Background and purpose — The accelerated wear of poorly functioning metal-on-metal (MoM) hip implants may cause elevated whole-blood cobalt (Co) and chromium (Cr) levels. Hematological and endocrinological changes have been described as the most sensitive adverse effects due to Co exposure. We studied whether there is an association between whole-blood Co/Cr levels and leukocyte, hemoglobin, or platelet levels.Patients and methods — We analyzed whole-blood Co and Cr values and complete blood counts  from 1,900 patients with MoM hips. The mean age at the time of whole-blood metal ion measurements was 67 years (SD 10). The mean time from primary surgery to whole-blood metal ion measurement was 8.2 years (SD 3.0). The mean interval between postoperative blood counts and metal ion measurements was 0.2 months (SD 2.7).Results — The median Co value was 1.9 µg/L (0.2–225), Cr 1.6 µg/L (0.2–125), mean leukocyte count 6.7 × 109/L (SD 1.9), hemoglobin value 143 g/L (SD 13), and platelet count 277 × 109/L (SD 70). We did not observe clinically significant correlations between whole-blood Co/Cr and leukocyte, hemoglobin, or platelet counts.Interpretation — Elevated whole-blood Co and Cr values are unlikely to explain abnormal blood counts in patients with MoM hips and the reason for possible abnormal blood counts should be sought elsewhere. The abnormal wear of poorly functioning MoM implants may cause elevated whole-blood cobalt (Co) and chromium (Cr) levels  and DePuy Orthopaedics recalled their ASR MoM hip replacement systems  between October 2009 and March 2018.For the complete blood counts, 3 mL of blood was drawn into an EDTA tube. Leukocyte count, hemoglobin, hematocrit, erythrocyte count, mean corpuscular volume, and mean hemoglobin concentration as well as platelet count were then analyzed. Reference values for blood count values provided by our hospital laboratory were used. The complete blood counts were measured between October 2009 and June 2018. We used the complete blood count closest to the latest Co and Cr values. As the data was not prospectively collected and the whole blood Co and Cr measurements were not performed on the same day as the blood count, we chose 6 months as an arbitrary cut-off to exclude patients with long interval between the measurements. Of the 2,260 patients with whole-blood metal ion levels available, 1,900 had their blood count measured less than 6 months before or after the whole-blood metal ion measurement and were therefore included in the study. 572 of the patients went through a revision and for them last pre-revision measurements were used. For 314 patients there was no complete blood count available within 6 months of the latest whole-blood Co and Cr values, and thus an older metal ion measurement fulfilling the condition of blood count measured within a 6-month interval was used. For 258 patients leukocyte differentials were available and they were analyzed as a subgroup.As the distributions of whole-blood Co and Cr values are skewed, median values (ranges) are reported. Blood hemoglobin value, leukocyte, and platelet counts are normally distributed, so mean values (SD) are used. Spearman’s correlation coefficients were calculated to test whether there is correlation between whole-blood metal ion levels and either hemoglobin, leukocyte, or platelet counts. For Spearman’s correlation coefficient, we calculated 95% confidence intervals (CI). We chose a correlation coefficient of 0.225 (or –0.225) as the limit for clinically significant, equaling roughly 5% of variance for a dependent variable explained by an independent variable. If the observed CIs for correlation coefficients did not include 0.225 the correlation coefficient was considered as clinically not significant. We also calculated the change in blood count components for those 1,873 (99%) patients with preoperative blood counts available.The analyses were performed for all patients regardless of the implant brand and manufacturer or whether they had unilateral or bilateral MoM hip implants even though the blood concentrations are different among them, because, when considering the systemic effects of Co and Cr, it is irrelevant whether the Co and Cr in the blood are from 1 or 2 hips and which implant brand is used.Permission for the study was obtained from the institutional review board (permission number R17524S). This work was supported by the competitive research funds of Pirkanmaa Hospital District, Tampere, Finland, representing governmental funding. No conflicts of interest declared except for AE, who is paid lecturer for Zimmer Biomet, and receives institutional research support (not related to current study) from DePuy Synthes and Zimmer Biomet.Of the 1,900 patients included in the study, 81% had unilateral and 19% had bilateral MoM replacement . 58% of 9/L (SD 1.9), hemoglobin 143 g/L (SD 13), and platelet count 277 × 109/L (SD 70). Mean preoperative leukocyte count was 6.8 × 109/L (SD 2.5), hemoglobin 140 g/L (SD 14), and platelet count 245 × 109/L (SD 71). The mean time between preoperative and last postoperative blood count was 8.3 years (SD 3.0). The mean change in leukocyte (Δleukocytes) count was 0.1 × 109/L (SD 2.4), in hemoglobin (Δhemoglobin) –3.0 g/L (SD 13), and in platelet count (Δplatelets) –32 × 109/L (SD 61).The median value for whole-blood Co concentration was 1.9 µg/L (0.2–225) and for Cr 1.6 µg/L (0.2–125). Mean postoperative leukocyte count was 6.7 × 10Supplementary data). None of the 95% CIs for ρ included predefined 0.225 or –0.225 , indicating that clinically significant associations can be excluded.Spearman’s correlation coefficients (ρ) between whole-blood Co/Cr and complete blood count parameters are presented in Supplementary data). For all other analyses, clinically significant associations were excluded.Leukocyte differentials including lymphocytes, neutrophils, eosinophils, basophils, and monocytes were available for 258 patients. This subgroup included 53% men. The mean age at the time of blood metal ion measurement was 67 years (SD 10), mean time from primary surgery to metal ion measurement was 7.8 years (SD 2.7), and interval between metal ion and leukocyte differentials 0.9 months (SD 3.0). The Spearman’s correlation coefficients (ρ) between whole-blood Co and blood eosinophil count  and between Cr and monocytes count  were the only ρ between Co/Cr and leukocyte differential counts in which 0.225 was not excluded from 95% CI  in a patient with an intensively worn CoCr head due to ceramic fragments from the previous fractured implant embedded in a new polyethylene liner resulting in extremely high  serum Co levels  and 44 with metal-on-polyethylene (MoP) arthroplasty reported a higher percentage of HLA DR + CD8+ T-cells and lower percentage of B-cells in the MoM group  and 0.42 . The reference values for blood eosinophils at our laboratory are 0.01–0.45 × 10To our knowledge there are no case reports or studies concerning abnormal platelet values that would have been linked to implant metals. An in vitro study concerning CoCr stent materials stated that platelets adhere to and are activated by CoCr  form, which does not pose a health risk to patients. Our results supplement the previous results with clinical data from a hip arthroplasty population. The reasons for possible abnormal blood counts in patients with MoM hip and their Co and Cr below several hundred µg/L should be primarily sought elsewhere.  Click here for additional data file.


Trypanosoma cruzi, and the main cause of morbimortality from cardiovascular problems in endemic areas. Although efforts have been made to understand the signaling pathways and molecular mechanisms underlying CCC, the immunological signaling pathways regulated by the etiological treatment with benznidazole (Bz) has not been reported. In experimental CCC, Bz combined with the hemorheological and immunoregulatory agent pentoxifylline (PTX) has beneficial effects on CCC. To explore the molecular mechanisms of Bz or Bz+PTX therapeutic strategies, C57BL/6 mice chronically infected with the T. cruzi Colombian strain (discrete typing unit TcI) and showing electrocardiographic abnormalities were submitted to suboptimal dose of Bz or Bz+PTX from 120 to 150 days postinfection. Electrocardiographic alterations, such as prolonged corrected QT interval and heart parasite load, were beneficially impacted by Bz and Bz+PTX. RT-qPCR TaqMan array was used to evaluate the expression of 92 genes related to the immune response in RNA extracted from heart tissues. In comparison with non-infected mice, 30 genes were upregulated, and 31 were downregulated in infected mice. Particularly, infection upregulated the cytokines IFN-γ, IL-12b, and IL-2  and the T-cell chemoattractants CCL3 and CCL5 . Bz therapy restored the expression of genes related to inflammatory response, cellular development, growth, and proliferation, and tissue development pathways, most probably linked to the cardiac remodeling processes inherent to CCC, thus mitigating the Th1-driven response found in vehicle-treated infected mice. The combined Bz+PTX therapy revealed pathways related to the modulation of cell death and survival, and organismal survival, supporting that this strategy may mitigate the progression of CCC. Altogether, our results contribute to the better understanding of the molecular mechanisms of the immune response in the heart tissue in chronic Chagas disease and reinforce that parasite persistence and dysregulated immune response underpin CCC severity. Therefore, Bz and Bz+PTX chemotherapies emerge as tools to interfere in these pathways aiming to improve CCC prognosis.Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe form of Chagas disease, a neglected tropical illness caused by the protozoan  Trypanosoma cruzi, responsible for an estimated 6 to 7 million people infected and approximately 75 million at risk of infection (Chagas disease (CD) or American trypanosomiasis is an infectious disease caused by the protozoan parasite nfection . Chagas nfection . Howevernfection . Health nfection . Chagas nfection , usuallynfection . Trypanonfection ; the mainfection .T. cruzi parasite-host immune response and disruption of intrinsic heart factors  is the most frequent and severe form of CD, being one of the main causes of morbidity and mortality due to heart diseases in endemic areas . CCC ons factors , as CCC  factors . In CD,  factors , leading factors . The pro factors , making T. cruzi and other protozoa, thus producing reactive metabolites that have trypanocidal effect in the intracellular and extracellular forms of the parasite  and nifurtimox, are effective when administered in the acute phase of infection . Howeverparasite . In addiparasite . The BENparasite . Thus, iparasite  to reducparasite . On the parasite . Thus, fparasite . Additioparasite . Treatmeoduction . PTX hasoduction . Moreoveoduction . HoweverT. cruzi–infected C57BL/6 mice showed that genes related to immune/inflammatory response and chemokine/cytokine receptor activity were the most activated pathways  strain  strain , we invehttps://www.mctic.gov.br/mctic/opencms/institucional/concea) and the Federal Law 11.794 (8 October 2008). The Institutional Committee for Animal Ethics of Fiocruz (CEUA-Fiocruz L004/09 and LW10/14) approved all experimental procedures used in the present study. All presented data were obtained from two independent experiments .This study was carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation  mice were intraperitoneally infected with 100 blood-derived trypomastigotes (BDTs) of T. cruzi Colombian strain (DTU TcI) in 0.2 ml of vaccine-grade sterile buffered saline . After 120 days postinfection (120 dpi), all animals were in the chronic phase and presenting clinical signs of CCC . Immediately after arrival, mice were housed in polypropylene cages under specific pathogen-free, randomly grouped in three mice per cage. The cages were maintained in microisolators, in standard conditions , noise and light (12-h light-dark cycle) control, and mice received grain-based chow food and water s of CCC , receiviMice were tranquilized with diazepam (10 mg/kg), and transducers were placed subcutaneously (DII). The traces were recorded for 2 min using a digital Power Lab2/20system connected to a bio-amplifier at 2 mV for 1 second . The filters were standardized between 0.1 and 100 Hz, and the traces were analyzed using Scope software for Windows V3.6.10 . The ECG parameters were analyzed as previously described .2) per section (three sections per heart) were evaluated as previously described , and the hearts were removed, embedded in tissue-freezing medium , and stored in liquid nitrogen. Serial cryostat sections, 3-μm thick, were fixed in cold acetone and subjected to polyclonal rabbit antibody recognizing mouse fibronectin (FN)  used at the dilution of 1:800 followed by indirect immunoperoxidase anti-rabbit immunoglobulin and peroxidase-streptavidin complex  staining revealed with liquid DAB+substrate chromogen system . Appropriate controls were prepared by replacing primary antibodies with immunoglobulin or normal rabbit serum. All antibodies and reagents were utilized in compliance with the manufacturer’s instructions. Images were digitized using a color view XS digital video camera adapted to a Zeiss microscope and analyzed with the Nis-Elements BR 4.0 software . The FN-positive areas in 25 fields (12.5 mmescribed .T. cruzi satellite DNA was done by using the specific primers (6 BDTs (Colombian strain), obtained from VERO cells infection, in 30 mg of non-infected heart tissue, proceeding DNA extraction, and making a 1:10 serial dilution of the eluted DNA in TE buffer, ranging from 106 to 0.1 parasite equivalents. Real-time PCR reactions were carried out on Applied Biosystems ViiA 7 real-time PCR  thermocycler, using the cycling conditions: 50°C for 2 min, 94°C for 10 min, followed by 40 cycles at 95°C, and 58°C for 1 min, where fluorescence was collected after each cycle. All samples were run in duplicate, and threshold was set at 0.02 for both targets.Genomic DNA was extracted from 10 to 20 mg of mouse hearts using High Pure PCR Template Preparation Kit , following the manufacturer’s instructions. Before extraction, tissues were withdrawn from RNA later and disrupted in 500 µl of tissue lysis buffer, using a TissueRuptor II  on maximal speed for 30 s. This homogenate was submitted to DNA extraction, according to the manufacturer’s recommendations. At the last step of the protocol, DNA was eluted from the silica column in 100 µl of elution buffer and stored at −20°C until further analysis. Amplification of  primers  Cruzi1 (® ND2000 (ThermoFisher), and integrity was analyzed in a Bioanalyzer 2100  using RNA Nano 6000 kit. Only samples with RIN ≥ 7.5 were used in this study.Mouse hearts were withdrawn from RNA later and disrupted in 500 µl of lysis buffer using TissueRuptor II  on maximal speed for 30 s. Total RNA was extracted using mirVana™ miRNA Isolation Kit (Life Technologies), according to the manufacturer’s recommendations. Total RNA quantification and purity were assessed in a NanoDropg for 2 min. Real-time PCR reactions were carried out on Applied Biosystems ViiA 7 Real-Time PCR  thermocycler, using the cycling conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Fluorescence was collected after each cycle, at the annealing/extension step. Raw data files were pre-processed using QuantStudio™ Real-Time PCR Software  with threshold and baseline corrections for each sample, and gene expression results were analyzed using Expression Suite v1.0.3 . Threshold was set at 0.01 for all targets. After the stability score analysis of the reference gene candidates, using the Expression Suite Software, GAPDH and HPRT targets were selected as the most-stable reference gene pair. Gene expression was estimated by the ΔΔCt method , were used for the gene expression analysis of 92 immune response and 4 endogenous genes according to the Applied Biosystems protocols. Reverse transcription was performed from 2.5 µg of total RNA using SuperScript™ IV VILO™ Master Mix with ezDNase , according to manufacturer’s instructions. Quantitative real-time RT-qPCR was done in 96-well pre-made TaqMan™ Array Mouse Immune Response . Each plate contained FAM/NFQ-MGB labeled TaqMan probes specific to 92 immune response genes and 4 endogenous reference gene candidates for data normalization and relative quantification. Ten microliters of Mastermix and 2 µl sample were loaded on each well, sealed, and centrifuged at 500t method , using u® Pathway Analysis (IPA) software    was used® Assays were used, according to the manufacturer’s recommendations: IFN-γ (assay ID Mm00801778_m1), CD3e (assay ID Mm00599683_m1), IL-2 (assay ID Mm00434256_m1), CSF2 (assay ID Mm00438328_m1), IL-7 (assay ID Mm00434291_m1), C3 (assay ID Mm00437858_m1), CD19 (assay ID Mm00515420_m1), and Selectin (assay ID Mm00441278_m1). As reference genes, GAPDH (assay ID Mm99999915_g1) and HPRT (assay ID Mm00446968_m1) were used. Two µg of total RNA was treated with DNAse I (Sigma) following manufacturer’s recommendations, and reverse transcription was subsequently performed using Superscript IV (Invitrogen) with random primers. Real-time quantitative RT-PCR was carried out in a 10-µl reaction containing 5 µl 2× TaqMan Universal PCR Master Mix (Applied Biosystems), 0.5 µl 20× TaqMan probe, 2 µl cDNA (at the concentration of 1 ng/µl), and 2.5 µl of nuclease-free water. Real-time PCR reactions were carried out on Applied Biosystems ViiA 7 Real-Time PCR System  using the cycling conditions: 10 min at 95 °C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Fluorescence was collected after each cycle at the annealing/extension step. All samples were run in duplicate, and threshold was set at 0.02 for all targets. Results were analyzed using ExpressionSuite v1.0.3 , GAPDH and HPRT targets were selected as endogenous controls, and gene expression was estimated using the ΔΔCt method  all pairwise multiple comparison  with SigmaPlot for Windows version 12.0 . Results were expressed as means and standard deviations (SDs), differences were considered significant if p < 0.05 as described in each figure legend.), and a prolonged QTc interval when compared with the non-infected (NI) mice group , which is a highly pathogenic parasite strain. This mouse model of CCC was first established by  disease . At 150 T. cruzi infection or drug therapies groups, and they showed high overlap, with 41 altered genes being shared in between groups  from ed group . The apped group . Thus, med group Table S1T. cruzi chronic infection  induce the production of pro-inflammatory cytokine IL-12 contributing to an amplification of Th1 activation by inducing the production of IFN-γ. IFN-γ production is also induced by transcription factors T-bet, Stat1, and Stat4 in the nucleus. Notably, increased expression IFN-γ by distinct molecular mechanisms can be associated with upstream regulators found in this study.The canonical pathways revealed a long list of significant activated pathways for the vehicle-treated group based of the expression profile of immune genes. Among the most upregulated pathways (orange-shade bars), we can highlight “Th1 pathway,” “type I diabetes mellitus signaling,” “crosstalk between dendritic and natural killer cells,” “dendritic cell maturation,” and at a lower degree, “natural killer cells signaling,” “apoptosis signaling,” and “cytotoxic T lymphocyte apoptosis of target T-cells and interferon signaling”  but had their expression restored (fold change below 2.0) with therapy using Bz or Bz+PTX. We were able to identify 13 genes that fulfill this criterion in the Bz-treated group  when compared to the non-infected group (not-detected), which was partially abrogated after Bz (5.66 ± 4.28), PTX (6.90 ± 4.16), and Bz+PTX therapies (10.88 ± 8.24)  when compared with the non-infected control (1.24 ± 0.42). In this situation, therapies with Bz (4.41 ± 2.08), PTX (4.42 ± 1.67), and Bz+PTX (4.72 ± 1.684) were unable to down-regulate the expression of this cytokine  compared with the non-infected group (0.68 ± 0.21). Interestingly, CSF2 expression was reversed through therapy with Bz (2.57 ± 1.51), PTX (2.18 ± 1.50) and partially reversed by Bz+PTX combined therapy (4.15 ± 2.12)  responsible for antigen recognition and T-cell activation, although previous study expression profiling of hearts obtained of transplanted CCC patients found TCR upregulation . These dd in CCC . In the d in CCC , and CCRd in CCC , corroboted mice . Moreoveted mice .T. cruzi infection.Altogether, the upregulation found for these genes in the present study might substantiate the main controlled pathways found for the vehicle-treated group, such as “inflammatory response,” “organismal injury and abnormalities,” “connective tissue disorders,” “cellular development,” “cellular growth and proliferation,” “tissue development,” and “immune cell trafficking” associated with the upregulation of several Th1-driven response molecules, and the lack of regulatory pathways to oppose this strong pro-inflammatory response thereof culminating in tissue remodeling causing the establishment of CCC as extensively shown in previous studies, , strengtT. cruzi-specific effector T cells also diminishes, therefore, causing a rearrangement in Th1-driven immunological response contributing to a more regulatory milieu in humans  and T helper cells (CD4+ naive T-cells) reinforcing that upon elimination of the etiologic agent, i.e., T. cruzi, there are less antigens available to trigger immune response  approved all experimental procedures used in the present study.in vivo experimental model of CCC. SR and KB performed the network pathway analysis and IA contributed to the data set and manuscript analysis. OM and JL-V were responsible for experimental design and data analysis. OM was responsible for the final manuscript revision. All authors contributed to the article and approved the submitted version.PF performed the majority of the experiments, analyzed data sets, and wrote the manuscript. GV-P and IP contributed to the This study was financed in part by the Coordination for the Improvement of Higher Education Personnel  - Finance Code 001. This work also received financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Rio de Janeiro (FAPERJ). OM is a researcher fellow of CNPq1D (311539/2020-3) and FAPERJ . JL-V is fellow of CNPq1B (BPP 306037/2019-0) and FAPERJ . IA partially supported by grant 5U54MD007592 from the National Institute on Minority Health and Health Disparities (NIMHD), a component of the National Institutes of Health (NIH).