N6 of A37 (i6A37) of certain tRNAs, are among a minority of enzymes that modify cytosolic and mitochondrial tRNAs. Pathogenic mutations to the human IPTase, TRIT1, that decrease i6A37 levels, cause mitochondrial insufficiency that leads to neurodevelopmental disease. We show that TRIT1 encodes an amino-terminal mitochondrial targeting sequence (MTS) that directs mitochondrial import and modification of mitochondrial-tRNAs. Full understanding of IPTase function must consider the tRNAs selected for modification, which vary among species, and in their cytosol and mitochondria. Selection is principally via recognition of the tRNA A36-A37-A38 sequence. An exception is unmodified tRNATrpCCA-A37-A38 in Saccharomyces cerevisiae, whereas tRNATrpCCA is readily modified in Schizosaccharomyces pombe, indicating variable IPTase recognition systems and suggesting that additional exceptions may account for some of the tRNA-i6A37 paucity in higher eukaryotes. Yet TRIT1 had not been characterized for restrictive type substrate-specific recognition. We used i6A37-dependent tRNA-mediated suppression and i6A37-sensitive northern blotting to examine IPTase activities in S. pombe and S. cerevisiae lacking endogenous IPTases on a diversity of tRNA-A36-A37-A38 substrates. Point mutations to the TRIT1 MTS that decrease human mitochondrial import, decrease modification of mitochondrial but not cytosolic tRNAs in both yeasts. TRIT1 exhibits clear substrate-specific restriction against a cytosolic-tRNATrpCCA-A37-A38. Additional data suggest that position 32 of tRNATrpCCA is a conditional determinant for substrate-specific i6A37 modification by the restrictive IPTases, Mod5 and TRIT1. The cumulative biochemical and phylogenetic sequence analyses provide new insights into IPTase activities and determinants of tRNA-i6A37 profiles in cytosol and mitochondria.The tRNA isopentenyltransferases (IPTases), which add an isopentenyl group to  tRNA isopentenyltransferases (IPTases) are tRNA modification enzymes that are conserved in bacteria and eukaryotes. They add an isopentenyl group to the Adenosine base at position 37, adjacent to the anticodon of specific subsets of tRNAs that decode codons that begin with Uridine. This modification stabilizes the otherwise weak adjacent codon-anticodon base pair and increases the efficiency of decoding of the corresponding codons of the genetic code. IPTases belong to a group of enzymes that modify both cytoplasmic and mitochondrial tRNAs of eukaryotic cells. Interestingly, during evolution there were changes in the way that IPTases are targeted to mitochondria as well as changes in the relative numbers and identities of IPTase tRNA substrates in the cytoplasm vs. mitochondria. The latter is consistent with phenotypic consequences of IPTase deficiencies in fission and budding yeasts, and mammals. Pathogenic mutations to human IPTase (TRIT1) cause mitochondrial insufficiency and neurodevelopmental disease, principally due to decreased modification of the mt-tRNA substrates. In this study, we identify the way human TRIT1 is targeted to mitochondria. We also show that TRIT1 exhibits a tRNA anticodon identity-specific substrate sensitivity. The work leads to new understanding of the IPTases and the variable anticodon identities of their tRNA substrates found throughout nature. N6 of adenosine, found only on tRNAs that decode UNN codons, at position 37 (i6A37) directly 3' to the anticodon. Bacterial i6A37 is hypermodified to 2-methylthio-N6-isopentenyl-A37 (ms2i6A37), whereas eukaryotic cy-tRNA-i6A37 is not. Also, while the mt-tRNAs of S. cerevisiae and S. pombe are not hypermodified, mammalian mt-tRNAs i6A37 are  + 0.41[Pgc] + 81.5 − Pm − (B/L) − 0.65, where M is the molar salt concentration, Pgc is the percent G+C content in the oligonucleotide DNA probe, Pm is the percentage of mismatched bases, if any, B is 675, and L is the oligonucleotide DNA probe length , 100 μg/ml sheared salmon sperm DNA (supplier) at the oligonucleotide DNA probe hybridization temperature for 1 to 2 h. A 32P was confirmed by Phosphor Imager analysis. In general, multiple ACL probings are done followed by the body probes. Before the next hybridization, blots were stripped with 0.1 × SSC, 0.1% SDS at 85–90°C, and the nearly complete removal (≥90%) of 6A37 modification is [1-(ACLtit1-Δ (transformed with IPTase constructs)/BPtit1-Δ (transformed IPTase constructs))/(ACLtit1Δ (empty vector)/BPtit1Δ (empty vector))] x 100, where % modification in yNB5 (tit1-Δ) = 0, “ACL” indicates the ACL probe and “BP” indicates the body probe. In cases where MT-8 cells were used rather than yNB5, mod5Δ would substitute for tit1Δ. As described, the formula is internally normalized by the tit1Δ or mod5Δ samples.The formula used to calculate % iS1 FigA) Translation initiation contexts of ATG codons; the first two lines show consensuses derived for S. cerevisiae  and the last line shows a consensus derived for vertebrate [Xho1 site of S. pombe expression vectors pRep4X or pRep82X (pRepXho1). Line 7 shows the native TRIT1 M1 ATG context. B) Nucleotide sequence of MOD5 including its M1 and M12 ATG sites and the M12A substitution mutation in the Mod5MTS-M12A-TRIT1 construct. C) Western blot of TRIT1 protein developed using anti-TRIT1 Ab, from cells with constructs containing the strong (pRep4X) or weak (pRep82X) nmt1+ promoter (see text), indicated above the lanes as 4X or 82X. Lanes are numbered below. MW markers are indicated in kDa. The shared band below 25 kDa is an internal control. Lower panel shows β-actin as loading control used for quantitative normalization. D) Determination of TRIT1 levels in C by quantitative Odyssey CLx imaging (Methods); numbers above bars indicate TRIT1/β-actin levels in each sample; The pRep vectors used are indicated as 4X and 82X are indicated along the X-axis. E) Northern blot of 2 cy- and 2 mt- tRNAs by TRIT1 and Mod5MTS-TRIT1 each from the strong promoter, pRep4X and weak promoter pRep82X, as indicated above the lanes, as 4X and 82X. The top four panels show the ACL probings as indicated to the left, and the bottom four panels show the corresponding body probings. F) Quantitation of % i6A37 modification of the mt-tRNAs and the cy-tRNAs; the pRep vectors used are indicated as 4X and 82X along the X-axis. G) Clover leaf representations of S. pombe cy-tRNATrpCCA and mt-tRNATrpCCA as encoded by the nuclear and mitochondrial DNA and folded by tRNAscan-SE [rtebrate . Lines 3Ascan-SE  Table 1A Transla(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigTrpCCA (A), cy-tRNACysGCA (B), and cy-tRNATyrGUA (C), in the eukaryotes indicated; the 32 and 37 positions are numbered and the horizontal bar indicates the AC. The empty boxes reflect that no genes for this tRNA were indicated for this species [ref Sequence alignments of the ACLs of cy-tRNAcies ref .(TIF)Click here for additional data file.


Wx-B1) was found to be associated with low amylose content and good chapatti quality. For validation, near-isogenic lines (NILs) of this allele were created by marker assisted backcross (MAB) breeding. Background screening indicated 88.2 to 96.7% background recovery in 16 selected BC3F5 NILs. Processing quality and sensory evaluation of selected NILs indicated improvement in chapatti making quality. Traits that showed improvement were mouthfeel, tearing strength and softness indicating that the Wx-B1 may be one of the major genes controlling chapatti softness.Chapatti (unleavened flatbread) is a staple food in northern India and neighboring countries but the genetics behind its processing quality are poorly understood. To understand the genes determining chapatti quality, differentially expressed genes were selected from microarray data of contrasting chapatti cultivars. From the gene and trait association studies, a null allele of granule bound starch synthase (GBSS;  Wheat is an important crop worldwide. In India, Nepal, Bangladesh, Pakistan, Sri Lanka, East Africa, and the Caribbean, it is mainly consumed in the form of an unleavened flatbread–the chapatti. Limited studies have been carried out to understand the genes/QTLs involved in chapatti making quality . A good Other than proteins, starch (65–70% of the endosperm) also plays an important role in end-product quality. Different components of the starch, amylose  and amylopectin  and their ratios (AP/AM) influence properties like pasting, gelatinization and cooking quality –14. LoweMost reports on the end-product quality of wheat are related to bread, biscuit, and noodles. Chapatti, despite being the important food in several countries, has attracted limited attention ,20–22. PIn this study, we have screened the previously published microarray data generated from contrasting chapatti quality wheat cultivars . From thFor screening of differentially expressed genes and selection of candidate genes, seed microarray data of good (C306 and LOK1) vs. poor  chapatti quality wheat cultivars (cv.), were collected at three developmental stages , containing 61,290 probe sets representing about 25 K unigenes was used for screening [Based on microarray data granule bound starch synthase (GBSS), HMW-GS and puroindoline genes were selected as candidates for further analysis. Fourteen wheat cultivars with known chapatti quality  were screened for allelic diversity. Polymorphism in the GBSS and puroindoline genes were screened by PCR based markers . HMW-GS Seeds of all the fourteen cultivars were also screened for amylose content. The wheat starch granules were isolated according to reference . The per3), were followed by seven selfing generations. The BC3 were named as C3 (from the cross C306/4*PBW343) and C6 (from the cross C306/4*PBW621). Individual selections (plants/plots) from the same cross were number from A to Z e.g. C3A to C3H from cross C3 and C6A to C6H from cross C6. The foreground selection (FGS) was carried out using SSR marker WMC313 followed by co-dominant marker Wx-B1. The background screening (BGS) was carried out using 400 deletion bin-based primers, spread across 42 chromosomes [The NILs were developed by crossing the good chapatti quality wheat cv C306 as the donor, with poor chapatti quality but high yielding cultivars PBW343and PBW621 as recipients, through the backcross breeding method . All wheomosomes .Sodium dodecyl sulfate sedimentation (SDSS) test was performed on a small scale using 1 g flour . Dough e2), L—initial chapatti length (m) and ΔL—change in length (extensibility).Sensory evaluation of fresh chapattis was done by a panel of 10 members. The sensory attributes of chapatti were evaluated in terms of dough stickiness, rollability, puffing height, black spots, color, taste, aroma, mouth feel, tearing strength and softness by a panel consisting of ten members using the 9 point-Hedonic scale with a score of 9—extreme liking, 8—like very much, 7—like moderately, 6—like slightly, 5—neither like nor dislike, 4—dislike slightly, 3—dislike moderately, 2—dislike very much and 1 for extreme disliking . Donor cWx-B1, starch content.Results were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s-b test using IBM SPSS Statistics 21.0. The Principal Component Analysis was performed by XLSTAT 2020 for the analysis of chapatti quality, PIN genes, HMW-GS, null For candidate gene screening differential expression microarray data of good vs. poor chapatti quality lines was utilized . This daPina and Pinb), all the tested hard wheat lines  and negatively correlated with amylose content (-0.765) and Pina-D1a (-0.785)  . As Pina1 plants from each cross were screened by co-dominant gene specific marker for heterozygosity of the Wx-B1 gene (foreground selection) and a linked SSR marker. Selected plants were marked and used for backcrossing. Around 70 plants from BC2 and BC3 were screened. Total of 150 plants were screened at BC3F2 stage. 