerland.w/v, 1:2) at 4 °C for 24 h. The supernatant was drained, and the rice grains were ground with a blender  and passed through 100 mesh screens. The slurry was centrifuged  at 3000 rpm for 15 min. The supernatant was discarded, and the starch layer was resuspended with a triple volume of deionized water and centrifuged at 3000 rpm for 15 min. This process of washing the starch was repeated 4 times and the final pH was adjusted to 7 with hydrochloric acid (1 N). The starch slurry was further centrifuged at 3000 rpm for 15 min, which was followed by drying in a hot air oven  at 60 °C for 24 h. The dried starch cake was ground by mean of grinder , which was followed by sieving through 100 meshes , vacuum packed  in high-density polyethylene (HDP), and stored in a desiccator at 25 °C for further use. The rice flour (RF) was prepared by milling rice samples (RD 31) with a pin mill  and sieved through a 100-mesh screen . The native rice starch (NRS) was extracted following the method described by Qin et al.  with sliw/v) ratios. The mixture of rice starch and HCl solution was warmed at 40 °C for 3 h. After adjusting the final pH to 6.5 using NaOH solution (10% w/v), the lintnerized starch was washed with distilled water and centrifuged at 1000 rpm for 5 min. The washed sample was dried in a hot air oven at 40 °C, cooled down, ground by using a grinder  into powder form, and passed through 100-mesh sieves . The dried lintnerized starch was vacuum packed into the high-density polyethylene (HDP) pouch and stored in desiccators for further use.Lintnerization (acid hydrolysis) of the native rice starch sample was conducted by following the method, as described by Nasrin and Anal  with sliw/w). The starch sample was gelatinized by heating in a water bath  maintained at 85 °C for 30 min. The pregelatinized starch sample was then autoclaved  at 135 °C for 30 min, cooled down, and stored at 4 °C for 24 h. This process of autoclaving and cold storing was repeated three times for each sample. The lintnerized-autoclaved starch was then dried in a hot air oven at 50 °C, cooled down, ground by using a grinder , sieved through 100 meshes , vacuum packed in an HDP pouch, and stored in the desiccators. Native rice starch and lintnerized rice samples (acid hydrolyzed at different concentrations) were suspended in water (1:10 w/v) was heated in boiling water for 30 min, which was followed by cooling down and freezing the samples at −20 °C for 24 h. The frozen sample was thawed in a water bath at 30 °C for 90 min and dried at 60 °C in a hot air oven until it was dry. The dried samples were milled by means of a grinder  and sieved through 100 meshes , vacuum packed in HDP bags, and stored in a desiccator for a further experiment. Freeze-thawing of native starch was conducted by following the method described by Wang et al.  with somAmylose content of the sample was determined by colorimetric measurement of the blue amylose iodine complex following the method of Juliano  with sli1 = absorbance of resistant starch sample - absorbance of blank, ΔE2 = absorbance of non-resistant starch sample - absorbance of blank, F = 100 (µg of D-glucose) divided by the glucose oxidase-peroxidase-amino antipyrine (GOPOD) absorbance for this 100 µg of D-glucose, W = dry weight of sample analyzed. Resistant starch content of the sample was determined enzymatically using the Megazyme Resistant Starch assay procedure test kit . A sample (100 mg) with the mixture (4 mL) of pancreatic α-amylase (10 mg/mL) and amyloglucosidase (3 U/mL) was incubated in a shaking water bath at 37 °C for 16 h. Ethanol  was added to the mixture to terminate the enzymatic reaction, which was followed by centrifugation (3000 rpm for 10 min). The supernatant was separated and used for determining non-resistant starch. The resistant starch in the pellet was mixed with ethanol , stirred, and centrifuged (3000 rpm for 10 min), and the supernatant was decanted. The pellet was then mixed with KOH solution  with continuous stirring (300 rpm) in an ice water bath for 20 min followed by the addition of sodium acetate buffer  and amyloglucosidase . The mixture was incubated in a water bath (50 °C for 30 min) and centrifuged (3000 for 10 min). The supernatant (0.1 mL) was added to glucose oxidase-peroxidase-amino antipyrine (GOPOD) (3 mL) and incubated (50 °C for 20 min). The absorbance of the mixture was measured using spectrophotometer  at 510 nm. The resistant starch content, non-resistant starch, and the total starch content were calculated using an equation from the kit manual. Total starches were calculated as the sum of resistant starch and non-resistant starch.The