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CYP or UGT) or transporters  in the pharmacokinetics and safety of dexketoprofen to explore whether dosing adjustments based on genetic polymorphism would be beneficial for its prescription. For this regard, 85 healthy volunteers enrolled in three bioequivalence clinical trials were genotyped for 46 SNPs in 14 genes. Women showed lower AUC adjusted by dose/weight (AUC/DW) and higher Vd/F and Cl/F than men . CYP1A2*1B allele, CYP2B6 IM/PM and CYP2D6 IM/PM phenotypes were related to drug accumulation (AUC/DW or Cmax/DW) compared to the CYP1A2*1 allele, CYP2B6 NM/RM and CYP2D6 NM/UM phenotypes . ABCB1 C1236TT, C3435TT and G2677A/TA/T alleles were related to lower Cmax/DW compared to C, C, and G alleles . ABCB1 C1236TT allele was also related to lower AUC/DW . The remaining studied transporter genes  and metabolizing enzyme genes  were unrelated to dexketoprofen pharmacokinetic variability. We conclude that dexketoprofen pharmacokinetics can be influenced by several polymorphisms, although there is not a clear pharmacogenetic predictor that would justify individualization of therapy based on its genotyping. Further studies should be conducted to confirm the role of SNPs in CYP2B6, CYP2D6, CYP1A2 and ABCB1 on the pharmacokinetic variability of dexketoprofen. Current evidence on dexketoprofen pharmacogenetics does not justify its inclusion in pharmacogenetic guidelines.Dexketoprofen is the (S)-(+)-enantiomer of racemic ketoprofen, a nonsteroidal anti-inflammatory drug used for the management of different types of pain. To the best of our knowledge, no article was published to date on dexketoprofen pharmacogenetics. Thence, in this work, we aimed to explore the influence of sex, race and several single nucleotide polymorphisms (SNPs) in genes encoding metabolizing enzymes (e.g.  Racemic ketoprofen is a chiral 2-arylpropionic acid derivative nonsteroidal anti-inflammatory drug (NSAID) with anti-inflammatory and antipyretic properties. The (S)-(+)-enantiomer (dexketoprofen) is active, while (R)-(−)-ketoprofen is completely inactive . DexketoMaximum daily dose is 75 mg, being 25 mg/8 h, 12.5 mg/4 h or 12.5 mg/6 h the recommended doses. In elderly patients, or those with hepatic or renal impairment, the maximum daily dose should be reduced and patients should be closely monitored. Administered as a water-soluble salt (dexketoprofen trometamol), dexketoprofen reaches the maximum concentration (Cmax) 30 min (15–60 min) after drug intake; absorption is delayed by the presence of food. It binds extensively to plasmatic proteins (99%) with a mean volume of distribution of 0.25 L/kg. It is extensively glucuronized to conjugates and eliminated in urine in this form .To the best of our knowledge, no article was published to date on dexketoprofen pharmacogenetics. However, understanding predictors of the effectiveness and safety of dexketoprofen would help to personalize treatment, as reported for other NSAIDs like ibuprofen or meloxicam . The mosCYP or UGT) or transporters  in the pharmacokinetics and safety of dexketoprofen to explore whether dosing adjustments based on genetic polymorphism would be beneficial for its prescription.On March 2020, the Clinical Pharmacogenetics Implementation Consortium published their guideline on Nonsteroidal Anti-inflammatory Drugs and CYP2C9 . This inn = 96) signed their informed consent for their participation in the clinical trials, among which, 85 signed a specific informed consent for the pharmacogenetic study.Healthy volunteers enrolled in three dexketoprofen bioequivalence clinical trials  conducted in the Clinical Trial Unit of Hospital Universitario de La Princesa (UECHUP), Madrid, (Spain) participated in the pharmacogenetic study. Hospital’s Research Ethics Committee and the Spanish Drugs Agency (AEMPS) revised and approved the study protocols; research was conducted complying with European and Spanish legislation and under Good Clinical Practice Guidelines; the Declaration of Helsinki (revised) was fully endorsed. All healthy volunteers  out of the 18.5–30 kg/mThe three clinical trials were phase I, oral single-dose, open-label, crossover and randomized, organized in 2 sequences and 2 periods evaluating bioequivalence between dexketoprofen 25 mg test formulations and Enantyum 25 mg . Volunteers were hospitalized either the night before or 1 h before drug intake in the morning and remained hospitalized 12 h after dosing. Between drug intake (0 h), and 12 h after dosing, 17 or 18 blood samples were extracted for pharmacokinetic profiling, which was blinded. Formulations were administered by oral route with 240 ml of water under fasting conditions. Blood was collected in EDTA-K2 tubes; plasma was separated immediately after each blood extraction by centrifugation and samples were frozen until their shipment to the analytical laboratory. The method involved a liquid-liquid extraction procedure with tert-butyl methyl ether; dexketoprofen and internal standard were measured by reversed phase high performance liquid chromatography coupled to a tandem mass spectrometer (LC/MS/MS). Method complied with the European Medicines Agency (EMA) requirements for bioequivalence demonstration.t). By means of linear regression of the log-linear part of the concentration-time curve, the terminal rate constant (ke) was calculated. The AUC from t = 12 to infinite was calculated as Ct/ke (AUCt-∞). AUC∞ was calculated as: AUCt + AUCt-∞. By dividing the dose (D) by AUC∞ and weight, drug clearance adjusted for bioavailability (Cl/F) was calculated. By dividing Cl/F by ke, the volume of distribution adjusted for bioavailability (Vd/F) was calculated. Half-life (t1/2) was estimated as–ln 2/ke. The maximum concentration (Cmax) and the time to reach the Cmax (tmax) were directly obtained from concentration-time curves.The CERTARA Phoenix WinNonlin software, most recent version at the moment of clinical trial performance  was used for the non-compartmental analysis of pharmacokinetic parameters. The area under the curve (AUC) from pre-dose to the last time-point (12 h) was calculated following the trapezoidal rule  was carried out at predose and 1 or 2 h after drug intake. Simultaneously, vital signs were monitored . Volunteers were monitored for the occurrence of adverse events (AEs) by an open question, and, in addition, those reported spontaneously by themselves were noted. The Spanish Pharmacovigilance System  algorithCYP1A2*1C (rs2069514), *1F (rs762551), *1B (rs2470890), CYP2A6*9 (rs28399433), CYP2B6*9 (rs3745274), *5 (rs3211371), rs4803419, rs2279345, rs2279343, CYP2C8*2 (rs11572103), *3 (rs10509681), *4 (rs1058930), CYP2C9*2 (rs1799853), *3 (rs1057910), CYP2C19*2 (rs4244285), *3 (rs4986893), *4 (rs28399504), *17 (rs12248560), CYP2D6*3 (rs35742686), *4 (rs3892097), *6 (rs5030655), *7 (rs5030867), *8 (rs5030865), *9 (rs5030656), *10 (rs1065852), *14 (rs5030865), *17 (rs28371706), *41 (rs28371725), CYP3A4 *22 (rs35599367), rs55785340, rs4646438, CYP3A5*3 (rs776746), *6 (rs10264272), ABCB1 C3435T (rs1045642), G2677 T/A (rs2032582), C1236T (rs1128503), ABCC2 c.1247G>A (rs2273697), rs717620, SLCO1B1*1B (rs2306283), *5 (rs4149056), c.-910G > A (rs4149015), rs11045879, SLC22A1*2 (rs72552763), *3 (rs12208357), *5 (rs34059508), and UGT1A1*80 (rs887829). A CYP2D6 copy number variation assay (CNV) was performed following the methodology previously reported  was used for the extraction of DNA form peripheral blood collected in an EDTA-K2 tube. 46 SNPs in 14 genes were analyzed: reported . For thereported .CYP2D6 , CYP2C19 , SLCO1B1 , CYP2B6 (*5 and *9) and CYP2C9  variants were used to infer the enzymatic phenotype based on CPIC guidelines  variants were used to infer the activity score and phenotype as described in previous publications  to correct the differences in weight between sexes or races which can produce pharmacokinetic variability. For distribution normalization, pharmacokinetic parameters were logarithmically transformed. Firstly, a univariate analysis was performed. The means of pharmacokinetic parameters or the incidence of adverse drug reactions (ADRs) were compared based on categorical variables . For mean comparison, a T test (variables with two categories) or an ANOVA test followed by a Bonferroni post-hoc (variables with three or more categories) were used. For the comparison of the incidence of ADRs according to categorical variables, a Chi2 test was used. Afterward, each pharmacokinetic parameter or ADR were individually analyzed with a multivariate analysis. By linear or logistic regression, pharmacokinetic parameters or ADRs were explored, respectively. As independent variables, any variable with p < 0.05 in the univariate analysis was explored ; in addition, pharmacokinetic parameters were introduced as independent variables in the logistic regression for the evaluation of ADR occurrence.The SPSS software was used for statistical analysis . AUCp < 0.0001, p < 0.0001, and p = 0.030, respectively) (p < 0.003).Study population was composed by 42 women (49.41%) and 43 men (50.59%). Men’s height, weight and body mass index (BMI) were significantly superior than that of women (ctively) . Caucasictively) . Caucasi∞ was 4,031.88 ± 819.35 ng*h/mL  and mean Cmax was 2,745.20 ± 690.81 ng/ml . After DW correction, women showed lower AUC/DW compared to men  and the differences in Cmax/DW disappeared .Dexketoprofen mean AUCappeared . The Blaucasians ; howeverCYP1A2*1B/*1B genotype was related to higher Cmax/DW (ANOVA p = 0.035) and to lower Tmax  compared to the *1/*1 genotype (p = 0.050). CYP2B6 intermediate (IM) and poor (PM) metabolizers were related to higher Cmax/DW compared to normal (NM) and rapid (RM) metabolizers . These associations were lost after multivariate analysis. Moreover, CYP2B6 intermediate and poor metabolizers were related to lower Tmax compared to RMs . CYP2D6 IMs/PMs were related to higher AUC∞/DW compared to NMs (p = 0.017). This association was not confirmed by multivariate analysis.Some genetic polymorphisms were associated with dexketoprofen pharmacokinetic variability . The CYPgenotype  and to hABCB1 C1236T/T carriers showed lower AUC/DW , and Cmax  compared to C/T and C/C carriers; C/C carriers showed lower Cl/F compared to C/T + T/T . Moreover, ABCB1 C3435T T/T carriers showed lower Cmax/DW compared to C/C (ANOVA p = 0.021). This association was not confirmed by multivariate analysis. Finally, G2677T/A TT/AA carriers were related to lower Cmax/DW compared to G/G and GT/GA   . The remAll the AEs reported during the three clinical trials were