20 selected plants each from crosses C3 and C6 were advanced to BC3F4 stage . As it was BC3 based progeny, around 93.75% background recovery was expected. Among the 16 lines analyzed, background recovery was found to be between 88.2 and 96.7%. Based on the above-mentioned parameters, 2 BC3F7 NILs, one from each cross with maximum recipient parent recovery (96.7% and 96.3%) were chosen for next year field performance and quality study (Year 2 data). The yield (ton/hectare) of NILs viz., C3C (2.68) and C6H (4.77) were significantly higher than donor C306 (1.3) and lower than the recipient parents PBW343 (3.3) and PBW621 (5.8) (Around 70 BCF4 stage . FGS the21 (5.8) . The tho21 (5.8) .In year 1, SDSS of 8 selected NILs from each cross ranged between 2.43 to 3.5 for C3 NILs and 3.63 to 7.07 for C6 NILs. For donor C306 it was 2.27 and for recipients PBW343 and PBW621, it was 6.6 to 9.7, respectively . In yearDough extensibility test in the year 1, revealed that the separation distance, peak positive force and area to positive peak were significantly higher in recipient cultivars as compared to the donor with the highest values observed in PBW621. The NILs showed transgressive segregation i.e., some of NILs had values even lower than C306 and others higher than PBW621 . In yearRecipient parents had significantly higher SRC than C306 in all the four types of solutions, but SRC of NILs followed a random pattern, neither aligning with donor or recipients .We observed that the To, Tp, Tc of donor line C306 and NILC3C were significantly higher than PBW343. But there was no significant difference observed in ΔH of all the three lines. The To and Tc values of PBW621 were lower than C306 and NILC6H. There was no significant difference observed in ΔH of all the three lines. . OverallIn year-1, most of the NILs showed significantly better chapatti sensory evaluation than their recipient parents (C306>NILs>Recipients) . NILC3C,TM was significantly lower in C306 compared to recipient parents . The TM This study utilized microarray data to shortlist genes related to chapatti making quality based on differential expression data of contrasting lines. Out of several differentially expressed genes, a few genes with more than 10-fold differential expression along with previous literature support were shortlisted for further validation. Several studies on differential expression of contrasting trait lines have been reported, and these studies have identified several differentially expressed genes belonging to diverse pathways and highlighted complex mechanisms involving regulation at epigenomic, transcriptional, translational and post-translational levels –42, but Pina-D1a and Pinb-D1a) made very poor chapattis. Effect of grain hardness on chapatti quality has not been reported much in scientific publications [Glu-A1 (0–1 subunits) on chromosome 1A, Glu-B1 (1–2) on chromosome 1B and Glu-D1 (2) on chromosome 1D and previous studies have shown co-relation of their allelic variation with bread-making quality [Glu-D1xy-5+10 [Glu-B1x-20 [Glu-D1xy-2+12 and other Glu-B1xy alleles. But conflicting results indicating no association of individual HMW-GS with chapatti making quality have also been reported [In this study, we shortlisted three genes  and their isoforms, screened them on previously known chapatti quality lines, selected GBSS-4A, prepared its NILs and determined its influence on chapatti softness. These three are major genes reported for their association with wheat processing into different food products like bread, biscuits, noodles etc. One of these three, the puroindoline genes are responsible for grain texture. The soft wheat with low protein content is used for biscuits and cakes and hard wheat with high protein content is preferred for bread and noodles. We could find an association of grain hardness with chapatti quality, as soft wheat chapattis with both functional puroindoline alleles Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Circular RNAs (circRNAs) have been reported to play key roles in the development of various cancers. However, the biological functions and clinical significance of most circRNAs are still elusive. The purpose of this study was to explore the function and mechanism of a certain circRNA named circCDKN2B-AS1 in cervical cancer development and its potential value in the clinic.18FDG-microPET/CT imaging and lactic acid (LA) and pyruvic acid (PA) content detection assays were used to detect the level of glucose metabolism in subcutaneous tumors from nude mice.qRT-PCR was used to verify the expression level of circCDKN2B-AS1. CCK-8, Transwell, and flow cytometry (FCM) assays were performed to detect cellular proliferation, migration, and apoptosis, respectively. A Seahorse XFe96 Analyzer was used to measure glycolysis metabolism level. RNA pull-down, RNA immunoprecipitation (RIP), actinomycin-D addition assays and Western blotting were used to screen and elucidate the potential mechanisms involved. BALB/c nude mice and zebrafish embryos  were used as animal models to investigate tumorigenesis capability. CircCDKN2B-AS1, a circular isoform of the long noncoding RNA (lncRNA) CDKN2B-AS1, was upregulated in cervical cancer and precancerous tissues. We found that circCDKN2B-AS1 associated with the IMP3 protein depending on a specific binding site and regulated the stability of Hexokinase 2 (HK2) mRNA, the rate-limiting enzyme of the aerobic glycolysis pathway. The expression level of circCDKN2B-AS1 fated the binding of IMP3 to the 3′ untranslated region (UTR) of HK2 mRNA, consequently affecting the malignant cell phenotype and aerobic glycolysis in cervical cancer in vitro and in vivo. Mutant circCDKN2B-AS1, lacking the IMP3 binding site, did not have such effects. Utilization of an inhibitory peptide to block the interaction between circCDKN2B-AS1 and the IMP3 protein impeded the binding of IMP3 to the 3’UTR of HK2 mRNA and suppressed aerobic glycolysis in cervical cancer cells.Our findings demonstrate that circCDKN2B-AS1 facilitates aerobic glycolysis by sponging the IMP3 protein to stabilize HK2 mRNA, consequently promoting the malignant phenotype in cervical cancer, which may provide a potential approach for cervical cancer therapeutics. Cervical cancer is the fourth most common cancer among women globally, leading to an estimated 530,000 new cases and 270,000 deaths each year , 2. ScreCircular RNAs (circRNAs), formed by a covalently closed loop, are a class of noncoding RNAs , 9. CircIMP3, also known as IGF2BP3, is an RBP. A previous study revealed that IMP3 is highly expressed in various cancers and can regulate the expression of various genes at the posttranscriptional level –24. HK2 In our previous circRNA sequencing analysis in normal and cervical cancer tissues, we identified a circular isoform of the long noncoding RNA (lncRNA) CDKN2B-AS1, named circCDKN2B-AS1. We verified that circCDKN2B-AS1 was upregulated in HPV16-positive cervical cancer and precancerous tissues compared with normal tissues. We further identified that circCDKN2B-AS1 facilitates aerobic glycolysis by interacting with the IMP3 protein to stabilize HK2 mRNA, consequently promoting the malignant phenotype in cervical cancer cells. To better explore the potential therapeutic role of circCDKN2B-AS1, we selected the above two animal models and verified the capability of circCDKN2B-AS1 to regulate HK2 expression and glycolysis by binding IMP3 protein in vivo. Our study may provide a potential approach for cervical cancer therapeutics.In total, 46 normal cervical epithelium tissues, 41 high-grade squamous intraepithelial lesions (HSILs), and 75 cervical cancer tissues were collected for qRT-PCR assays. Cervical cancer samples were obtained from patients who underwent radical hysterectomy, HSIL samples were obtained from patients who underwent colposcopy biopsy, and normal cervical tissues were collected from patients who underwent hysterectomy because of benign gynecological diseases. All the samples were collected from September 2015 to September 2020 at Women’s Hospital, Zhejiang University School of Medicine, China. Patients provided informed consent to obtain samples, and the study was subject to approval by the Hospital Ethical Committee. Patient information for all specimens is shown in Additional file The HPV type of tissue samples was identified using the 21 HPV GenoArray Diagnostic Kit (Innovation Technologies of Hybribio). With the use of PCR principles to amplify extracted HPV DNA from cervical samples, amplified DNA amplicons were then hybridized with immobilized specific HPV probes on a HybriMem membrane under the patented flow-through hybridization technique. The enzyme immunoassay method was applied for color development to obtain test results. HPV-negative specimens used for RNA sequencing were amplified with the common primer my09/11 PCR of HPV L1 to further determine the HPV-negative status.RNA was extracted using TRIzol reagent  according to the manufacturer’s instructions. For tissue samples used to validate circRNA expression, the RNA integrity number (RIN) was assessed. qRT-PCR analyses were performed using the PrimeScript RT Reagent Kit and SYBR Premix Ex Taq . All the primers are presented in Additional file 2, whereas the cervical cancer cell line CaSki and the human embryonic renal cell line HEK293 were cultured in RPMI-1640  containing 10% FBS.The human cervical cancer cell line SiHa and the human embryonic renal cell line HEK293 were obtained from the American Type Culture Collection . The human cervical cancer CaSki cell line was obtained from the Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences (China), where it was tested and authenticated. It was not cultured continuously for more than 3 months. The SiHa cell line was cultured in DMEM  supplemented with 10% FBS and maintained at 37 °C in 5% COCY3-labeled probes targeting the junction point sequence were used to visualize circCDKN2B-AS1 in situ  and transferred to PVDF membranes . The antibodies used were as follows: IMP3 (EMD Millipore), HK2 (Abcam), GAPDH (Diagbio) E-cadherin (CST) and β-Catenin (CST).The human circCDKN2B-AS1 linear sequence was inserted into the plasmid vector pLent-EF1a-circRNA-CMV-RFP-P2A-Puro, and lentiviruses stably overexpressing circCDKN2B-AS1 were constructed . A nonfluorescent circCDKN2B-AS1 overexpression plasmid was constructed for apoptosis detection using the plasmid vector . The full-length products and the junction point of the circCDKN2B-AS1-overexpressing products were determined by Sanger sequencing after RT-PCR. Short hairpin RNAs (shRNAs) specific to circCDKN2B-AS1 were inserted into the lentiviral vector GV334 . Stable cell lines were obtained by puromycin resistance. The human HK2 overexpression plasmid was purchased from GeneChem. All of the small interfering RNA (siRNA) and primer sequences used in this study are presented in Additional file 4 cells were resuspended in serum-free Opti-MEM medium, and cell invasion/migration was examined in Transwell cell culture chamber filters coated on the upper side with/without Matrigel (Corning Biocoat) as previously described [In vitro cell viability was detected using Cell Counting Kit-8 assays . Transwell assays were performed at 48 h after transfection: 1 × 10escribed . The wou2 produced by the mitochondria.Assays were performed using a Seahorse XFe96 analyzer  according to the manufacturer’s instructions. The ECAR was measured using a Seahorse XF Glycolytic Rate Assay Kit  and a Seahorse XF Glycolysis Stress Test Kit . The glycolytic capacities of cells were analyzed with the Seahorse XF Glycolysis Rate/Stress Test Report Generator package. The %PER from glycolysis was calculated by subtracting the acidification from COWe inhibited the glycolysis level of cervical cancer cells in vitro by adding 2-DG .Biotin-labeled RNA pull-down assays were performed using a Pierce™ Magnetic RNA-Protein Pull-down Kit . Briefly, cell lysates were prepared using standard lysis buffers . Biotin-labeled DNA probes  according to the instructions of the manufacturer.To assess the half-life of HK2 mRNA, actinomycin-D  was added to block mRNA synthesis. Total RNA was