in-vitro glycemic index (GI) of the sample was determined following the method described by Goni et al.  with slio = absorbance without extract, Ae = absorbance with extract.According to Sadiq et al. , antioxiFerric ion reducing antioxidant power assay (FRAP) was measured following Sadiq et al.  with a sTotal phenolic content was analyzed following Folin and Ciocalteu  using a The pasting properties of starch samples were analyzed by a Rapid Visco Analyzer . A sample (2.5 g) was mixed uniformly with distilled water (25 mL). The sample suspension was subjected to four settings including preheating at 50 °C for one min, heating until temperature rise to 95 °C, holding at 95 °C for 3.2 min, and cooling to 50 °C. Samples were mixed and homogenized at 960 rpm for 10 s at the beginning of the test, and then speed was reduced to 160 rpm and continued throughout the test. The total analysis time was 13 min. Peak viscosity, trough, breakdown viscosity, final viscosity, setback viscosity, peak time, and pasting temperature were obtained by the rapid visco analyzer (RVA).The swelling power and solubility of the tested samples were calculated following Shrestha et al.,  with sliThe water holding capacity of the sample was determined, according to the method of Rodriguez-Ambriz et al.  with sliThe microstructural images of the sample were taken using a Scanning Electron Microscope (SEM) . The sample particles were sprayed over the adhesive carbon tape fixed with a circular brass stub and placed in a vacuum chamber. After reaching a vacuum of 8 Pa and plasma current of 15 mA, the gold particles were splattered for 90 s. The stub with gold splattered samples was then kept inside the Scanning Electron Microscope (SEM) chamber for imaging. The images were analyzed at 6000× resolution.−1 wave numbers. Each spectrum was the average of eight scans. All measurements were conducted under ambient conditions.The infrared spectra of the sample were measured using an Attenuated Total Reflectance (ATR) FTIR  with a spectra-Tech HATR accessory. The sample was used directly for measuring the spectrum from 500 to 4000 cmColor spectra of the sample were determined by using a Hunter-Lab spectrophotometer . The sample (10 g) was placed in the glass container and put over the slit of the instrument. The average value of 10 measurements was reported and showed as L* (lightness), a* (redness), and b* (yellowness) values. The color intensity (B) was calculated by following Gavahian et al.  and usinAll the experiments were conducted in triplicate and means ± standard deviations were reported. The analysis of variance was performed by one-way ANOVA procedures of MSTAT-C software . Comparisons among the samples were further analyzed by Tukey’s honestly significant difference (HSD) test at a 95% confidence level. The chemical composition of RD 31 rice flour, native rice starch, and modified starch were investigated . The totThe amylose content of the lintnerized or acid hydrolyzed starch (35.57 ± 0.73 to 41.48 ± 0.48%) and lintnerized. This was followed by autoclaved starch (21.70 ± 0.66% to 28.96 ± 0.26), which was observed to decrease significantly. The earlier reports also suggested the decrease in amylose content of starch, including the decrease in amylose content of native culled banana starch from 39.88% to 34.05%  with aciHowever, the amylose content of starch resulting from autoclaving (45.97 ± 0.79%) and freeze thaw treatment (43.59 ± 0.34%) was significantly similar to the native starch. During autoclaving treatment, starch was gelatinized at a temperature of 135 °C under pressure whereby the starch granules become fully disrupted. Upon cooling, the amylose chains can associate to form hydrogen bond stabilized double helices . Moreovep < 0.05) with the lintnerization treatment so that resistant starch content increased with a rise in the concentration of hydrochloric acid starting from 1 N (13.73 ± 0.48%), 2 N (22.99 ± 0.48%), and 3 N (40.19 ± 2.96%). This increase in resistant starch content with the lintnerization treatment might be due to the disruption of amorphous regions by the acid leading to an increase in the ratio of crystalline parts, which are more difficult for enzymes to access [The formation of resistant starch is influenced by several factors, including amylose content, molecule chain length, autoclaving temperature, storage time, and starch gel temperature . The reso access . The resistant starch content was further observed to increase significantly with the lintnerization method, which was followed by the autoclaving treatment. However, the highest value of resistant starch was exhibited by the autoclaved