unrelated to dexketoprofen administration. No ADR was reported during any of the three clinical trials. No clinically relevant alteration of vital signs or ECG was observed.CYP2C9  metabolism. Should it undergo this route, it would be secondary, as its primary route of metabolization is glucuronidation. Nor is there much information available on whether this drug is a substrate of solute carrier transporters (SLC) or of the ATP Binding Cassette (ABC) family. Hence, we aimed to explore whether sex, race, CYP2C9 and other CYP phenotypes as well as polymorphism in relevant pharmacogenes had an impact on dexketoprofen tolerability or pharmacokinetics.Despite belonging to the same NSAID family (propionic acid derivatives) than flurbiprofen and ibuprofen, there is no conclusive evidence that dexketoprofen undergoes CYP . Intereescribed . The difhttps://www.pharmgkb.org/chemical/PA166049175) indexes cero clinical or variant annotations for dexketoprofen. Thence, we decided to genotype our healthy volunteers for a set of pharmacogenes that includes important transporters and metabolizing enzymes.One of the greatest challenges when conceiving this work was the strategy for the selection of polymorphisms. On the one hand, the available dexketoprofen pharmacokinetic information was scarce, i.e., it is conjugated predominantly to glucuronide derivatives that are excreted in urine in around 80%. However, little or no information is published on which enzymes perform the glucuronidation and whether dexketoprofen is a substrate of CYP enzymes and of ABC or SLC transporters. On the other hand, no other candidate-gene pharmacogenetic study was published previously and the Pharmacogenomics knowledgebase (PharmGKB)  were in all cases <15% and disappeared after multivariate analysis. Therefore, even if these differences are real, they may unlikely have a clinically relevant effect.To our knowledge, there is no evidence that dexketoprofen undergoes CYP1A2, CYP2B6, or CYP2D6-mediated metabolism. However, we observed that ABCB1 polymorphisms were studied: C1236T, G2677T/A and C3435T , nor its polymorphism was examined in relation to the pharmacokinetic variability of dexketoprofen. Here, the three most relevant d C3435T . Interestoxicity . In conttoxicity  and to itoxicity  comparedABCC2, SLC22A1, and SLCO1B1) and metabolizing enzyme genes  were unrelated to dexketoprofen pharmacokinetic variability, which is consistent with previous knowledge on dexketoprofen pharmacokinetics  dexketoprofen effectiveness, 2) long-term pharmacokinetics and tolerability. It is possible that UGT2B7 nor UGT2B15 genes are involve in the elimination of dexketoprofen but they could not be evaluated because they were not included in the array used at our center. We were also unable to analyze the influence of Black and Asian race on dexketoprofen pharmacokinetics because there was only one subject of each race. Furthermore, it is important that these results are interpreted with caution given the small sample size. In contrast, bioequivalence clinical trials offer a controlled setting for the evaluation of pharmacokinetic variability based on genetic polymorphism or demographics as confounding factors are avoided. These results can be considered preliminary and further studies are needed to confirm the hypotheses that were raised in the study.n = 85) with a comprehensive genotype screening strategy based on a robust dataset, which lays the foundation for exploring dexketoprofen pharmacogenetics in the future. We conclude that dexketoprofen pharmacokinetics can be influenced by several polymorphisms, although the effect is small and there is not a clear pharmacogenetic predictor that would justify individualization of therapy based on its genotyping. Further studies should be conducted to confirm the role of SNPs in CYP2B6, CYP2D6, CYP1A2, and ABCB1 on the pharmacokinetic variability of dexketoprofen. Current evidence on dexketoprofen pharmacogenetics does not justify its inclusion in pharmacogenetic guidelines.This candidate gene study is, to our knowledge, the only one published to date (


Various tannins are commercially available nowadays for use in such application fields. We have analysed a representative selection of commercially available condensed and hydrolysable tannins for structural features and purity. Using a combination of quantitative  Giovannetti investigated the interactions between iron solutions and substances called ‘astringents’ in the middle of the 17th century, marking as such what is today seen as the onset of tannin research. In 1772, researchers identified the presence of an acid in these compounds. This acid was subsequently isolated by Scheele and shown to be gallic acid. At the end of the 18th and the beginning of the 19th century, the tannins were officially recognised as a discrete group of molecules that differ with respect to the gallic acid content . One defThe rather wide range of functions comes together with a wide variety of tannin structures, commonly divided in tannins such as gallotannins and ellagitannins, that are soluble and cleavable into their components in hot water or in the presence of a tannin-specific enzyme, e.g., tannase, and that were thus termed ‘hydrolysable’ tannins, and non-hydrolysable tannins or flavones, a tannin species without carbohydrate moieties, termed proanthocyanidins, polyflavonoid tannins, catechol-type tannins, and pyrocatecholic-type tannins that are stable under these conditions and thus called ‘non-hydrolysable’ or ‘condensed’ tannins ,2,9. Aim1H-13C}-HSQC measurements, quantitative 31P NMR and 1H NMR spectroscopy, MALDI-ToF analysis and gel permeation chromatography (GPC).The design of novel products and processes aimed at improving the exploitation of the huge potential of tannins requires their use both in their natural form as well as in the form of chemically altered derivatives. It is therefore necessary, in order to develop targeted selective chemical derivatization protocols, to analyse the tannin structures, as such verifying also the information available for the commercialised samples. To this end, commercially available tannins from different origins have been structurally characterized using the toolbox shown in Wet-chemical analysis such as the Scalbert test for the elucidation of cyanidin contents and the Folin–Ciocalteau assay for the determination of the total phenol content concluded the fingerprinting. On the basis of the results of the various analyses, it was possible to unequivocally assign the tannin structures. This is especially relevant for two reasons: (i) commercial tannins might be mixtures or even different from what is declared, and (ii) a detailed knowledge of structural features is key for an eventual chemical derivatisation of tannins.As commercially available materials, eleven representative tannins from two different suppliers were selected : condensThe analysis techniques summarised as the toolbox shown in 1H NMR signals are therefore not very diagnostic. On the contrary, 31P NMR of suitably in situ labelled samples allows direct straightforward identification and quantification of all the different phenolic groups present in the tannin sample. -benzoyl}-d-glucopyranose that could be seen as the ‘default’ TA. Several other hydrolysable tannins exist, of course, varying in terms of the core carbohydrate, the repeat number of esterified galloyl units, the form and degree of additional oxidative linkages between the aromatic moieties and eventual oligomeric gallic acid-stemming impurities.In case of commercially available hydrolysable tannins, it is worthwhile to understand the nature of the tannin, especially also with respect to the core sugar moiety. Tannic acid  is commercialised as a typical tannic acid, and so are Tanal 02 and Tanal 04, with the differences lying in molecular size and phenolic content. Such differences are principally compatible with a ‘typical’ tannic acid structure in the sense that such a ‘typical’ tannic acid does not exist, but that the number of galloyl units attached to the centre carbohydrate may vary. It was thus initially expected to confirm within the structural analysis the presence of glucose as common central sugar in all three Tanal samples under study.Tanal 01 (Ta-01) and Tanal 02 (Ta-02) turned out to be tannic acids; i.e., displaying a glucopyranose core esterified with 10 galloyl units , a commercialised tara tannin. HSQC analysis clearly indicates the presence of a quinic acid as the core carbohydrate  is a commercialised chestnut tannin. The batch analysed in this work turned out to be a mixture of castalgin and vescalgin, as indicated in 31P NMR data confirm this interpretation as well. The latter analysis additionally indicates also in this case noteworthy amounts of carbohydrate impurities.VEGETAN CN POLVERE . One hundred microliters of the standard solution, prepared using N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (e-HNDI) at a concentration of 0.1 m in the above-mentioned solvent mixture mixed with 50 mg/mL of chromium(III) acetylacetonate as spin-relaxing agent were added, followed by 50 µL of 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (Cl-TMPD). After 1 h stirring at room temperature, the functionalized mixture was quantitatively transferred to a standard NMR tube for analysis. 31P NMR spectra were recorded on a Bruker 400 MHz spectrometer at 20 °C using an inverse gated decoupling sequence with a delay of 10 s between successive pulses. Chemical shifts were expressed in parts per million from 85% H3PO4 as an external reference. All chemical shifts reported are relative to the peak for the reaction product of water with Cl-TMDP 132.2 ppm in the used conditions. NMR data were processed with MestreNova .followed ,28,29,301H-13C HSQC measurements: Samples of around 50 mg were dissolved in 600 μL of DMSO-d6 (providing NMR sample solutions with concentrations of around 83 mg/mL); chromium(III) acetylacetonate was added as a spin-relaxing agent at a final concentration of ca. 1.5–1.75 mg/mL. HSQC spectra were recorded at 27 °C on a Bruker 700 MHz instrument equipped with TopSpin 2.1 software. Spectra were referenced to the residual signals of DMSO-d6 (2.49 ppm for 1H and 39.5 ppm for 13C spectra). 