collected at different time points and subjected to RT-qPCR analysis. The expression level of HK2 mRNA was normalized to that of 18S rRNA and plotted as a percentage of the value at time 0 (set at 100%).The inhibitory peptides for blocking the interaction between circCDKN2B-AS1 and IMP3 were designed and synthesized by ChinaPeptides Co. Ltd. . The inhibitory peptides were synthesized by linking the biotin-labeled 11-amino acid cell-penetrating peptide YGRKKRRQRRR of the Tat protein transduction domain with the core amino acids of IMP3 at the N-terminus (purity greater than 95%).Biotin-labeled peptides were incubated with streptavidin magnetic beads  for 4 h at 4 °C. Then, the bead-peptide complex was incubated with total RNA at 4 °C overnight. Beads were extensively washed, and RNAs that precipitated were measured by qRT-PCR.7 stable SiHa cells were resuspended in 100 μL PBS and injected subcutaneously under the left armpit of 4-week-old nude mice (n = 10 per group). The length and width of the subcutaneous tumor were measured once a week for 5 weeks, and the volume of the subcutaneous tumor was calculated according to the following formula: volume (cm3) = (length×width2)/2. Five weeks after injection, 6 mice in each group were randomly selected and sacrificed by intravenous injection of pentobarbital (100 mg/kg), and subcutaneous tumors were harvested. A portion of the tumor was stored in RNA storage solution to extract RNA, and the remainder of the tumor was fixed with paraformaldehyde for immunohistochemistry and hematoxylin and eosin (HE) staining. The other 4 mice in each group were subjected to microPET/CT scans. Another 20 nude mice (4 weeks old) were randomly divided into 4 groups (n = 5 per group) and injected subcutaneously under the left armpit with 107 stable SiHa cells. siRNAs targeting a negative control (NC) or HK2 were mixed with FECT and Opti-MEM medium and injected into the subcutaneous tumor every week. The volume of the subcutaneous tumor was calculated according to the following formula: volume (cm3) = (length×width2)/2. Four weeks after the injection of cells, mice were sacrificed, and subcutaneous tumors were harvested, weighed, and stored at − 80 °C for RNA extraction, lactic acid (LA)/pyruvic acid (PA) detection and RIP assays.This study was conducted in full accordance with the ARRIVE guidelines for reporting animal research . All ani18F-FDG via the abdominal cavity and scanned at 120 min after injection. Nude mice were subjected to a 10-min microCT scan and then to a 15-min microPET scan. Scans were performed under isoflurane (1–1.5%) inhalation to maintain anesthesia. Images of nude mice were reconstructed manually. The PET/CT camera Vista explore was used to calculate the average standardized uptake volume (SUVAVG). Then, the mice were sacrificed by intravenous injection of pentobarbital (100 mg/kg).MicroPET/CT imaging of nude mice was performed using the SuperArgus PET/CT 4R system  5 weeks after cell injection. Briefly, 4 tumor-bearing nude mice in each group were anesthetized by inhalation of isoflurane gas (2–3%). Then, nude mice were injected with 7.4 MBq (200 μCi) of Subcutaneous tumors were digested with 10 mg type 1 collagenase  at 37 °C for 60 min. The digestion products were centrifuged at 1200 r for 5 min, and the precipitate was subjected further to RIP assays and RNA extraction.The levels of LA and PA in 10 mg subcutaneous tumors from each mouse were detected by using an LA detection kit  and a PA detection kit .Zebrafish  embryos were obtained from the Core Facilities, Zhejiang University, School of Medicine, cultured in Petri dishes with an appropriate amount of water and placed in a 28 °C incubator under constant temperature and light. Then, 0.003% 1-phenyl-2-thiourea (PTU) (Sigma) was added within 24 h after the embryo was produced. In total, 200 SiHa cells were harvested after transfection and resuspended in PBS. Plasma membranes were stained with red-fluorescent Alexa Fluor® 594 wheat germ agglutinin of the Image-iT™ LIVE Plasma Membrane and Nuclear Labeling Kit . Zebrafish embryos were anesthetized with 0.016% tricaine (Sigma). Then, the cells were injected into the yolk sac of zebrafish embryos. Then, we obtained images with a fluorescence microscope at 48 h after injection. The area of red fluorescence in the zebrafish yolk sac was calculated using ImageJ software . Next, tImmunohistochemical staining with an antibody specific to HK2  and quantitative evaluation were performed as previously described .Statistical significance was calculated by using GraphPad Prism 5 software. All experiments were repeated at least three times.To explore circRNA role in cervical cancer development, we analyzed aberrantly expressed circRNAs among 6 HPV16 positive cervical squamous-cell carcinoma, 6 HPV16 positive normal cervical epithelium, and 6 HPV negative normal cervical epithelium tissues in our circRNA sequencing data, available in the Gene Expression Omnibus (GEO) database (GSE147009) . However, the length of this circCDKN2B-AS1 annotated in CircPrimer software was 380 nt instead of 1719 nt . A previIn addition, we detected the level of circCDKN2B-AS1 expression by qRT-PCR assay in 46 normal cervical tissues, 41 HSIL, and 75 cervical cancer tissues regardless HPV positive or negative, and found that circCDKN2B-AS1 expression was upregulated in HSIL and cervical cancer, and the level of circCDKN2B-AS1 expression in cervical cancer and HSIL tissues was 15.16 and 5.48-fold, respectively, compared to normal cervical tissues  predicted by RBPmap. We verified the interaction between circCDKN2B-AS1 and IMP3 through the specific binding site in HEK293 cells, and we found that IMP3 was recruited by upregulated WT circCDKN2B-AS1 but not by upregulated mutant circCDKN2B-AS1 in HEK293 cells  and found that HK2, ENO1, PGK1 and PKM were the potential downstream molecules of circCDKN2B-AS1 and IMP3. Among these molecules, HK2 mRNA was the most significantly downregulated in SiHa cells following circCDKN2B-AS1 knockdown  and the CLIP-Seq analysis of IMP3 in the POSTAR2 database , elevaCircRNAs are noncoding RNAs that are more stable than their linear isoforms. It has been reported that circRNAs are dysregulated and play a tumor suppressive or oncogenic role in cancers, such as breast cancer, ovarian cancer and bladder cancer , 43, 44.As circCDKN2B-AS1 is mainly localized in the cytoplasm of cervical cancer cells, we suspected that circCDKN2B-AS1 binds to microRNAs or RBPs, as most circRNAs do –48. ThenThus, we hypothesized that circCDKN2B-AS1 stabilizes HK2 mRNA and facilitates aerobic glycolysis in cervical cancer by recruiting the IMP3 protein to the 3’UTR of HK2 mRNA Fig. . SupportFurthermore, we designed an inhibitory peptide, IIP, to block the interaction between circCDKN2B-AS1 and IMP3. We designed IIP based on the evidence that RRM1 of IMP3 is the key domain involved in binding CA-rich RNA elements , 23. OurThere were some limitations of our study. For instance, it needs to be determined whether some of the potential circCDKN2B-AS1 binding proteins, such as IMP2, are involved in the gene regulatory network that promotes aerobic glycolysis in cervical cancer cells. In addition, the role of circCDKN2B-AS1 in other tumors and other diseases also needs to be investigated in the future.In the study, we found, for the first time to our knowledge, that circCDKN2B-AS1 acted as a promoter that facilitated aerobic glycolysis by sponging IMP3 protein to stabilize HK2 mRNA, consequently promoted malignant phenotypes in cervical cancer in vitro and in vivo, and the synthesizing inhibitory peptide IIP could effectively block the interaction between circCDKN2B-AS1 and IMP3 protein. Our findings may provide a new approach for cervical cancer therapeutics.Additional file 1: Table S1. Patient information for all specimens.Additional file 2: Table S2. Sequences of primers, probes, siRNAs and peptides.Additional file 3: Fig. S1. Identification of circCDKN2B-AS1, circFNACB, circEPSTI1 and circOBSL1. (A) Sequencing analysis of products amplified by the divergent primers showed the junction in circFNACB. (B) Sequencing analysis of products amplified by the divergent primers showed the junction in circEPSTI1. (C) Sequencing analysis of products amplified by the divergent primers showed the junction in circOBSL1. (D) Linear and circular isoforms of CDKN2B-AS1 were amplified from cDNA or gDNA from SiHa cells with the convergent primers and divergent primers, respectively. (E) The backsplicing point-specific probe was used in Northern blot analysis to detect endogenous circCDKN2B-AS1 in cervical cancer cell lines. Left lane: RNA molecular weight markers ; right lane: circCDKN2B-AS1-specific probe.Additional file 4: Table S3. Sanger sequencing results of PCR products.Additional file 5: Fig. S2. CircCDKN2B-AS1 knockdown suppresses EMT of cervical cancer cells. (A) E-cadherin and β-Catenin protein expression levels in SiHa (left) and CaSki (right) cells were analyzed by Western blotting after transfection with two circCDKN2B-AS1 backsplicing-specific siRNAs or a negative control siRNA. GAPDH served as a loading control.Additional file 6: Fig. S3. CircCDKN2B-AS1 overexpression facilitates the growth and vitality of cervical cancer cells. (A) The expression levels of circCDKN2B-AS1 in SiHa (left panel) and CaSki (right panel) cells with or without circCDKN2B-AS1 overexpression were determined by qRT-PCR . (B) The growth curve of SiHa and CaSki cells transfected with circCDKN2B-AS1 overexpression plasmids or negative control plasmids was determined with CCK-8 assays. Upper panel: SiHa cells; lower panel: CaSki cells . (C) The apoptosis level of SiHa and CaSki cells transfected with circCDKN2B-AS1 overexpression plasmids or negative control plasmids. Upper panel: SiHa cells; lower panel: CaSki cells. Right panel: quantification results of early apoptosis . (D) Representative images (left panel) and quantification (right panel) of migration assays showing the migration capability of SiHa and CaSki cells transfected with circCDKN2B-AS1 overexpression plasmids or negative control plasmids . (E) Representative images (left panel) and quantification (right panel) of Matrigel invasion assays showing the invasion capability of SiHa and CaSki cells transfected with circCDKN2B-AS1 overexpression plasmids or negative control plasmids . *P < 0.05, **P < 0.01, ***P < 0.001.Additional file 7: Fig. S4. CircCDKN2B-AS1 cooperates with IMP3 to promote glycolysis in cervical cancer. (A) Analysis of differential bands in Fig. n = 6, one-way ANOVA). (E) Quantification of the %PER from the glycolysis of SiHa (left panel) and CaSki (right panel) cells transfected with negative control plasmids, circCDKN2B-AS1-overexpressing plasmids and mutant circCDKN2B-AS1-overexpressing plasmids . (F) The ECAR was measured in SiHa (left upper) and CaSki (left lower) cells transfected with the circCDKN2B-AS1 overexpression vector or the negative control vector with Seahorse XFe assays. Right panel: quantification of glycolytic capacity and glycolysis in SiHa (right upper) and CaSki (right lower) cells . *P < 0.05, **P < 0.01, ***P < 0.001.Additional file 8: Table S4. All proteins verified by mass spectrometry analysis.Additional file 9: Table S5. All proteins predicted by RBPmap.Additional file 10: Fig. S5. CircCDKN2B-AS1 regulates the stability of HK2 mRNA by recruiting IMP3. (A) The expression levels of HK2, ENO1, PGK1 and PKM in SiHa cells with or without circCDKN2B-AS1 knockdown were determined by qRT-PCR . (B) HK2 mRNA (lower) and HK2 protein (upper) levels in SiHa cells stably transfected with sh-circCDKN2B-AS1 or negative control and HK2 overexpression plasmids . *P < 0.05, **P < 0.01, ***P < 0.001.Additional file 11: Fig. S6. The expression levels of circCDKN2B-AS1 and IMP3 protein in in transplanted tumor tissues. The levels of IMP3 protein expression (left) in transplanted tumor tissues . Right: the association between circCDKN2B-AS1 and IMP3 protein expressions .