starch (64.95 ± 0.26%). During autoclaving treatment, the starch granules become fully disrupted, which, upon cooling, form double helices stabilized by hydrogen bonds. Thus, the repeated autoclaving-cooling treatments lead to the formation of resistant starch type 3 crystallites, which are resistant to starch hydrolyzing enzymes due to their tightly packed structure . The cryIn this work, analysis of the data trends indicate that more resistant starch could be generated at the higher concentrations of HCl. The lintnerization followed by autoclaving starch had higher resistant starch content than the starch treated by lintnerization alone. As shown in p < 0.05) lower when compared to the native starch (66.32 ± 0.04%). Furthermore, compared with lintnerized rice starch (51.94 ± 0.01–58.94 ± 0.07%), the autoclaved rice starch showed a significantly lower glycemic index value (46.12 ± 0.01 to 56.83 ± 0.07%). All samples, except the starch treated with 1 N hydrochloric acid and the freeze thawed one, could be categorized as low glycemic index starch. The lower GI value of rice flour is due to the presence of various bioactive compounds including dietary fiber, resistant starch, and oligosaccharides. The presence of dietary fiber has been associated with a decrease in glucose, insulin, and serum lipid concentrations in both diabetic and non-diabetic persons [The in vitro glycemic index (GI) of the rice flour, native starch, and the treated starch was investigated . The GI tarch 66. ± 0.04%.p < 0.05) exhibited the highest resistant starch and the lowest glycemic index. In relation to resistant starch content, the higher the resistant starch content is, the slower the digestion of rice and the lower the glycemic index is. Resistant starch decreases postprandial glucose and insulin responses and, hence, decreases the glycemic index of food [The autoclaved starch significantly ( of food . Conside of food .4/100 g) due to the presence of the phenolic compounds in the aleurone layer and the pericarp layer of the rice kernel. The antioxidant properties of rice flour, native starch, and the treated starch were evaluated and expressed in terms of total phenolic content (TPC), 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and the FRAP (Ferric ion reducing antioxidant power) ssay . The antp < 0.05), which might be due to removal of the rice bran layer. Additionally, the acid hydrolysis, autoclaving, and freeze-thawing treatments caused a significant decrease in the total phenolic compounds as well as in antioxidant activity. Fengmei [p < 0.05) in TPC, DPPH, and FRAP was observed between lintnerization, which was followed by autoclaving treatment and freeze-thaw treatment with the lowest value in lintnerized-autoclaved starch samples. Several research studies have reported that free phenolic compounds are more susceptible to loss due to hydrothermal treatment and heat treatment [However, immediately after rice starch extraction, the antioxidant properties reduced significantly  gel formed to its dry weight. It depends on the processing conditions  and is thought of as its water binding capacity. The native rice starch with autoclave treatment (NARS) exhibited the highest swelling power among the samples (19.32 ± 1.06 g/g), while the acid hydrolyzed starch (2 N HCl), which was followed by autoclave treatment (LA 2 NA) showing the lowest swelling power (1.00 ± 0.00 g/g) at 90 °C . SwellinWater holding capacity is greatly influenced by the physical conditions of the starch molecule including dietary fiber, protein, and amylose content . The watRice starch granules are the smallest among the other cereal grains starch and exit as discrete particles. The morphology of RD 31 rice flour, native rice starch, and all the treated starches were photographed under a scanning electron microscope (SEM) at a magnification of 6000X . The surThe shape and size of starch granule were observed to change with acid hydrolysis and heat treatment. The morphology of acid hydrolyzed starch appeared to be rougher than the native starch, which indicated that acid treatment caused the erosion of the core part of the starch granules d. This d−1. The peak intensity and shape of lintnerized factors. Lintnerization was followed by autoclaving. Autoclaved and freeze thawed RD 31 rice starches were significantly different than that of rice flour and native rice starch. The change in peak intensity and shape, therefore, indicates the modification in the starch granular structure and the bonding of the starch substrates with the different treatment.The changes in chemical finger printing of native starch with acid hydrolysis, autoclaving, and