1H-13C HSQC spectra were obtained using 32 scans obtained using the standard Bruker pulse program (hsqcegtpsisp2) with the following parameters for acquisition: TD = 2048 (F2), 512 (F1); SW = 13.0327 ppm (F2), 160 ppm (F1); O1 = 4200.54 Hz; O2 = 14083.02 Hz; D1 = 2 s; CNST2 = 145; acquisition time F2 channel = 112.34 ms; F1 channel = 8.7102 ms. Processing: SI = 1024 , WDW = QSINE, LB = 1.00 Hz(F2), 0.30 Hz (F1); PH_mod = pk; baseline correction ABSG = 5 , BCFW = 1.00 ppm, BC_mod = quad (F2), no (F1); linear prediction = no (F2), LPfr (F1). The integration ranges as previously reported were applied. The NMR data were processed with MestreNova .2,6-DHB) matrix solution (10 mg/mL in acetone). For non-ionic analytes, to enhance ion formation, sodium chloride (NaCl) was added to the 2,6-dihydroxy-benzoic acid  solution (10 mg/mL in distilled water). The sample and the matrix solutions were mixed as follows: three parts matrix solution, three parts sample solution, one part NaCl solution; approximately 2.5 µL of the resulting mix was placed on the MALDI sample holder. After drying overnight in the dark, the samples were analysed using settings specifically optimised for each sample type. Assignments of mass peaks were achieved using (combinations of) the molecular mass(es) of the structural units shown in MALDI-ToF analyses were performed using a Voyager-DE™ PRO Biospectrometry™ Workstation operated using the Voyager operating software (version X). Samples were dissolved in water/acetone , and the solutions were mixed with the 2,6-dihydroxy-benzoic acid  was used as an eluent at 70 °C column temperature. The run time at 0.25 mL min−1 flow rate was 20 min. Molecular weights were calculated from a linear calibration constructed with poly(styrene sulfonic acid) polymers (4300−2.6 × 106 g mol−1); analyses were run in duplicate.Approximately 3 mg of tannin sample were dissolved in 1 mL DMSO containing 0.1% lithium chloride. A Shimadzu instrument was used consisting of a controller unit (CBM-20A), a pumping unit (LC 20AT), a degasser (DGU-20A3), a column oven (CTO-20AC), a diode array detector (SPD-M20A) and a refractive index detector (RID-10A); the system was controlled by Shimadzu LabSolutions (Version 5.42 SP3). Three analytical GPC columns (each 7.5 × 30 mm) in series were used for analyses: Agilent PLgel 5 µm 10,000 Å, followed by Agilent PLgel 5 µm 1000 Å, followed by an Agilent PLgel 5 µm 500 Å. HPLC-grade DMSO (Chromasolv4·7 H2O in 500 mL HCl/n-C4H9OH (2/3)) were added to 0.5 mL of the aqueous tannin solution (1 mg/mL). The mixtures were heated at 95 °C during 15 min. The absorbance was read at λ = 530 nm employing a Shimadzu UV-1800 spectrophotometer, operated via UV Probe, Version 2.42. The results are expressed in cyanidin equivalents (CyaE) as mg per g of dry tannin material  (ε mol (cyanidin) = 34,700 L mol−1cm−1) using Equation (1) with A—absorbance measured, V—volume, D—dilution factor, MW—molecular weight of cyanidin, ε—molar extinction coefficient cyanidin, l—path length, m—mass dry bark.The standard procedure as described elsewhere was adapted : 5.0 mL 2/mL). After 2 min, 2.0 mL of sodium carbonate (7.5% (m/m)) were added. The mixture was heated at 50 °C for 5 min. The absorbance was read at λ = 765 nm employing a Shimadzu UV-1800 spectrophotometer, operated via UV Probe, Version 2.42. Calibration was achieved following this approach using a solution of gallic acid in distilled water (0.5 mg/mL), diluted to eight calibration points covering the range between 0 and 0.01 mg/mL for which a linear calibration curve was obtained. The results are reported as mg of gallic acid equivalent (GAE) per mg of dry tannin (mg GAE/mg tannin).The total phenolic content of the extracts was determined by the Folin–Ciocalteu method : 2.5 mL 31P NMR [31P NMR allows the direct structural assignment of tannins not only as pure compounds but also when present in complex matrices highly polluted by impurities. Based on the structural information obtained, the tannins can be used for chemical derivatisation and biological testing, as will be reported in due course.By employing a combination of analysis techniques well established in connection with the structural characterisation of tannins ,22, such31P NMR , it was


Life-threatening hemoptysis presents an immediate diagnostic and therapeutic challenge, especially during the perinatal period.2 inhomogeneous tumorous lesion in the right superior mediastinum and a right main bronchus obstruction along with atelectasis of the right lung. Bronchoscopy showed a tumorous protrusion blocking the right main bronchus with active hemorrhage, and malignancy was suspected. Bronchial artery embolization (BAE) was performed to control the bleeding. The arteriogram revealed tortuosity, dilation and hypertrophy of the right bronchial arteries and aneurysms of the internal thoracic artery (ITA). The bleeding completely stopped after BAE. Bronchoscopy was performed again to remove residual blood clots. The patient recovered soon after the procedure and was discharged.A 28-year-old perinatal woman with no significant past medical or surgical history presented with repeating hemoptysis and respiratory failure. Computed tomography revealed a 2.1 × 3.2  cmLife-threatening hemoptysis concomitant with ITA aneurysms, which may have a misleading clinical diagnosis and treatment options, has not been reported previously in perinatal women. BAE could be used as a first-line treatment irrespective of the underlying causes. Hemoptysis is considered life threatening if, regardless of the degree, it is associated with respiratory failure from airway obstruction or hypotension . BronchiA 28-year-old G3P2A1 woman with no significant past medical or surgical history was admitted on postpartum day 4 and presented with repeating hemoptysis and increasing dyspnea. She coughed up 10–20 mL fresh blood for 4 days and complained of dyspnea that increased on exertion and was relieved by rest after spontaneous vaginal delivery. Her pregnancy and delivery were uneventful, with a healthy female child at 38 weeks and 1 day. Her first pregnancy was miscarried at 2 months, and her second pregnancy went well and gave birth to a girl by vaginal delivery. The girl is 3 years old and doing well. Social and family history were unremarkable.9/L, Platelet count 223 × 109/L, Hemoglobin 88 g/L, Red blood cell count 4.05 × 1012/L; mean corpuscular volume (MCV) 78.8 fL, mean corpuscular hemoglobin (MCH) 21.7 pg, and mean corpuscular-hemoglobin concentration (MCHC) 276.0 g/L. The absolute counts and percentages of other blood cells were within the normal ranges. Arterial blood gas analysis on 5 L nasal cannula showed pH 7.385; pO2 78 mmHg; pCO2 32.5 mmHg, HCO3 22 mmol/L; O2 saturation 95.8 %, PaO2/FiO2 ratio 190. The rest of the laboratory results, including the comprehensive metabolic panel, inflammation markers, and pro-B-type natriuretic peptide (pro-BNP), were all within normal limits. Autoimmune and vasculitis panels were negative. Complement levels and immunoglobin levels were all unremarkable. Chest radiographs suggested right lung atelectasis  5.82 g/L, thrombin time (TT) 11.5 s, prethrombin time ratio (PTR) 0.86, activated partial thromboplastin time (APTT) 26 s, prothrombin time (PT) 11 s, international normalized ratio (INR) of PT 0.88; D-dimer 620 μg/L; white blood cell count 11.2 × 10Life-threatening hemoptysis is a medical emergency with high mortality presenting with several difficult diagnostic and therapeutic challenges. Identification of the underlying causes is important. A prospective study with 606 patients was performed to investigate the etiology of hemoptysis and revealed that compared with CT scans (77.3% of cases) and bronchoscopy (48.7% of cases), the combination of CT scan and bronchoscopy was able to diagnose the etiology of the hemorrhage in 83.9% of cases . HoweverITA aneurysms are rare and commonly misdiagnosed as malignant tumors –9. They Pregnancy has been reportedly associated with the development of aneurysms, presumably due to the associated hemodynamic and hormonal changes , 11. How


The exact Cmin sampling time was used for Bayesian estimation. A significantly higher early treatment response was observed in patients with a day 2 AUC ≥ 400 µg·h/mL than those with <400 µg·h/mL, and a significantly higher early nephrotoxicity rate was observed in patients with a day 2 AUC ≥ 600 µg·h/mL than those with <600 µg·h/mL. These AUC cutoff values constituted independent factors for each outcome. In sub-analysis, the discrimination ability for early clinical outcomes using these AUC cutoffs was confirmed only in patients with q12 vancomycin administration. A significant difference in early treatment response using the 400 µg·h/mL cutoff was obtained only in patients with low-risk infections. The usefulness of the vancomycin AUC target to decrease nephrotoxicity while assuring clinical efficacy was even confirmed with a single Cmin measurement. However, assessment with two samples might be required in patients with q24 administration or high/moderate-risk MRSA infections.Area under the concentration–time curve (AUC)-guided vancomycin treatment is associated with decreased nephrotoxicity. It is preferable to obtain two samples to estimate the AUC. This study examined the usefulness of AUC estimation via trough concentration (C Staphylococcus aureus (MRSA) infections [min) has been used as a surrogate marker of the AUC. However, Neely et al. [min < 15 μg/mL. Thus, Cmin-guided dosing targeting a Cmin ≥ 15 μg/mL may lead to excessive vancomycin exposure. Phillips [min alone. A revised consensus guideline recommended AUC-guided dosing with the use of Bayesian methods [min-guided monitoring. In addition, they reported that a high AUC/MIC ratio  was associated with a significantly lower treatment failure rate, and a high AUC  significantly increased the risk of nephrotoxicity [Vancomycin remains a first-line therapy for methicillin-resistant fections . The ratfections ,3,4,5. Ty et al.  found thPhillips  reported methods  conductetoxicity .min alone may be sufficient to estimate the AUC in some patients. For the wide distribution of AUC-guided dosing in clinical practice, further validation of an AUC estimation using only the Cmin is required. When a population PK model based on richly sampled data is used as a Bayesian prior, Cmin-only data can be used to generate accurate AUC estimates. In contrast, a population PK model based on limited samples was worse at predicting the true AUC from Cmin-only data, and two-point measurements may be required as a better Bayesian prior [min produced moderate and unignorable bias in patients with vancomycin q12h and q24h administration, respectively. Considering the decrease in accuracy, we suspected that AUC estimation using only the trough concentration might be avoided in patients with difficult-to-treat MRSA infections and in patients with kidney dysfunction who are likely to be prescribed once daily dosing.Although it has been considered preferable to obtain two pharmacokinetic (PK) samples to accurately estimate the AUC using the Bayesian approach, updated therapeutic drug monitoring (TDM) guidelines  suggestean prior . Previouan prior . The AUCThe primary purpose of this study was to confirm the correlation between clinical outcomes and pre-described AUC cutoff levels , using AUC estimation by one-point sampling. As a subgroup-analysis, the relationship was separately evaluated in patients with vancomycin q12h and q24h administration, and those with high/moderate-risk and low-risk MRSA infections. If a clear correlation was obtained only in patients with vancomycin q12h administration or in patients with low-risk MRSA infections, the result might support previously mentioned hypotheses.Among 444 patients with MRSA infections, 312 patients met the inclusion criteria (reasons for