On February 17, 2019, a male patient aged 13 years with no underlying medical conditions was evaluated in an Idaho hospital emergency department for a 1-day history of fever (103°F [39.4°C]), dry cough, sore throat, headache, and weakness. A respiratory specimen was collected and tested positive for influenza A by rapid influenza diagnostic test (RIDT). The patient was treated with oseltamivir as an outpatient and recovered. As part of routine surveillance, a second specimen collected during the emergency department visit on February 17 was forwarded to the Idaho Bureau of Laboratories (IBL), where CDC’s influenza reverse transcription–polymerase chain reaction (RT-PCR) diagnostic panel detected both pandemic influenza A and H3, which suggested an influenza A(H3N2) variant virus of swine origin. The specimen was sent to CDC’s influenza diagnostic laboratory for confirmation, and the patient was interviewed.During the week preceding illness onset, the patient did not travel and reported no animal exposure; he had not received a 2018–19 seasonal influenza vaccine. One household member developed respiratory symptoms on February 23, 2019, and sought care at an outpatient clinic, where a respiratory specimen tested positive for influenza A by RIDT. No specimen was available for additional testing, and no other exposures were identified. No additional household members reported respiratory symptoms.Next generation sequencing at CDC revealed a new seasonal human influenza A(H3N2) and A(H1N1)pdm09 reassortant virus, rather than an influenza A(H3N2) variant virus of swine origin. Reassortment occurs when two influenza viruses infect a single host cell and exchange gene segments, creating a new virus. Sequencing data suggested that the patient was not coinfected and that the reassortment event likely occurred in another person. Phylogenetic analysis determined that the hemagglutinin genes belonged to human H3 subclade 3C.3a and neuraminidase genes belonged to human N2. Gene segments PB2, PB1, PA, NP, M, and NS displayed genetic similarity to human-origin influenza A(H1N1)pdm09 viruses. Genetic markers that would confer reduced susceptibility to oseltamivir, peramivir, and zanamivir were not detected. Viruses in H3 subclade 3C.3a react poorly by focus reduction assay with ferret antisera raised against A/Singapore/INFMH-16–0019/2016(3C.2a1), signifying that the 2018–19 Northern Hemisphere influenza vaccineAs part of enhanced surveillance, the hospital where the patient sought care forwarded an additional 45 specimens that tested positive by RIDT for influenza A, collected during January 1–April 27, 2019, to IBL. Using the CDC influenza RT-PCR diagnostic panel, IBL determined that 23 (51.1%) were influenza A(H1N1)pdm09, 13 (28.9%) were influenza A(H3N2), and influenza was not detected in nine (20.0%) specimens. IBL sent 17 of the 45 specimens to CDC for sequencing; no additional reassortant viruses were identified.,At the time of the patient’s illness onset, influenza A(H1N1)pdm09 and A(H3) were cocirculating in Idaho , increas


According to the European directive Euratom 2013/59 in these materials, the “ACI” must be < 1 to be suitable for their use in construction. In this paper, data on the activity concentration of natural radionuclides in cement-based materials  as well as their chemical composition are presented. Radioactivity measurements have been determined by using gamma spectroscopy the chemical compositions have been determined by X-Ray Fluorescence. Data for cements measured shown that white cements present a lower concentration of activity than conventional CEM I. In addition, the CAC  present high activity concentration in the 232Th series. Regarding additions, FA (Fly Ash) are those that present the highest concentration of activity in the 238U and 232Th series, while olive biomass ashes are those supplementary cementitious materials that show the highest concentration of activity for 40K. Some pigments used in mortar and concrete technology were also characterized. Granitic and volcanic rocks, potentially used as aggregates present much higher activity concentration than the siliceous aggregate.Cement based materials may contain varying levels of radionuclides, mainly  A brief description of the materials accompanies these data.1.1.1Chemical composition of 16 different cements (Portland and other types of cements) is shown in 1.1.2Fly Ash); b): S (Slags- where S1 to S3 are blast furnace slags and S4-S5 are steel slags) and c): other SCMs  used in the preparation of mortars and concretes have been chemically characterised. Results are shown in 1.2238U, 235U and 232Th series as well as and 40K given in Bq kg−1 in all measured cement based materials are shown below. The uncertainties of the activity concentrations shown in The activity concentrations of 1.2.1238U, 235U and 232Th series, and 40K in commercial cements are shown in Activity concentrations of 1.2.2238U, 235U and 232Th series and 40K series for 37 Supplementary Cementitious Materials (SCM) are presented in Results obtained from gamma spectroscopy in 1.2.3238U, 235U and 232Th series and 40K in seven different pigments are shown in Activity concentrations of 1.2.4238U, 235U and 232Th series and 40K for aggregates are shown in Activity concentrations of 22.1In this work, a total of 56 samples of components used in cement-based materials have been studied.Commercial Cements: 16 cements with different chemical and mineralogical composition.aSix Type I cements (CEM I 42.5R y CEM I 52.5R) from different cement factories (samples C1-C6). according to European Standard EN-197-1-2011 [3]bThree blended cements: One CEM II/A-L with a maximum of 20% of limestone (sample C7), one CEM II/B-V with coal fly ash (FA) content around 21–35% (sample C8) and one CEM III/B with high content (around 66–80%) of blast furnace slag (sample C9). All of them according to European Standard EN-197-1-2011 c3A) (sample C10) [3]One CEM I 52,5 S/R, sulphate-resistant, which therefore has a lower proportion of tricalcium aluminate (Cd2O3 content and without TiO2. as per standard UNE 80305:2012 [4]Two white cements (samples C11 and C12) with a very low FeeThree calcium aluminate cements (CAC) from different factories, with very different chemical and mineralogical composition compared to a Portland cement, whose main component is monocalcium aluminate (CA) (samples C13-C15). This type of cement complies with European standard EN 14647-2006 [5]f2 content while higher sulphate proportion.One calcium sulfoaluminate cement with a very different chemical composition and with a very low SiOSupplementary Cementitious Materials: 27 SCMs from very different sources, some of them commonly used in the preparation of cement-based materials.aFly ash (FA): Eleven coal fly ashes from seven different thermoelectric power plants, with different composition and proportion of unburned material (samples FA1-FA11)bSlags: Three vitreous blast furnace slags with different origins (samples S1-S3) and two steel slags (samples S4 and S5). Slags are by-products of iron and steel-making production.cOne silica fume (sample SF1) which is a by-product originated in the reduction of quartz with coal, in electric arc furnaces for the production of silicon and ferrosilicon.d) from urban glass recycling waste.One waste glass (sample WG1eOne limestone (sample L1)fOne red mud (sample RM1) that is a solid waste with high iron content, generated in the industrial process to produce alumina from bauxite.gTwo samples of metacaolin (sample MK1 and MK2) resulting from the calcination of kaolinitic clays.hThree pozzolans, with different origin and chemical composition (samples PZ1-PZ3)iTwo olive biomass samples prepared from the mixture of various biomass bottom ashes (sample OBBAM1) or biomass fly ashes (OBFAM1).Pigments: Seven pigments (samples P1-P7) commonly used in construction, with different colour and chemical composition. One of them (P6) has a high organic matter content.aOne standardized siliceous aggregate according to EN 196-1 (sample A1).bThree granitic aggregates of different geographical origin (samples A2-A4).cTwo volcanic aggregates (samples A5 and A6).Aggregates: Six aggregates with maximum particle size of 2 mm.2.22.2.1The chemical characterization of all the materials was determined on a S8 Tiger Bruker X-ray fluorescence (XRF) spectrometer. Loss on ignition calculated as per UNE-EN196-2:2014 2.2.2Radiological measurements of the samples were performed by gamma spectrometry with a counting time of 80,000 s, using three High-purity Germanium detectors .60Co and intrinsic efficiencies from 30% to 100%. They are protected with 15 cm iron shield. Main characteristics of both types of Ge detectors are shown in The detectors have an active surface from 26 to 38 cm 2 and 2 keV resolution for The gamma detectors are connected to two electronic chains consisting of: two amplifiers, two Analog-to-Digital Converters (ADCs), 2 High Voltage Power Supplies and 2 AIM modules all from Canberra Industries. The AIMs modules have the function of communicating the electronic chain with the PC allowing it to: (1) control the parameters of the detector and electronic chain and (2) acquire the spectra. Once the spectra were acquired they were analysed with Genie 2000 software 222Rn loses. Each container was totally filled with the sample and the measurements were carried out after 21 days to ensure secular equilibrium between radionuclides and their progeny All samples were measured in specific polypropylene sample container  sealed to avoid 238U (226Ra), 232Th and 40K were determined in the samples.Radionuclides occurring in natural decay chains headed by 238U serie measurements were based on the detection of emissions of their daughter nuclides, 234Th (63 keV), 226Ra (186.1 keV), 214Pb (351 keV) and 214Bi .232Th determination was determined on emissions of their daughter nuclides, 228Ac (911 and 969 keV), 212Pb (238 keV) and 208Tl (583 keV) respectively, since their own gamma emitting lines are not of sufficient intense (232Th at 63.8 keV) or present significant interferences with other naturally occurring radionuclides.235U (144 and 163 KeV) and 40K (1460.8 keV) were directly measured by their emissions lines.