freeze-thaw treatments were analyzed by the Fourier Transform Infrared (FTIR) spectrum  and the −1 is related to the pyranose ring in the glucose unit. The anhydrous glucose ring O=C stretching is represented by the peak between 990 cm−1 and 1030 cm−1 [−1. The intensities of the peak of the acid hydrolyzed starch are the feature peaks of the C-O-C and C-O bond of the glycosidic linkage in the starch [−1. The most intense peak of glucose is characteristic of the C-O stretch vibration [−1, which were similar to findings of Ashwar et al. [−1 in autoclaved rice starch. This increased intensity indicates that the autoclaving treatment causes more efficient packing of double helices within the crystalline lamella. Similarly, a research study by Basilio-Cortés et al. [−1 in the spectra of modified corn starch, which signifies the changes in the starch crystallinity resulting from an interaction of hydrochloric acid-starch-succinic anhydride. In all spectra, a composite variation mode in the region below 800 cm030 cm−1 . The OH-e starch . Neverthibration . The sifr et al.  who repos et al.  showed tThe color value of RD 31 rice flour, native rice starch, and modified starch were studied by the colorimeter and expressed in terms of the CIE L*a*b* color system . The natp ˂ 0.05) with the lintnerization treatment and lintnerization followed by the autoclaving treatment with respect to the native starch. In comparison to non-autoclaved counterpart, lintnerization followed by autoclaving treatment starch appeared to be darker in color (p ≥ 0.05) differences in comparison with the native starch.The “L” value was observed to decrease, while “a*” value and “b*” value increased significantly (in color . The effw/w) with the lowest glycemic index (46.12%), which was followed by lintnerized-autoclaved starch. The viscosity of the native rice starch was observed to decrease with lintnerization and autoclaving treatment. The acid hydrolyzed and autoclaved starch showed the highest solubility tendency and swelling power with reduced water holding capacity. The FTIR analysis proved that autoclaving and lintnerization treatments modified the native rice starch at a molecular level. The surface of the modified starch by lintnerization appeared to be rougher, whereas more compact surfaces were observed with the autoclaved starch. The results of this study indicate the potential of the application of the lintnerized and autoclaved Pathumthani 80 rice starch as a source of resistant starch for developing functional foods with a low glycemic value. This study revealed that dual autoclaving and the lintnerization treatment can cause the reorganization of the amylose and amylopectin chains of starch, which significantly improved its physicochemical characteristics and digestibility, and, thereby, lowered glycemic properties. The Pathuthani 80 (RD 31) rice cooked with autoclaving gave the highest resistant starch (64.95%


This article has been retracted: From the The First Hospital of China Medical University Professor Committee, in response to the Journal’s request:The First Hospital of China Medical University Professor Committee convened the 14th plenary meeting to investigate Yu-Ji Li’s paper published in Oncotarget , in response to the Journal’s editorial board’s request. A thorough investigation and open discussion was held in the meeting that included all department members in charge of scientific affairs.At the meeting, Dr. Yu-Ji Li reported all events in the production of his paper. He admitted the problems in the publication that were identified by the Journal and acknowledged that he was responsible for the incidents. The chairman of Dr. Li’s department also detailed on the incidents and pointed out the loopholes in the paper. Inquiries were made and documents were carefully checked by the Committee members. The Professor Committee agreed that Yu-Ji Li was negligent in his research attitude and was unable to offer complete original materials. After the discussion, the decision was made as the following:1) Due to repeated and manipulated use of figures that misled the scientific conclusion, we ask for retraction of the paper by Dr. Yu-Ji Li from the Journal. (Dr. Li himself previously attempted to retract but his plea was unclear in expression).2) Apologies must be issued to the Journal and its readerships for the authors’ negligence and lack of rigorous academic integrity.3) The University will take strict measures against the misconduct by the authors of the paper according to relevant institutional regulations.https://doi.org/10.18632/oncotarget.8289Original article: Oncotarget. 2016; 7:31993-32005.