exclusion: less than 18 years (33), intermittent hemodialysis (71), and continuous renal replacement therapy (28)), but 52 of these patients were excluded for the following reasons: pregnancy (6), the previous use of antimicrobial agents with anti-MRSA activity (31), and MRSA infections with a vancomycin MIC = 2 mg/L (15). Thus, 260 patients were included in the study. Vancomycin was administered with q12h in 202 patients and q24h in 58 patients, and vancomycin was used in 105 patients with high/moderate-risk infections and in the remaining 155 patients with low-risk infections.2, and no patients with eGFR < 70 mL/min/1.73 m2. Once daily administration was employed in 10 patients with eGFR ≥ 70 mL/min/1.73 m2 and 48 patients with eGFR < 70 mL/min/1.73 m2. For 202 patients with vancomycin q12h administration, 69, 64, and 34 patients were treated using regimens A, B, and C, respectively. A loading dose was used in 98 patients  and 25 mg/kg (70)), and the maintenance doses were 17.5–22.5 mg/kg twice daily in 54 patients, 12.5–17.5 mg/kg twice daily in 133 patients, and 7.5–12.5 mg/kg twice daily in 15 patients.The baseline demographics of the enrolled patients are presented in n = 0), 9–10 (n = 12), 10–11 (n = 46), 11–12 (n = 111), 12–13 (n = 29), and ≥13 h (n = 4) in patients with vancomycin q12h administration and were <20 (n = 0), 20–21 (n = 4), 21–22 (n = 10), 22–23 (n = 16), 23–24 (n = 24), 24–25 (n = 3), and ≥25 h (n = 1) in patients with vancomycin q24h administration. The median AUC values of day 1, day 2, and the steady-state for the initial vancomycin regimen AUC were 382.7, 403.9, and 422.4, respectively  had the previously recommended Cmin target of 15–20 μg/mL [min is far from an adequate surrogate maker of AUC.The exact sampling times after the previous dose in the initial TDM were <9 . In regard to safety, a significantly higher early nephrotoxicity rate was observed in patients with a day 2 AUC ≥ 600 µg·h/mL than those with <600 µg·h/mL .p = 0.014). The risk factors for a decreased early treatment response were collagen disease , intensive care unit stay , and an APACHE II score >10  (p < 0.001) and concomitant piperacillin/tazobactam use  were independent factors for early nephrotoxicity  . In additoxicity .p = 0.001) and early nephrotoxicity (p < 0.001) was confirmed only in patients with q12 vancomycin administration . The institutional review board waived the requirement for informed consent from patients included in this study. This retrospective study was conducted between April 2011 and May 2020. The study included adult patients who were treated with vancomycin for MRSA infections, who underwent TDM, and who received at least 3 days of vancomycin treatment. The exclusion criteria were as follows: hypersensitivity to vancomycin; pregnancy; age < 18 years, thrice-daily vancomycin administration; intermittent hemodialysis or continuous renal replacement therapy; receipt of any concomitant antibiotics with anti-MRSA activity; receipt of antibiotics with anti-MRSA activity for >24 h within the previous 3 days; receipt of concomitant nephrotoxic antimicrobial agents; and isolation of strains with a vancomycin MIC = 2 μg/mL.Regimens A–C were adopted in patients with normal renal function (eGFR ≥70), and dosage reduction of each regimen was performed in patients with reduced renal function. Regimen A (15 mg/kg twice daily without a loading dose) was recommended between April 2011 and December 2015. Regimen B (loading dose of 25 mg/kg and maintenance dose of 15 mg twice daily) was provided between January 2016 and May 2018, and regimen C (loading dose of 25–30 mg/kg and maintenance dose of 20 mg/kg twice daily) was administered between June 2018 and May 2020.min-guided dosing was conducted during the study period. The target Cmin was 10–15 μg/mL during initial TDM, and dosing adjustment to achieve Cmin 15–20 μg/mL was performed only in patients with complicated MRSA infections or those without clinical responses [min sample was obtained before the fifth dose. The diagnosis of each type of infection, excluding respiratory tract infection, was based on the definitions of the guidelines issued by the National Healthcare Safety Network . Infections with at least one of the following signs were analyzed: core temperature > 37.8 °C, total peripheral white blood cell (WBC) count > 10,000/mm3, or C-reactive protein (CRP) > 3.0 mg/dL. The presence of pneumonia was identified by chest X-rays or CT scans consistent with pneumonia and at least two of the following signs or symptoms: new-onset or worsening cough; purulent sputum or increased suctioning requirements; auscultatory findings of pneumonia; dyspnea, tachypnea, or respiratory rate ≥ 30 min; hypoxemia; worsening gas exchange; and at least one inflammatory sign [Only Cesponses . An initory sign . The MIChttps://pharmacokinetic-simulation.shinyapps.io/app-ver1/, accessed on 7 October 2021). The Shiny package, which is an R function for the development of a web application , was used as the main user interface, which can actively render the results of calculations from R to the html format in accordance with the input. A previously reported Japanese population PK model [min for day 1, day 2, and the steady-state were estimated by the Bayesian software. The cutoff for the AUC value was defined as 400 µg·h/mL for the treatment response and 600 µg·h/mL for nephrotoxicity, and the AUC of day 2 was used to evaluate the relationship with an early clinical response and nephrotoxicity [The vancomycin concentration was measured using a commercial reagent kit . The coefficient of the dynamic range was 0.8–50 μg/mL (as specified by the manufacturer). The individual PK of vancomycin was retrospectively analyzed using the Bayesian estimation software PAT  . PAT wasPK model  was usedPK model . The exatoxicity .As an adverse effect, nephrotoxicity was evaluated. Nephrotoxicity was defined as an increase in serum creatinine levels of >0.5 mg/L or 50% versus baseline. The early occurrence of nephrotoxicity at 48–72 h after the start of therapy was evaluated. The rate of nephrotoxicity during vancomycin therapy was also evaluated. An early clinical response at 48–72 h after the start of therapy was investigated. We defined patients as responders if they had a ≥30% decrease in the total peripheral WBC count or CRP level, a reduction in fever (defined as a daily maximum temperature decrease of >0.3 °C for at least 2 consecutive days in febrile patients), no worsening clinical features, and no death within 96 h ,18. Treamin in patients with vancomycin q24h administration produced unignorable bias compared with the reference AUC based on eight samples [The type of infection or the source of infection are important parameters to evaluate treatment efficacy. Because the population is highly heterogeneous regarding the source of infection, we divided the patients into high/moderate and low-risk infections. High/moderate risk infections consisted of bloodstream infections and complicated infections  . Low-ris samples , and subt-test, whereas nonparametric variables were analyzed using the Mann–Whitney U-test or Fisher’s exact test. Multivariate analyses were performed to determine the adjusted odds ratio (OR) for early clinical responses and early nephrotoxicity. Univariate analysis was estimated for each variable with the chi-squared test, and potential confounders were examined via cross-tabulation. Variables selected in univariate analysis (p < 0.1) were subsequently entered into a stepwise logistic regression model to estimate the magnitude of association ). The level of significance was set at p < 0.05. SPSS ver. 24  was used to perform these analyses.Parametric variables were analyzed using the Student’s min measurement. However, assessment with a two-point sample might be required in patients with renal dysfunction who are likely to be prescribed once daily dosing or have bacteremia/complicated MRSA infections.The usefulness of the previously reported vancomycin AUC target to decrease nephrotoxicity while assuring clinical efficacy was confirmed with a single C


The grains of foxtail millet are enriched in carotenoids, which endow this plant with a yellow color and extremely high nutritional value. However, the underlying molecular regulation mechanism and gene coexpression network remain unclear.The carotenoid species and content were detected by HPLC for two foxtail millet varieties at three panicle development stages. Based on a homologous sequence BLAST analysis, these genes related to carotenoid metabolism were identified from the foxtail millet genome database. The conserved protein domains, chromosome locations, gene structures and phylogenetic trees were analyzed using bioinformatics tools. RNA-seq was performed for these samples to identify differentially expressed genes (DEGs). A Pearson correlation analysis was performed between the expression of genes related to carotenoid metabolism and the content of carotenoid metabolites. Furthermore, the expression levels of the key DEGs were verified by qRT-PCR. The gene coexpression network was constructed by a weighted gene coexpression network analysis (WGCNA).SiHDS, SiHMGS3, SiPDS and SiNCED1 were more highly expressed in the panicle of foxtail millet. The expression of SiCMT, SiAACT3, SiPSY1, SiZEP1/2, and SiCCD8c/8d was significantly correlated with the lutein content. The expression of SiCMT, SiHDR, SiIDI2, SiAACT3, SiPSY1, and SiZEP1/2 was significantly correlated with the content of β-carotene. WGCNA showed that the coral module was highly correlated with lutein and β-carotene, and 13 structural genes from the carotenoid biosynthetic pathway were identified. Network visualization revealed 25 intramodular hub genes that putatively control carotenoid metabolism.The major carotenoid metabolites in the panicles of DHD and JG21 were lutein and β-carotene. These carotenoid metabolite contents sharply decreased during the panicle development stage. The lutein and β-carotene contents were highest at the S1 stage of DHD, with values of 11.474 μg /100 mg and 12.524 μg /100 mg, respectively. Fifty-four genes related to carotenoid metabolism were identified in the foxtail millet genome. Cis-acting element analysis showed that these gene promoters mainly contain ‘plant hormone’, ‘drought stress resistance’, ‘MYB binding site’, ‘endosperm specific’ and ‘seed specific’ cis-acting elements and especially the ‘light-responsive’ and ‘ABA-responsive’ elements. In the carotenoid metabolic pathways, Based on the integrative analysis of the transcriptomics and carotenoid metabonomics, we found that DEGs related to carotenoid metabolism had a stronger correlation with the key carotenoid metabolite content. The correlation analysis and WGCNA identified and predicted the gene regulation network related to carotenoid metabolism. These results lay the foundation for exploring the key target genes regulating carotenoid metabolism flux in the panicle of foxtail millet. We hope that these target genes could be used to genetically modify millet to enhance