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Influenza vaccination is annually recommended for specific populations at risk, such as older adults. We estimated the 2018/2019 influenza vaccine effectiveness (IVE) overall, by influenza subtype, type of vaccine, and by time elapsed since vaccination among subjects 65 years old or over in a multicenter prospective study in the Valencia Hospital Surveillance Network for the Study of Influenza and other Respiratory Viruses . Information about potential confounders was obtained from clinical registries and/or by interviewing patients and vaccination details were only ascertained by registries. A test-negative design was performed in order to estimate IVE. As a result, IVE was estimated at 46% : ), 41% ), and 45% ) against overall influenza, A(H1N1)pdm09 and A(H3N2), respectively. An intra-seasonal not relevant waning effect was detected. The IVE for the adjuvanted vaccine in ≥75 years old was 45%  and for the non-adjuvanted vaccine in 65–74 years old was 59% . Thus, our data revealed moderate vaccine effectiveness against influenza A(H3N2) and not significant against A(H1N1)pdm09. Significant protection was conferred by the adjuvanted vaccine to patients ≥75 years old. Moreover, an intra-seasonal not relevant waning effect was detected, and a not significant IVE decreasing trend was observed over time. Seasonal influenza is a common acute respiratory viral infection (ARVI) that can lead to serious complications and vaccination is key for its prevention . InfluenAccording to the data collected by the European Centre for Disease Prevention and Control (ECDC) across the WHO European Region, 99% of circulating influenza viruses in the 2018/2019 season were type A, with A(H1N1)pdm09 (57%) prevailing over AH3N2) 43%), and 1% were type B. Almost all (1882/1885) genetically characterized A(H1N1)pdm09 viruses were subclade 6B.1, represented by the vaccine virus A/Michigan/45/2015. Most of the A(H3N2) circulating viruses in Europe belonged to subgroup 3C.2a1b (66%) represented by A/Alsace/1746/2018 and to clade 3C.3a (25%) represented by A/England/538/2018, both discordant with the egg-propagated A/Singapore/INFIMH-16-0019/2016 vaccine virus strain as The Francis Crick Institute, WHO Collaborating Centre, reported [N2 (43%),3%, and 1Interim results from different studies across Europe for the 2018/2019 season showed a low vaccine effectiveness in hospitalized subjects aged 65 or more years old for both influenza A subtypes, A(H1N1)pdm09 and A(H3N2) [In Spain, the Influenza Surveillance System reported that the 2018/2019 influenza season was characterized by the circulation of influenza A and very few cases of influenza B (less than 1%). The distribution by influenza A subtype was 45% A(H1N1)pdm09 and 55% A(H3N2). The peak of the season was reached in the week 2019-04, three weeks later than the previous two seasons .The declining of effectiveness, also known as waning immunity, is considered a major concern around influenza vaccines. Our group, among other authors, have shown that the protection conferred by the influenza vaccine may wane ,9, althoIn this study, we report the 2018/2019 end-of-season influenza vaccine effectiveness (IVE) in hospitalized patients 65 years old or over from the Valencia Region of Spain. Estimates are provided by influenza subtype and type of vaccine. We also explored the presence of a declining in the vaccine effectiveness over time.The study was carried out in 4 hospitals in the Valencia Region: Hospital General Universitario de Castellón , Hospital Universitario y Politécnico La Fe , Hospital Universitario Doctor Peset  and Hospital General Universitario de Alicante . Those hospitals provided healthcare to 1,118,732 (22%) inhabitants of the Valencia Region. The study encompassed the whole year although we restricted the analysis to the influenza season, defined by the time period between the first of two consecutive weeks with 2 or more influenza cases and the previous week to the first of two consecutive weeks with no influenza cases. The study methodologies have been described in previous publications . Full-tiThe Valencia Region Vaccine Information System (VRVIS) is a population-based registry that records all information related to vaccination. We can link this registry to sociodemographic information, Primary Care visits and hospitalizations through a unique personal identification number.®  and Chiromas® , the second one included in its composition the adjuvant MF59C.1 [® was recommended for individuals 65 to 74 years old and Chiromas® for individuals 75 years old and above and for institutionalized patients 65 years old or more [The VRVIS covers both public and private healthcare facilities. The sensitivity and specificity of the VRVIS were estimated previously for the influenza vaccine as 90% and 99% . We obta MF59C.1 ,16. Infl or more , but onlTwo different swabs, nasopharyngeal and pharyngeal , were obtained within the first 8–48 h of admission from all patients fulfilling the inclusion criteria. Both swabs were combined in one tube of viral transport media  and frozen until shipped refrigerated to a centralized Virology laboratory at FISABIO-Public Health. One third of the viral transport media volume was extracted for total nucleic acids using an automated silica-based method . Extracted nucleic acids were tested for influenza viruses by multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR), following WHO protocols  with thehttps://github.com/tseemann/snippy) [Molecular characterization of influenza A(H1N1)pdm09 and A(H3N2) viruses was performed by whole-genome sequencing (WGS). All isolates from hospitalized cases with enough viral load (Ct < 27) were systematically selected and a universal viral whole-genome amplification protocol  was used/snippy) .www.gisaid.org). An alignment of reference sequences with sample sequences was generated with the Clustal W algorithm integrated in the BioEdit software ver. 7.2.5 (https://archive.org/details/bioedit). Phylogenetic trees were inferred using Maximum-likelihood methods and the best-fitting nucleotide substitution model with RAxML, and branch reliability was evaluated by approximate likelihood-ratio tests [Genetic characterization of influenza A(H1N1)pdm09 and A(H3N2) viruses was performed by comparison of the obtained consensus sequences for the complete HA coding region from the clinical isolates with representative and reference HA sequences  obtainedio tests .p-values under 0.05 were considered statistically significant. An analogous descriptive analysis was performed comparing vaccinated and unvaccinated individuals. IVE was estimated following the test-negative design [We explored the differences between laboratory-confirmed influenza (LCI) and non-LCI hospitalized patients in terms of age, sex, presence of chronic conditions (including pulmonary and heart diseases), admissions in the previous 12 months, number of General Practitioner (GP) visits in the previous 3 months, smoking habits, socioeconomic class according to occupation , obesitye design . Cases we design ,31. We iWe explored the presence of an intra-seasonal waning of the vaccine effectiveness by using the number of elapsed days between vaccination and symptoms’ onset. We defined tertiles for that variable and we compared those categories with the reference category of no vaccination. For the waning effect analysis, we dropped out those patients who were not immunized when the influenza season started, in other words, we deleted patients not immunized at the onset of symptoms of the first detected influenza positive.We proceeded as in the IVE analysis, adjusting a multivariate logistic regression by all potential confounders. The IVE for each time interval was calculated as (1 − aOR) × 100% and the 95% CI was also provided.To better understand the impact of previous vaccination on current IVE we performed all the analysis restricting the sample size to those patients who were previously vaccinated in any of the prior two seasons.All statistical analyses were carried out in Stata version 14  and R .From 6836 eligible patients aged ≥65 years old, we included 992 patients in the IVE analysis after applying the exclusion criteria .The influenza period spanned from week 2019-01 to 2019-16. The first influenza wave was A(H1N1)pdm09, from 2019-01 to 2019-09, and the second and wider wave, from 2019-03 to 2019-16 was A(H3N2). The A(H1N1)pdm09 peaked in week 2019-04 and A(H3N2) peaked in 2019-08 and 2019-10 .From the 992 included patients, 164 (16.53%) were LCI: 46 (28.05%) A(H1N1)pdm09, 116 (70.73%) A(H3N2), and 2 A not subtyped . Influenp-value < 0.001). No differences between LCI and non-LCI were detected in terms of sex, presence of chronic conditions, pulmonary or heart diseases, admissions in the previous 12 months, smoking habits, socioeconomic class according to occupation, obesity, days from onset to swabbing, functional impairment status based on the Barthel Index and previous vaccination status. Positives and negatives for influenza differed in their vaccination status: 58.54% of positives were vaccinated vs. 68.36% of negatives . Patients with pulmonary or heart diseases had higher vaccination coverage . Males were more commonly vaccinated than females . No differences between vaccinated and unvaccinated individuals were found regarding age, admissions in the previous 12 months, GP visits in the previous 3 months, smoking habits, socioeconomic class, obesity, days from onset to swabbing and functional impairment status  were vaccinated. Vaccinated individuals presented more chronic conditions than not vaccinated pdm09 viruses sequenced belonged to the A/Michigan/45/2015 (vaccine virus) clade 6B.1 which is defined by the amino acid substitutions S84N, S162N  and I216T .Within clade 6B.1, viruses clustered into the 6B.1A subclade defined by additional substitutions S74R, S164T  and I295V. All viruses carried the substitution S183P, potentially linked to increased binding to α-2,6 sialic acid-linked receptors.n = 31) clustered to subclade 6B.1A5, characterized by N260D; with all of them close to A/Swansea/9504/2018 (clade 6B.1A5 (*), N129D + T185I). There was no evident grouping of isolates from the same collection month, or from vaccinated individuals within any of the observed subclades.Within subclade 6B.1A several subclades showed different amino acid substitutions as compared to the vaccine virus. Three isolates clustered within A/Paris/2533/2018, subclade 6B1.A7 (characterized by K302T), and (HA2) I77M + N169S + E179D and one to A/Brisbane/02/2018 (2019-20 vaccine virus) subclade 6B1.A1. Finally, most of the isolates (n = 35) and 3C.3a A/England/538/2018 (n = 38)  viruses in the VAHNSI network and reference viruses indicated that all the 73 sequenced viruses fell within two clades: 3C.2a Hong Kong/4801/2014 ((n = 38) .There was no evident grouping of isolates from the same collection month, or from vaccinated individuals within any of the two clades. Only one 3C.2a isolate corresponded to subclade 3C.2a2 A/Switzerland/8060/2017. The 3C.2a2 viruses were defined by L3I, N128T , T131K, R142K, N144S , N145S, F159Y, K160T , P198S, F219S, R261Q, N225D, and (HA2)D160N.n = 34) fell within subclade 3C.2a1, with no virus clustering in the same 3C.2a1 A/Singapore/INFIMH-16-0019/2016 (vaccine virus) branch; instead, all 3C.2a1 viruses further diversified on the closely-related A/Alsace/1746/2018 3C.2a1b subgroup. These viruses were characterized by substitutions E62G K92R, N121K/E, R142G, N171K, H311Q and (HA2)I77V, G155E, and G160E. Within this 3C.2a1b subgroup, two potentially differentiated subclades were observed: 3C.2a1b (*), (n = 19) with T131K + (HA2)V200I with or without HA2(V18M) and 3C.2a1b (**), (n = 15) with T135K  together or not with T128A . The 38 collected A(H3N2) 3C.3a viruses were in the A/England/538/2018 subgroup , and were defined by the substitutions L3I, S91N, T128A, A138S, most R142G, N144K , most F193S, K326R and (HA2)D160N. We could distinguish two differentiating subgroups within 3C.3a isolates: 3C.3a (*), (n = 9) carried additional substitutions (HA2)M17L + A201V; and 3C.3a (**), (n = 6) carried additional substitutions (HA2)M17L + A201V + V176I.Most 3C.2a viruses . In particular, the IVE against all influenza types for the adjuvanted vaccine in those patients aged ≥75 was 44.81%  and for the non-adjuvanted vaccine in patients 65–74 years