the carotenoid content in the future.The online version contains supplementary material available at 10.1186/s12870-022-03467-2. Setaria italica (L.) Beauv.] belongs to the Setaria genus of Poaceae grass family, and it is widely planted in Eurasian arid and semiarid areas as a kind of C4 crop that endures drought stress and infertile soils [Xiaomi’ and ‘huagu11’ [Foxtail millet [le soils . The ancle soils . At preshuagu11’ –6. The chuagu11’ , 8. Foxthuagu11’ . Previouhuagu11’ , 11. Carhuagu11’ . Bioforthuagu11’ –15. HencThe carotenoid metabolism pathway in plants is well known. Carotenoids and their derivatives are composed of isopentenyl pyrophosphate (IPP) and its double bond isomer and dimethylallyl diphosphate (DMAPP). In plant cells, these IPP precursors are mainly synthesized by the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways and the precursors of carotenoid synthesis mainly come from the MEP metabolic pathway , 17. FinPSY in dark environment and also participates in the formation of chloroplasts [PSY/PDS/LCYB [Transcription factors play an important regulatory role in the carotenoid metabolism pathway. Phytochrome-interacting factor 1 (PIF1) regulated for carotenoid biosynthesis by inhibiting the expression of roplasts . An bZIProplasts . RecentlPDS/LCYB . So far,PSY and CRTL genes in rice, a new edible rice variety named “golden rice” was successfully developed [crtRB1 and lcyE had a significant effect on the β-carotene content (7.9-fold and 2.1-fold higher) compared to.Many previous studies on genes involved in carotenoid biosynthesis and regulated networks in plants have been reported. Through introducing eveloped . In maizthe unfavorable genotype . In tomaFoxtail millet varieties JG21 and DHD were planted in the experimental field of Shanxi Agricultural University in April 2019 . The experiments were done in three biological replicates. Samples were collected from the middle part of the panicle at S1 , S2  and S3  . A YMC Carotenoid S-3 μm (150*4.6 mm) liquid chromatographic column was used. The injection volume was 2 μl, the column temperature was 40 °C, and the detection wavelength was 450 nm. The solvent systems contained mobile phase A (methanol: water (1:1)) and mobile phase B (acetonitrile (ACN): ethyl acetate (3:1)). All solvents used were HPLC grade and filtered through a 0.2-mm filter prior to use. The gradient was 30% A:70% B for 0.5 min, then increased in intervals to 0.1% A:99.9% B for 5.5 min and then to 30% A:70% B for the last 2 min. UPLC–MS/MS was performed by Sanshu Biotechnology Co., Ltd.  . The sequences of key genes in the carotenoid metabolism pathway of foxtail millet were obtained from homozygous genes in Arabidopsis thaliana. TBtools analysis tools were used for the gene sequence information analysis, collinearity analysis, and chromosome mapping analysis [http://pfam.xfam.org/search/) and subcellular location prediction (https://wolfpsort.hgc.jp/).The genes involved in the map00900 and map00906 metabolic pathways were analyzed and mapped to Kyoto Encyclopedia of Genes and Genomes Database  using Hisat2 (http://ccb.jhu.edu/software/hisat2/index.shtml) [The developing panicle of DHD and JG21 were separated at the S1/S2/S3 filling stages, and total RNA was isolated using a Quick RNA Isolation kit  . RNA quax.shtml) .P value < 0.05 as the thresholds [http://www.genome.jp/kegg) and GO  annotations of DEGs were further performed with GOseq and KOBAS software, respectively [https://meme-suite.org/meme/tools/fimo) according to default parameters.For gene expression quantification, HTSeq was used to count the read numbers mapped to each gene, and the FPKM (fragments per kilobase per million fragments) value was used to normalize the expression level of each gene. DEGs between different samples were identified using the R package DESeq with |log2FoldChange| > 1 and resholds . Volcanoectively . The FPKectively . A weighhttp://primer3.ut.ee/) . Purified RNA samples were reverse-transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser  according to the manufacturer’s protocol. Four transcripts were selected for the qRT-PCR assay. Gene-specific qRT-PCR primers were designed using Primer 3 software  Table S. qRT-PCR formula .For two foxtail millet cultivars, five carotenoid metabolites, lutein, β-carotene, zeaxanthin, violaxanthin, and neoxanthin, were detected in the spikes at the three panicle development stages by HPLC. The results showed that most of carotenoid metabolite contents were higher at the S1 stage than at the other stages of the two cultivars except zeaxanthin of DHD Fig. . MoreoveSiGGPPS7b, SiLCYB and SiNNCED4 gene structures did not have any introns. The shortest gene genomic sequence length was SiGGPPS7b at 1098 bp. The longest gene genomic sequence length was SiCYP97C1 at 18373 bp. These homologous genes for each gene family contained the same specific conserved protein domain  hormone cis-acting elements. Moreover, the promoters of all 54 genes had many MYB binding sites. Of them, the promoters of 51 and 14 genes had some ‘drought stress resistance’ and ‘light responsiveness’ cis-acting elements, respectively , 526 genes with expression differences between S2 and S3 (D2), and 3173 genes with expression differences between S1 and S3 (D3). Similarly, JG21 had 2209 genes with expression differences between S1 and S2 (J1), 1544 genes between S2 and S3 (J2), and 3412 genes between S1 and S3 (J3). Among them, the number of DEGs between D1 and D2 was at least 24, and the number of DEGs between D1 and D3 was at most 1784. D2 vs. D3, J1 vs. J2, J2 vs. J3, and J1 vs. J3 had 244, 187, 848 and 1184 differentially expressed genes, respectively. The number of differentially expressed genes shared by the D1, D2, and D3 stages was 209, while that of the J1, J2, and J3 stages was 175.DEGs between DHD and JG21 at different stages of ear development were analyzed Fig. S. There wThe GO enrichment analysis showed that these DEGs mapped to “cellular processes”, “environmental information processing”, “genetic information processing”, “metabolism” and “organismal system”. The KEGG enrichment analysis showed that these DEGs mainly mapped to the ‘phenylpropanoid biosynthesis’, ‘flavone and flavanol biosynthesis’ and ‘flavonoid biosynthesis’ pathways between DHD and JG21 at the three panicle development stages Fig. S. HoweverSiHDS gene was more highly expressed at the S1 stage of DHD and JG21 than the other genes, with TPM values of 60.3 and 60.4, respectively. However, the SiIDI3 gene had nearly no expression at the three panicle development stages of DHD and JG21. In the MVA pathway, the highest expression level was observed for the SiHMGS3 gene during panicle development of DHD and JG21, with TPM values ranging from 41.0 to 70.5. However, SiMVK had the lowest expression level compared to other genes. In the carotenoid biosynthesis pathway, only SiPDS had a higher expression level, with TPM values ranging from 54.3 to 70.7. The remaining genes had both lower expression levels, with TPM values ranging from 0.0 to 25.9. Moreover, only SiNCED1a had a higher expression level than the other genes during the panicle development stage of the two cultivars. The others had lower expression levels for the two cultivars.To investigate the differential expression levels of genes related to the carotenoid metabolism pathway, we analyzed 54 gene expression values from the RNA-seq of JG21 and DHD at three panicle development stages Fig. S. In the SiDXS3, SiMCT, SiHDR, SiIDI2, SiAACT1/3, SiMVK-like, SipMVK, SiPSY1, SiZDS and SiZEP1/2 were significantly positively correlated with the neoxanthin content at P < 0.05. However, SiCCD8b was significantly negatively correlated with the neoxanthin content at P < 0.05. Moreover, SiDXS3, SiMCT, SiAACT3, SiZEP1/2, and SiCCD8a/8c/8d were significantly positively correlated with the violaxanthin content at P < 0.05. SiMCT, SiAACT3, SiPSY1, SiZEP2, and SiCc8a/8d were significantly positively correlated with the lutein content at P < 0.05. Remarkably, SiZEP1/2 was significantly positively correlated with four carotenoid metabolites except zeaxanthin . However, SiPSY2 and SiNCED1b/4 were significantly positively correlated with the zeaxanthin content. SiCMT, SiHDR, SiIDI2, SiAACT3, SiPSY1 and SiZEP12 were significantly positively correlated with the β-carotene content. Moreover, SiBCH1 was significantly negatively correlated with the β-carotene content . Furthermore, SiPSY1 was significantly positively correlated with the contents of neoxanthin, lutein, and β-carotene at P < 0.05 . SiCCD8b was negatively correlated with all carotenoids except zeaxanthin .Based on Pearson’s correlation coefficient analysis, the relationship between the expression levels of genes related to carotenoid metabolism and major carotenoid metabolite contents during the panicle development stage of the two cultivars was investigated Fig. S. SiDXS3,SiMCT, SiIDI2, SiGGPPS7a, SipMVK-like, SipMVK, SiAACT1/3, SiPDS, SiPSY1, SiBCH1, SiZEP1/2 and SiCD8b  was significantly higher than that in JG21 . However, at the end of development, the contents of the two types of carotene in the white-grained variety DHD were 5.43 μg /100 mg and 2.59 μg /100 mg, respectively, which were lower than that of the yellow-grained variety JG21  during this period , which is a key transcription regulator of carotenoid biosynthesis, could be activated by phytochrome under red light signal treatment during the process of seedling deyellowing [AtPSY in the dark [SiZEP2 in the panicle of foxtail millet [In recent years, a number of studies have shown that some transcription factors directly or indirectly regulate the expression of carotenoid metabolism and metabolism genes and further control carotenoid metabolism flux in plants. To date, research reports on the interactive relationship between some transcription factors and genes related to carotenoid metabolism have been identified and proven in ellowing . PIF1 isthe dark , 64. PIFthe dark . Anotherthe dark , 66. Spathe dark –70. Hencl millet .PSY1 is not expressed [AtmiR156b, which repressed AtSPL3 expression [SiPSY1 gene interacts with the SPL18 gene as a potential regulator of carotenoid metabolism.In tomato, an SPL gene mutant with a colorless and immature tomato could not produce lycopene because xpressed . Overexppression , enhancepression , 75. HenCBP gene, including tomato AGAMOUS-like 1 (TAGL1), RIPENING INHIBITOR (RIN), and FRUITFULL1/2 (FUL1/2) [SlPSY1, SlPSY2, SlZDS, SlZ-ISO, SlCRTISO and SlBCH while negatively regulating the expression of SlLCYB and SlLCYE [SiMADS8 and SiMCT genes had a stronger coexpression relationship.Several MADS-box regulators affect the expression of the tomato (FUL1/2) . These Md SlLCYE –82. Our Based on these results, we obtained a gene coexpression network related to carotenoid metabolism and found that three gene pairs had a stronger interaction relationship. These results would lay the foundation for exploring the underlying molecular regulation mechanism of carotenoid metabolism in the panicle of foxtail millet.Additional file 1.


Lactuca sativa L.) is a winter-spring leafy vegetable, but the high demand for fresh products available year-round requires off-season production. However, the warm climate of the Mediterranean areas can impair the summer production of lettuce, thus requiring the adoption of genotypes tolerant to high irradiance as well as useful agronomic strategies like shading net installations. The aim of our research was to assess the leaf morpho-physiological and anatomical changes, in addition to productive responses, of four lettuce cultivars  grown under shading and non-shading conditions to unveil the adaptive mechanisms of this crop in response to sub-optimal microclimate (high irradiance and temperature) in a protected environment. Growth and yield parameters, leaf gas exchanges, chlorophyll fluorescence and morpho-anatomical leaf traits  were determined. Under shading conditions, the fresh yields of the cultivars ‘Ballerina’, ‘Opalix’ (‘Oak leaf’) and ‘Integral’ (‘Romaine’) increased by 16.0%, 26.9% and 13.2% respectively, compared to non-shading conditions while both abaxial and adaxial stomatal density decreased. In contrast, ‘Canasta’ under non-shading conditions increased fresh yield, dry biomass and instantaneous water use efficiency by 9.6%, 18.0% and 15.7%, respectively, while reduced abaxial stomatal density by 30.4%, compared to shading conditions. Regardless of cultivar, the unshaded treatment increased the leaf mass area by 19.5%. Even though high light intensity and high temperature are critical limiting factors for summer lettuce cultivation in a protected environment, ‘Canasta’ showed the most effective adaptive mechanisms and had the best production performance under sub-optimal microclimatic conditions. However, greenhouse coverage with a white shading net (49% screening) proved to be a suitable agricultural practice that ensured an adequate microclimate for the off-season growth of more sensitive cultivars ‘Ballerina’, ‘Oak leaf’ and ‘Romaine’.Lettuce ( Asteraceae; Lactuca sativa L.) is one of the most used and popular leafy vegetables globally, but its nutritional value is underestimated for its high water content (about 95%) . ,47. −2 s−2 s−1) . The dif−2 s−1) . Therefo−2 s−1) . In cont−2 s−1, confirming once again the high genetic variability of this species. Regardless of cultivar, unshaded plants univocally increased LMA , probably due to photoinhibition , except for ‘Canasta’, which grew faster . CalciumLactuca sativa L.) cultivars with different morphology of leaves (v/v) of peat and perlite. The pots were covered with a fine layer of perlite to prevent water evaporation from the substrate. Plants were arranged in double rows with a distance of 35 and 25 cm inter- and intra-rows, respectively, for a density of 11.5 plants m−2. Seedlings were irrigated with nutrient solution (NS) provided by a drip irrigation system consisting of a 16 mm polyethylene main pipeline equipped with 2 L h−1 drippers. The Hoagland NS had the following composition: 8.0 mM nitrate, 0.7 mM phosphorus, 2.5 mM potassium, 3.0 mM calcium, 1.0 mM sulfur, 0.7 mM magnesium, 1.0 mM ammonium, 1 mM sodium, 1 mM chlorine, 20 μM iron, 9 μM manganese, 0.3 μM cupper, 1.6 μM zinc, 20 μM boron and 0.3 μM molybdenum. The pH and EC of the NS were 6.0 ± 0.2 and 1.2 ± 0.1 dS m−1, respectively. Relative humidity and temperature were recorded continuously using WatchDog A150 data loggers  at canopy level at different points of the greenhouse. Climate data were collected at a 30-min interval. Periodic measurements of Photosynthetic Photon Flux Density (PPFD) were recorded from 7:30 AM to 6:30 PM using a handheld spectral radiometer . Average temperature, relative humidity and PPFD trend recorded during the growing season at the experimental site are shown in The experimental trial was conducted during the early summer season 2020 in a glass greenhouse located at the Department of Agriculture (DIA) of the University of Naples Federico II . The experimental protocol included a white shading net with a 49% light screening factor  and an unshaded treatment in factorial combination with four lettuce (f leaves . The gla−1) and separated into leaves and stems. Leaf area was quantified by digital image analysis with ImageJ v1.52a software . A subsample of leaf tissue was immediately stored at −20 °C for total ascorbic acid and pigment analysis. All harvested tissues were oven-dried at 70 °C until constant weight (~72 h) for dry biomass (g plant−1) and leaf dry matter (%) determination. Dried leaves were ground with an MF10.1 cutting-grinding head mill  and sieved with MF0.5 sieve  for total nitrogen and minerals determination.At 25 days after transplanting (DAT), the plants were harvested, weighed for fresh yield determination (g plant3 plant−1) according to the following equation:At 8, 14 and 21 DAT on three plants per plot, the plant growth trend was quantified through the growth index  measurements were taken on young fully expanded leaves with a handheld Minolta Chlorophyll Meter SPAD-502 . A single average SPAD value for each replicate was obtained by measuring ten leaves per plot.2 net assimilation rate , stomatal conductance  and transpiration  were measured using a Li-6400 portable leaf gas exchange analyzer . The measurements were performed at ambient CO2 concentration and photosynthetic active radiation of 1000 µmol m−2 s−1, as set in the leaf gas exchange analyzer chamber. Instantaneous water use efficiency (WUEi) was calculated as ACO2/E.On July 9 (24 DAT) between 11:00 AM and 2:00 PM, leaf gas exchange measurements and fluorescence emission were performed on healthy fully expanded leaves of three plants per plot. COv/Fm Meter, Opti-Sciences Inc., Hudson, NH, USA) on the same leaves used for leaf gas exchange measurements. According to Kitajima and Butler [v/Fm) was calculated as (Fm − F0)/Fm, where F0 was the ground signal induced by a blue LED internal light of 1–2 µmol photons m−2 s−1 and Fm was the maximal fluorescence level in the induced darkness by one second of saturating light pulse of 3000 µmol photons m−2 s−1.On the same date, on 10 min dark-adapted leaves, chlorophyll fluorescence measurements were taken with a portable fluorometer  H2O2, then was mineralized in a DK 20 Heating Digester . The mineralized sample was distilled in a UDK 140 distiller  by adding 33% of NaOH. Ammonia was trapped in H3BO3 by steam distillation and titrated with 0.1 N H2SO4. All reagents were purchased from Carlo Erba Reagents Srl .Total nitrogen content was determined according to the Kjeldahl method described by Bremner . Briefly® Advance EDI pure water system; Sartorius, Goettingen, Lower Saxony, Germany), incubated at 80 °C in a shaking water bath  for 10 min, centrifuged at 6000 rpm for 10 min  and then filtered by a syringe filter with a 0.45 µm pore size . For anions  determination, an IonPac AG11-HC 4 × 50 mm guard column and an IonPac AS11-HC 4 × 250 mm analytical column were used. For cations  determination, an IonPac CG12A 4 × 250 mm guard column and an IonPac CS12A 4 × 250 mm analytical column were used. All columns were purchased from Thermo Scientific™ Dionex™ .Mineral content in lettuce leaves was determined through ion chromatography  according to the method described by Rouphael et al. . Briefly−1 of fresh weight (FW), all minerals were expressed as mg g−1 of dry weight (DW). Total nitrogen was expressed as a percentage (%). Minerals and total nitrogen were analyzed in triplicate.Except nitrate expressed as mg kgThe LMA was evaluated on nine leaves per treatment as the ratio between leaf DW and leaf area. The number of epidermal cells and stomata were determined on the abaxial and adaxial sides of the same leaves used for leaf gas exchange and LMA measurements, as described by Cirillo et al. . Briefly2).Stomatal density and epidermal cell density were calculated as the ratio between the number of cells, and the area photographed for each image  of lettuce leaves were determined as described by Wellburn [a, chlorophyll b and carotenoid contents were determined through a UV-Vis spectrophotometer ONDA V-10 Plus  with an absorbance of 647, 664 and 470 nm, respectively.Pigments  using IBM SPSS Statistics version 20.0 . The mean effect of CV and GIC was compared according to one-way analysis of variance and High light intensity and high temperatures in Mediterranean regions pose a challenge to off-season lettuce production (spring-summer season), affecting growth and yield and resulting in quality losses. In this perspective, the combination of shading and genotypes tolerant to sub-optimal summer conditions is mandatory for off-season lettuce production. Our results showed that different genotypes revealed diverse responses to adverse microclimatic conditions. Among the four genotypes, ‘Canasta’ increased fresh yield and WUE in unshaded treatment . This wa