old IVE was 59.50%  .The IVE in ≥65 years old hospitalized patients in the Valencia Region for the 2018/2019 season was 40.65%  against influenza A(H1N1)pdm09 and the IVE against A(H3N2) was 44.90%  .The waning effect was explored according to the number of days elapsed between vaccination and onset of symptoms dates. The following tertiles were defined: 15–82 days (first), 83–122 days (second), and 123–177 days (third). IVE was 54.17% , 35.66% , and 49.83%  for patients in the first, second and third tertiles, respectively .Restricting the analysis to those patients previously vaccinated  the overall IVE was 33.78% . For the study of the influenza effectiveness declining, we obtained that IVE was 18.72% , 26.71% , and 55.48%  for those patients whose elapsed days between vaccination and symptoms’ onset were between 15 and 82; 83 and 122; and 123 and 177 days, respectively. IVE against A(H1N1)pdm09 was −26.70% , and 40.10%  against A(H3N2). The IVE for the adjuvanted vaccine in those patients 75 years old or over was 40.72%  and for the non-adjuvanted vaccine in those adults 65–74 years old was 42.47% .In this work, we report the 2018/2019 IVE overall, by influenza subtype, vaccine type and by time elapsed since vaccination in hospitalized older adults in a multicenter prospective study in the Valencia Region of Spain.We identified the AH3N2) subtype as predominant, representing 71% of total isolations compared to 28% for A(H1N1)pdm09. Contrasting results were reported by the Canadian Sentinel Practitioner Surveillance Network that registered 88% of A(H1N1)pdm09 and only 6% of A(H3N2), and the United States IVE Network that detected 63% of A(H1N1)pdm09 and 22% of A(H3N2) in outpatients of all ages ,33. HoweN2 subtypThe WHO recommended composition of the TIV for the northern hemisphere included an A/Michigan/45/2015 (H1N1)pdm09-like virus, an A/Singapore/INFIMH-16-0019/2016 H3N2)-like virus, and a B/Colorado/06/2017-like virus (B/Victoria lineage). In our study, 44% of the A(H3N2) viruses genetically characterized were similar to A/Alsace/1746/2018 (clade 3C.2a1b), and 55% of them to A/England/538/2018 (clade 3C.3a). The A(H3N2) vaccine component A/Singapore/INFIMH-16-0019/2016 is the clade 3C.2a1 representative, that replaced the clade 3C.2a A/Hong Kong/4801/2014 virus used in the previous 2016/17 and 2017/18 seasons. Despite genetic differences in the HA1 region, it is believed that 3C.2a and 3C.2a1 vaccine strains may be antigenically similar, but different to contemporary 3C.2a1b and 3C.3a viruses, with 3C.3a viruses showing greater antigenic differences ,40. UnfoN2-like vIn terms of IVE results, for hospitalized patients 65 years old or over from the Valencia Region of Spain we estimated an IVE of 46%. Interestingly, differences were found across regions or countries. Vaccine effectiveness against overall influenza was 53% 40%, 64%) in England and 35%  in Denmark in hospitalized older adults . Lower e%, 64% inConversely, we did not detect significant IVE against influenza A(H1N1)pdm09, probably due to low sample size when restricting the analysis, although our point estimate of 41% was close to the one reported in Denmark ) .Two different vaccines were administered in the Valencia Region during the 2018/2019 influenza season. We estimated the overall IVE for the adjuvanted vaccine in patients 75 years old or over, the target group for that vaccine. We found significant protection (45%) as well as other published works in older adults population from the United Kingdom both in hospitalizations and primary care with an IVE of 54%  and 62% , respectively ,41. On tMost studies reporting IVE estimates did not consider previous vaccinations in the regression model ,37,39,41We found a considerable heterogeneity in the results of published studies on repeated vaccination, most possibly due to different populations, variability of vaccine composition and circulating virus in the considered seasons . Older aThe presence of an intra-seasonal waning effect of the vaccine immunization was usually detected when exploring IVE against A(H3N2) ,47,48. WOur study was subject to different biases as it is usual in observational studies. We tried to avoid selection bias, including all patients fulfilling a well-established case definition. Vaccination status was ascertained by registries, influenza infection was laboratory-confirmed with a sensitive real-time RT-PCR assay, and only patients swabbed within 8–48 h of admission in hospital were included to avert misclassification bias. Unknown confounding can bias results in observational studies. We tried to control confounders by adjusting models by factors related to both vaccination status and influenza result. Nevertheless, residual confounding always remains as some confounder are not measurable. In some of the analysis, a larger sample size would allow us to provide more robust estimates.Our data revealed a moderate IVE, although an intra-seasonal not relevant waning effectiveness trend was observed, in hospitalized patients 65 years old or over in the Valencia Region of Spain during the 2018/2019 season. IVE by subtype ranged between 41 and 45%, although protection was not significant against A(H1N1)pdm09. The effectiveness of the adjuvanted vaccine was also proved in patients 75 years old or over, which represent the target population for receiving that vaccine.


BRCA1 (NBR1) that recognizes ubiquitinated MAVS and delivers it to autophagosomes for degradation. The degradation cascade mediated by PB1 facilitates H7N9 virus infection by blocking the RIG-I-MAVS-mediated innate signaling pathway. Taken together, these data uncover a negative regulatory mechanism involving the PB1-RNF5-MAVS-NBR1 axis and provide insights into an evasion strategy employed by influenza virus that involves selective autophagy and innate signaling pathways.Influenza A virus (IAV) has evolved various strategies to counteract the innate immune response using different viral proteins. However, the mechanism is not fully elucidated. In this study, we identified the PB1 protein of H7N9 virus as a new negative regulator of virus- or poly(I:C)-stimulated IFN induction and specifically interacted with and destabilized MAVS. A subsequent study revealed that PB1 promoted E3 ligase RNF5 to catalyze K27-linked polyubiquitination of MAVS at Lys362 and Lys461. Moreover, we found that PB1 preferentially associated with a selective autophagic receptor neighbor of  In 2013, H7N9 influenza viruses appeared in China and other countries resulting in 1, 567 human infections and 615 deaths. Understanding the cross-talk between virus and host is vital for the development of effective vaccines and therapeutics. Here, we identified the PB1 protein of H7N9 virus as a novel negative regulator that enhances the degradation of MAVS, an essential adaptor protein in the innate signaling pathway. Mechanistically, PB1 promoted the E3 ligase RNF5-mediated ubiquitination of MAVS and recruited the selective autophagic receptor NBR1 to associate with and deliver the ubiquitinated MAVS to the autophagosomes for degradation. Thus, the PB1-RNF5-MAVS-NBR1 axis inhibited innate immune antiviral response and facilitated virus replication by mediating MAVS degradation in an autophagosome-dependent manner. Our findings reveal a novel mechanism by which influenza virus negatively regulates the innate immune response. Influenza A viruses (IAVs) are important zoonotic pathogens involved in global pandemics and annual epidemics, which are continually challenging animal and human health. IAVs  have caused four influenza pandemics since 1918 and an unprecedented number of deaths .Orthomyxoviridae family. The IAV genome consists of eight gene segments  that encode at least 14 proteins .(A) PB1-F2 is silenced in the PB1 ORF. The upper panel shows the nucleotide acid and amino acid sequences of the PB1 (red) and PB1-F2 (blue) ORF in the PB1 ORF. The italicized nucleotide acids were mutated to the green-labeled nucleotide acids in the recombinant plasmids and virus. The lower panel shows the sequence of the recombinant plasmids. (B) Screening of viral proteins of SZ19 virus for their effects on SeV- and poly(I:C)-induced activation of IFN-β promoter. HEK293 cells were transfected with IFN-β promoter and plasmids encoding viral proteins  for 24 h. The cells were then infected with SeV or transfected with poly(I:C) for 12 h before luciferase analysis. (TIF)Click here for additional data file.S2 Fig(A) Schematic representation of the domain organization of MAVS and its interaction with PB1. -, no interaction; +, interaction. (B) PB1 from different subtypes of influenza A virus decreases the MAVS protein level. HEK293 cells were transfected with Myc-MAVS and Flag-PB1 from different subtypes of influenza A virus and NP from SZ19 virus for 24 h before immunoblot analysis. (C) HeLa cells were transfected with pRFP-GFP-LC3 and an empty vector or Flag-PB1. At 24 h post-transfection, cells were treated with EBSS or left untreated for the indicated times and then analyzed for autophagosome formation. The data shown represent three independent experiments.(TIF)Click here for additional data file.S3 FigP< 0.05(*), P < 0.01(**), P < 0.001(***), P < 0.0001(****)].(A) Knockdown of ULK1, ATG13, FIP200, and ATG101 has no marked effect on PB1-mediated MAVS degradation. HEK293 cells were transfected with siRNA for NC, ULK1, ATG13, FIP200, and ATG101 (100 nM/well) for 24 h, the cells then were transfected with indicated plasmids for another 24 h before immunoblot analysis with the indicated antibodies (upper panels). The lower chart shows the efficiency of siRNA for ULK1, ATG13, FIP200, and ATG101. HEK293 cells were transfected with siRNA for control, ULK1, ATG13, FIP200, and ATG101 (100 nM/well) for 24 h before qPCR analysis. (B) MAVS interacts with NBR1, OPTN, p62, and NDP52. HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analyses with the indicated antibodies. (C) Schematic representation of the domain organization of NBR1 and its interaction with MAVS. -, no interaction; +, interaction. The data shown represent three independent experiments; bars represent the mean ± SD of the three independent experiments (n = 3). .(A) HEK293 cells were transfected with Myc-MAVS and the indicated mutants in the presence or absence of Flag-PB1 for 24 h before immunoblot analysis. (B) The effects of MAVS-WT and MAVS-K362/461R on SZ19-ΔF2 virus replication. Wild-type and (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


C1RL), a prognostic biomarker in several kinds of tumours, has attracted increasing attention from oncologists. However, the role of C1RL in glioma remains unclear.Glioma is a deadly and immunosuppressive brain tumour. Complement C1r subcomponent like  analysis was conducted to investigate biological process enrichment. In addition, tumour purity, leukocyte infiltration and overall survival were evaluated based on C1RL expression.Through analysis of 2120 glioma patients from 5 public datasets, the relationships between C1RL expression was upregulated in glioblastoma (GBM), especially mesenchymal GBM and primary GBM. Increased C1RL expression accompanied the IDH1-wt phenotype in both lower grade glioma (LGG) and GBM. C1RL- associated genes were mainly enriched in biological processes related to the immune response. C1RL expression was also correlated with reduced tumour purity and increased M2 macrophage infiltration. Higher C1RL expression predicted unfavourable survival in patients with glioma and therapeutic resistance in GBM.We found that C1RL is involved in immunological activities and is an independent unfavourable prognostic biomarker in patients with glioma. C1RL is a potential clinical immunotherapeutic target for glioma treatment in the future.Our results imply that  PD-1 inhibition [Glioblastoma  and lower grade glioma  are incurable brain tumour. Existing therapeutic strategies only prolong the survival of glioma patients to a limited extent. Patients with glioma eventually die from tumour recurrence, even with aggressive treatment. Novel therapies that have been successful in other tumours, such as hibition , 3, havehibition , 5. The PD-L1 [PD-L1 on macrophages derived from healthy donors [The immunosuppressive microenvironment significantly contributes to the progression and therapeutic resistance of glioma. On the one hand, glioma cells induce a relatively weak immune response and enhance immunosuppression. Compared to other malignancies, glioma exhibits a lower mutational burden and fewer infiltrating T cells . GBM celPD-L1 . Glioma y donors , 9. Intry donors . On the y donors . Moreovey donors . Naïve Ty donors .C1RL) was found to be a prognostic marker in hepatocellular carcinoma [C1RL gene [C1RL also mediates the progression of Burkitt’s lymphoma [C1RL is a protein-coding gene associated with ovarian adenocarcinoma and leucorrhea. In terms of molecular function, the C1RL protein, which is homologous to C1r, is identified as the active form of serine hydrolase [C1RL protein cleaves prohaptoglobin in the endoplasmic reticulum [C1RL protein [C1RL in glioma remain unclear.Complement C1r subcomponent like . A microarray dataset containing 301 samples (CGGAmic) and an RNA-sequencing dataset containing 325 samples (CGGAseq) were downloaded from The Chinese Glioma Genome Atlas . A microarray dataset containing 276 samples (GSE16011mic) was downloaded from Gene Expression Omnibus .Five datasets including transcriptomic files and corresponding clinicopathological information for patients who were diagnosed with glioma (WHO II-IV) were downloaded. A microarray dataset containing 539 samples (TCGAmic) and an RNA sequencing dataset containing 702 samples (TCGAseq) were downloaded from The Cancer Genome Atlas  was applied to compare C1RL expression among three or more groups. P < 0.05 was considered significant.An unpaired t test was used in comparisons of C1RL and all other genes were calculated in RStudio 1.1.453 with the cor.test algorithm. C1RL-associated genes were defined as genes with an r value > 0.4 in the GBM dataset (TCGAmic) and r > 0.5 in the glioma datasets . All the C1RL-associated genes were introduced into DAVID (https://david.ncifcrf.gov/) for further GO analyses. The top 10 biological process terms of the GO analysis results are listed in Fig. Pearson correlation coefficients between C1RL and immunosuppressive genes  are shown in Fig. Moreover, the detailed correlations between https://cibersort.stanford.edu/) was used to evaluate leukocyte infiltration. Heatmaps were produced in MORPHEUS (https://software.broadinstitute.org/morpheus/) online. The colour shows the Z score  of all the expression data. The samples were ordered according to the expression of C1RL.The ESTIMATE algorithm package was used to analyse tumour purity. The CIBERSORT tool (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi).The log-rank test and Kaplan-Meier survival curves were used to describe survival differences between two groups. The survival analysis of the GSE16011 dataset was conducted in R2  superfamily of proteins. CD86 [LGALS9 [TGFB1 [C1RL expression exhibited positive relations with CD86, LGALS9, and TGFB1 , and standard therapy  to characterize cell composition of complex tissues from their gene expression profiles. In this study, both ESTIMATE algorithm and CIBERSORT algorithm were used to further assess the relationships between C1RL mRNA expression and 22 different immune cell populations. Increased amounts of immune cells, especially M2 macrophages, migrated into glioma tumours with relatively high C1RL expression (Fig. C1RL palys an immunosuppressive role in glioma.rognosis . Glioma rognosis . M2 macrrognosis . The ESTrognosis , 37. Besrognosis . The CIBion Fig. .A and B.C1RL in 2120 glioma patients from five datasets. The results indicate that C1RL is a negative biomarker for the patients with glioma. Furthermore, C1RL probably plays an immunosuppressive role in the pathogenesis of glioma by triggering the activation of haptoglobin and C1s.In conclusion, we analysed the immunological and clinicopathological characteristics of Additional file 1 Table S1. Clinical information of 2143 patients from the different datasets.Additional file 2 Table S2. C1RL associated genes.Additional file 3 Table S3. C1RL and WHO grade and glioma histopathology.


ABCB1 single-nucleotide polymorphism (SNP) 1236C>T with genotypes C/T or C/C (Jewish) and haplotypes AGCTT carrier, AGCGC heterozygote, or non-carrier (Caucasian), which have a predicted lower methadone dose requirement. In contrast, ABCB1 SNP 1236C>T with genotype T/T (Jewish); haplotypes AGCGC homozygote, AGCTT non-carrier (Caucasian), and ABCB1 3435C>T variant carrier; and haplotypes CGT, TTC, and TGT (Han Chinese) have a predicted higher methadone dose. For methadone plasma levels, ABCB1 diplotype non-CGC/TTT  predicted lower, and diplotype CGC/TTT , 3435C>T allelic carrier, haplotypes  (Han Chinese) predicted higher methadone levels. In terms of metabolism biomarkers, a lower methadone requirement was related to carriers of CYP2B6 genotypes *4(G/G) and *9(T/T) among Jewish patients, CYP2B6*9 genotype (T/T) and haplotypes (TA/TG); and CYP2C19. Higher methadone dose was observed in CYP2C19*1 allelic carriers (Han Chinese) and CYP2D6 ultrarapid metabolizer (Caucasian). Lower methadone levels were reported in CYP2B6 SNPs, haplotypes TTT, and AGATAA (Han Chinese), CYP2C19 genotype *1/*1 (Han Chinese), allelic carrier *1xN (Caucasian), and CYP3A4 genotype *1/*1 (Caucasian). Carriers of CYP2B6 genotype *6/*6 (Caucasian), CYP2B6 haplotypes ATGCAG and ATGCTG (Han Chinese), and CYP3A4 genotype *1/*1B (Caucasian) had predicted higher methadone plasma levels. Specific pharmacokinetics biomarkers have potential uses for personalized methadone treatment in specific populations.Methadone has a wide pharmacokinetic interindividual variability, resulting in unpredicted treatment response. Pharmacogenomic biomarkers seem promising for personalized methadone maintenance treatment. The evidence supports the use of  Methadone maintenance treatment (MMT) is a program widely available to treat opioid use disorder . MMT is The broad range of methadone dosage may be attributed to genetic polymorphisms. Numerous studies have shown the potential contribution of genetic polymorphisms to the variability of both methadone dose and concentration ,11,13-18(R)- and (S)-enantiomers in equal proportion. (R)-methadone has been reported to account for most opioid effects, including the primary analgesic effect [Methadone is a synthetic opioid that is available in a racemic mixture of c effect ,13. The c effect , respirac effect . Insuffic effect . Pharmacc effect ,22. In tc effect ,11,13-18(R)- and (S)-methadone are P-gp substrates [in vitro study reported the ability of methadone to inhibit both the wild-type and variant-type human P-gp; the former had a more potent effect than the latter - iability .ABCB1 genetic variants and methadone dose (ABCB1 single-nucleotide polymorphism (SNP) 1236C>T between lower- and higher-dose groups. Homozygotes with T allele had more than 6 times risk to require more than 150 mg/day stabilizing methadone dose than those who were heterozygotes or homozygotes for C allele (ABCB1 (AGCGC and AGCTT) were associated with methadone dose. These haplotypes were formed from 12 unique combinations from five common SNPs in the Caucasian population: 61A>G, 1199G>A, 1236C>T, 2677G>T, and 3435C>T. Possession of two copies of the AGCGC haplotypes was associated with a higher methadone dose compared to having a single copy or none. In contrast, the AGCTT haplotype carrier was associated with a significantly lower methadone dose requirement. Hung et al. [ABCB1 3435 C>T allelic variant carriers and non-carriers in terms of methadone dose in a Han Chinese population. A higher dose was required in carriers (homozygotes>heterozygotes) than non-carriers [ABCB1 haplotypes of CGT, TTC, and TGT, a combination of 1236C>T, 2677G>T/A, and 3435C>T had a significantly higher methadone requirement in the Han Chinese population [Multiple studies have reported the association between the one dose . In a stC allele . Multi-lg et al.  reportedcarriers . Moreovepulation .ABCB1 phenotypes of extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM) in a Caucasian population. Another study in the Caucasian population also reported no significant association between responders’ status and ABCB1 genotypes [In contrast, Fonseca et al.  reportedenotypes .A comparison of methadone dose was different between studies. Two studies with significant findings predefined the methadone dose group into (1) low (≤150 mg) and high dose (>150 mg)  and (2) ABCB1 CGC/TTT diplotype  was significantly associated with higher -methadone plasma levels compared to those who did not possess this diplotype. Other diplotypes, haplotypes, genotypes, or allelic variants had insignificant contributions towards methadone plasma levels. Similarly, a Caucasian population study found a similar negligible difference in (R)-, (S)-, and -methadone plasma levels between three phenotypes of ABCB1 3435 C>T polymorphism [ABCB1 61A>G and 3435C>T, and trough (R)-, (S)-, and -methadone plasma levels. Lower methadone levels were observed in carriers of allelic variants ABCB1 61A>G (A/G or G/G genotypes) and ABCB1 3435C>T (C/T or T/T genotypes). Higher methadone plasma concentrations were observed in carriers of ABCB1 haplotypes 61A/2677G/3435C and 2677G/3435C. No stereoselectivity in P-gp transport for methadone was reported [In a Malay population, Zahari et al.  reportedmorphism . In cont(R)-methadone isomer [(S)-methadone over the other isomer [S)-methadone. Other methadone-related CYPs include CYP2D6 and CYP2C9. The number of functional alleles of a CYP genotype determines the individual phenotype in the metabolism rate. CYP phenotypes include PM, IM, EM, and ultrarapid metabolizer (UM). In general, in the presence of functional enzymes, more methadone can be metabolized, resulting in reduced plasma concentration and perhaps a higher methadone dose. In contrast, reduced enzyme activity or enzyme non-functionality results in higher methadone plasma concentration and perhaps a lower dose of methadone (Methadone metabolism mainly occurs in the liver . CYP in e isomer . Inhibitr isomer . This shethadone . Numerou(S)-methadone in a preclinical study [(R)- and (S)-enantiomers with stereoselectivity towards the latter enantiomer [(S)-methadone is associated with adverse effects related to methadone. Ansermot et al. [(R)-methadone, who were previously on -methadone. Moreover, a study of methadone-related death found significantly higher methadone plasma levels in CYP2B6*6 allele carriers than CYP2B6*1 and *4. This result indicates the role of CYP2B6 in methadone metabolism with the carriers of the non-functional CYP2B6*6 representing the poor phenotype group [CYP2B6 is the primary enzyme responsible for methadone metabolism. CYP2B6 produces the highest EDDP metabolite compared to other CYP families and it showed stereoselectivity towards al study . A cliniantiomer . (S)-mett et al.  reportedpe group .CYP2B6*4 and CYP2B6*9 compared to those who were heterozygotes and non-carriers. The result was not attenuated even after controlling for other confounding factors such as age, sex, and the ABCB1 1236T/T genotypes. These variant alleles corresponded to CYP2B6*6. The associations between the additional SNPs of CYP2B6 and methadone dose were not significant [CYP2B6*9 carriers, who required a significantly lower dose than non-carriers [*4 (516G>T) and *9 (785A>G) with TA or TG, who had a significantly lower methadone dose requirement compared to non-carriers [Levran et al.  reportednificant . In the carriers . Moreovecarriers .CYP2B6 genotypes in methadone dose [CYP2B6*6 and methadone dose was different between two studies as one analyzed CYP2B6*4 and CYP2B6*9 genotypes separately. These alleles had been shown to have strong linkage disequilibrium and could represent CYP2D6*6 [CYP2B6*6 genotypes [Reports in the Caucasian population have found a negligible difference between one dose ,6. The cone dose  had moreone dose  and Fonsone dose  only excone dose , phenotyone dose . The anaCYP2D6*6 . Other senotypes ,6.(S)- and -methadone in homozygous carriers of the CYP2B6*6 allele variant than in heterozygotes and non-carriers in a Swiss MMT population. A similar finding was reported by Crettol et al. [(S)-methadone plasma levels and CYP2B6*6/*6 genotypes. In a Han Chinese population, a similar pattern was observed in carriers of the CYP2B6*6 (rs2279343 and rs3745274) allelic variants. Other SNPs  were also found to be significantly associated with (S)-methadone plasma levels. The SNP rs8100458 was significantly associated with trough (R)-methadone plasma levels. Moreover, TTT (a combination of rs8100458-rs10500282-rs10403955) and AGATAA (a combination of rs2279342-rs3745274-rs2279343-rs2279345-rs1038376-rs707265) haplotypes were associated with lower (S)-methadone plasma levels. ATGCAG and ATGCTG haplotypes were associated with higher (S)-methadone plasma levels [In terms of methadone concentration, Crettol et al.  found sil et al.  in anotha levels .(S)- and (RS)-methadone plasma levels were higher in homozygous carriers of CYP2B6*6. However, the result did not reach significant levels [CYP2B6*4  and CYP2B6*9  were also found not significantly associated with trough (R/S)-methadone plasma levels [(R)- and (S)-methadone plasma levels was not evaluated [In a Caucasian population, the means of both the trough CYP2C19 is another enzyme necessary for the metabolism of methadone. An in vitro study reported a similar production of EDDP by CYP3A4 with stereoselectivity towards (R)-methadone enantiomer [(R)-methadone stereoselectivity of CYP2C19 has been conflicting [antiomer ,36. In cflicting ,6,10.CYP2C19 phenotypes among the Han Chinese population [*1/*1) and IM  groups had significantly higher methadone dose than PM  group [CYP2B6 or CYP3A4, as well as the allelic combination of those CYPs included in the analysis, which allowed the formation of 6–12 different methadone dose ranges [Studies in a Han Chinese population reported a significant association between methadone dose and pulation ,18. Both3) group . The sigCYP2C19 genotype distribution between responders and non-responders, as well as between low-dose and high-dose groups among a Caucasian population in Switzerland. Similarly, another study in a Caucasian population in Spain reported negligible methadone dose difference between EM (*1/*1), IM (*1/*2), and PM (*2/*2) phenotypes of CYP2C19 [CYP2C19 [CYP2C19 metabolism, resulting in increased significant levels.In contrast, Crettol et al.  found no CYP2C19 . The con[CYP2C19 ,6. Wang [CYP2C19  performe(R)-methadone plasma levels and CYP2C19 among Han Chinese with negative urine morphine. Patients with the predicted phenotype of EM (*1/*1) reported a significantly lower trough (R)-methadone plasma levels than PM, but a negligible difference was found in terms of (S)-methadone plasma levels. The SNPs CYP2C19*2 and *3 represented the non-functional alleles while the SNP CYP2C19*1 represented the functional allele. Lack of functional allele variants in the genotype leads to reduced metabolism of (R)-methadone, resulting in higher methadone plasma levels [Wang et al.  reporteda levels .(R)-, (S)-, and -methadone plasma levels and CYP2C19 genotypes. Stereoselectivity towards (R)-methadone was not observed in two studies [in vitro study and in vivo study [In contrast, Crettol et al. , Crettol studies ,6, contrvo study ,31.*1/*1, *1/*2, and *1/*3) and PM (based on genotypes *2/*2). Wang et al. [*1/*1), IM (based on genotypes *1/*2 and *1/*3), and PM . The genotypes distribution between studies was also different, even between two homogenous populations. The genotype CYP2C19*2/*2, for example, had a frequency of 10.7% in the Han Chinese population [Classification of genotypes, phenotypes, and illicit opioid use may have contributed to the differing findings. Crettol et al.  classifig et al.  divided pulation  and Switpulation , respectCYP3A4 and CY3A5 belong to the CYP3A family . An initCYP3A4 and methadone dose in a Han Chinese population. Similarly, Levran et al. [CYP3A4 and methadone dose in a Jewish population. Despite similar findings in terms of methadone dose in both Han Chinese and Jewish populations, the majority of SNPs included in the final analysis were different, with only one similar SNP (rs2242480). These differences exist due to the differences in the selection method, references, software, methods, and ethnicity. The Caucasian population showed a negligible difference in methadone doses between EM, PM, and very PM phenotypes in terms of CYP3A5 [Chen et al.  reportedn et al.  found nof CYP3A5 .(S)-methadone plasma levels in the CYP3A4 (*1/*1B) genotype than the CYP3A4(*1/*1) genotype, with an insignificant difference in both trough and peak (R)-methadone plasma levels in the initial analysis. However, further analysis found no stereoselectivity towards (S)-methadone. Furthermore, no association was found between (R)-, (S)-, and -methadone plasma levels at either trough and peak with CYP3A5 genotypes [R)-, (S)-, and (RS)-methadone plasma levels and CYP3A5 phenotypes . A study in a Han Chinese population reported no significant difference in trough (R)- and (S)-methadone plasma levels between all selected CYP3A4 SNPs [*1B was included in the study because the variant is rare in the study population [CYP3A activity analysis using midazolam reported higher levels of all forms of methadone at both trough and peak in low CYP3A activity group in a Caucasian population . Crettolenotypes  reported3A4 SNPs . No allepulation .in vitro study, CYP2C9 was found to generate a measurable amount of EDDP only at 10 times the concentration at which major CYPs  were able to generate the measurable amount of methadone metabolites [In an abolites .CYP2C9 and methadone dose between low- and high-dose groups or between responder and non-responder groups. A study in another Caucasian population reported a higher methadone requirement in EM than IM and PM phenotypes. However, the differences did not reach significant levels [Crettol et al.  reportedt levels .(R)-, (S)-, and (RS)-methadone plasma levels and CYP2C9 phenotypes and genotypes [*2/*2, *2/*3, and *3/*3) and EM , and did not reach significance [*1/*1 and *1/*2), IM (*1/*3), and PM  phenotypes among a Caucasian population.The studies in the Caucasian population reported a negligible association between enotypes ,5. In thificance  reportedin vitro study. Clinical studies indicated a possible interaction of genetic polymorphisms in methadone metabolism [The contribution of CYP2D6 to methadone metabolism is minimal in comparison to other CYPs, as reported in the tabolism ,6. Up toCYP2D6 phenotypes in trough methadone plasma levels in a Caucasian population, with the former group requiring a significantly higher dose. The possession of at least one functional allele  was classified as EM in this study. UM was defined as those who possessed duplicates of *1 or *2 [Fonseca et al.  reported*1,*2, *9, and *10) with none, 1–2, and >2 classified as PM, EM, and UM, respectively [CYP2D6 SNPs and methadone dose.In contrast, de los Cobos et al.  in a stuectively . Similarectively  reportedectively  and Caucectively  reported were significantly higher in UM compared to EM in a study among a Caucasian population in Spain. We expected that plasma methadone levels should be lower in UM than EM because the expected UM activity is higher than EM. The findings might be due to the inhibition of CYP2D6 methadone metabolism because the majority (65%) of the study population had comedication, such as anti-depressants and antiretrovirals [(S)-methadone plasma levels in IM and EM groups than UM (*1/*xN) group and a negligible difference in trough (R)-methadone and peak of all forms of methadone plasma levels.All forms of trough methadone rovirals  reported(R/S)-methadone plasma levels between phenotypes. In that study, patients using concurrent medication known to inhibit CYP2D6 were not excluded from the analysis [Another study conducted in Spain reported no significant difference in trough analysis .The importance of CYP2D6 to methadone pharmacokinetics is limited because it is not the major CYP involved in methadone metabolism . HoweverABCB1 SNP 1236C>T with genotypes C/T or C/C (Jewish) [ABCB1 SNP 1236C>T with genotype T/T (Jewish) [ABCB1 3435C>T variant carrier; and haplotypes CGT, TTC, and TGT (Han Chinese) [ABCB1 diplotype non-CGC/TTT ; ABCB1 carriers of 61A>G, 3435C>T, and 2677G>T (Caucasian); and ABCB1 haplotypes of 61A/2677G/3435C and 2677G/3435C in Caucasian [ABCB1 3435C>T allelic carrier (Han Chinese); haplotypes CGT, TTC, and TGT (Han Chinese) [CYP2B6 genotypes *4 (G/G) and *9 (T/T) among Jewish patients [CYP2B6 genotype *9 (T/T) and haplotypes (TA/TG) [CYP2C19 [CYP2D6 EM compared to UM (Caucasian) [CYP2B6 genotypes *4  and *9 (G/G and G/T) among Jewish patients [CYP2C19*1 allelic carriers (Han Chinese) [CYP2D6 UM phenotypes  in Caucasian patients [CYP2B6 SNPs, haplotypes TTT and AGATAA (Han Chinese) [CYP2C19 genotype *1/*1 (Han Chinese) [*1xN [CYP3A4 genotype *1/*1 (Caucasian) [CYP2B6 genotype *6/*6 (Caucasian) [CYP2D6 UM phenotypes (*1xN and *2xN) [CYP3A4 genotype *1/*1B (Caucasian) [The evidence supports the use of (Jewish)  and hapl(Jewish) , which h(Jewish) ; haploty(Jewish) , and ABCChinese)  have a paucasian ; ABCB1 3Chinese)  predicte (TA/TG) ; and CYPChinese) ; and CYPChinese) , and CYPChinese) , allelicucasian) . Carrierucasian) .Specific pharmacokinetics biomarkers have potential uses for personalized methadone treatment in specific populations. The use of pharmacogenomics is essential because methadone has a wide interindividual variability and various possible drug–drug interactions with unpredictable treatment response. More studies are required to validate the use of these biomarkers.