Adverse effects of maternal vitamin D deficiency have been linked to adverse pregnancy outcomes. We investigated the relationship between maternal vitamin D status and newborn anthropometry measurements using a genetic approach and examined the interaction between genetic variations in involved in vitamin D synthesis and metabolism and maternal vitamin D concentrations on newborn anthropometry.The study was conducted in 183 pregnant Indonesian Minangkabau women. Genetic risk scores (GRSs) were created using six vitamin D–related single nucleotide polymorphisms and their association with 25-hydroxyvitamin D [25(OH)D] levels and newborn anthropometry (183 infants) were investigated.P > 0.05, for all comparisons). After correction for multiple testing using Bonferroni correction, GRS was significantly associated with 25(OH)D in the third trimester (P = 0.004). There was no association between GRS and newborn anthropometric measurements; however, there was an interaction between GRS and 25(OH)D on head circumference (P = 0.030), where mothers of neonates with head circumference < 35 cm had significantly lower 25(OH)D if they carried ≥4 risk alleles compared to those who carried ≤3 risk alleles.There was no significant association between maternal 25(OH)D concentrations and newborn anthropometry measurements (Our findings demonstrate the impact of vitamin D-related GRS on 25(OH)D and provides evidence for the effect of vitamin D-related GRS on newborn anthropometry through the influence of serum 25(OH)D levels among Indonesian pregnant women. Even though our study is a prospective cohort, before the implementation of vitamin D supplementation programs in Indonesia to prevent adverse pregnancy outcomes, further large studies are required to confirm our findings.The online version of this article (10.1007/s40200-019-00480-5) contains supplementary material, which is available to authorized users. As one of the tropical countries in Southeast Asia located at the equator, Indonesia has an abundant sunlight all year round. According to recent studies, vitamin D deficiency in Indonesian women ranges between 60 and 95% –5. Adequ  concentrations . Candida, DHCR7) , GWAS fo) (TYR)) –15, and YP27B1)) . Recent D levels , 17, andPrevious GWASs , 17 haveThe study was conducted among singleton pregnant women of West Sumatran Vitamin D Pregnant Mother (VDPM) cohort study in West Sumatra, from July 2017 to April 2018. The study was performed at community health centres in five cities  in West Sumatra, Indonesia. In this study, participants were followed up from the first trimester (T1) to third trimester (T3) of pregnancy and at delivery to determine newborn anthropometry measurements . This study was conducted in accordance with the declaration of Helsinki and approved by the Ethics Committees of Medical Faculty, Andalas University (No. 262/KEP/FK/2016). All women provided written informed consent prior to the start of the data collection.All participants were pregnant women who were recruited during their first antenatal care checks at the public health centres. Inclusion criteria included: 1) pregnant women willing to visit public health care at each site, 2) those who were in the T1 (<13 weeks) of their singleton pregnancy, 3) those who were healthy based on medical examination, and 4) those who were willing to participate by signing the informed consent and following the research procedures. Stratified random sampling was applied for the data collection that took place at two research locations: mountainous and coastal areas. Public health centers that had high numbers of the first-trimester pregnant mothers were chosen for the data collection. Women were excluded from the study if they had multiple pregnancies, some common complications of pregnancy such as preeclampsia, miscarriage or pregnancy loss, stillbirth, and they had chronic illness like diabetes, hypertension, cardiovascular disease, or hypothyroidism. Women who were taking drugs that can interfere with vitamin D metabolism such as antiepileptic agents, glucocorticoids, anti-oestrogens or antiretroviral drugs during pregnancy were excluded. Out of 239 women, 53 were dropped out for different reasons, including pregnancy loss, change of residence, not willing to continue research, and those who could not be contacted again. The number of pregnancy loss due to complications of pregnancy such as foetal inflammation, stillbirth, and abnormal foetal development was 25 (13.44%). There were 3 cases of preterm birth, 8 cases of stillbirth, and 14 cases of miscarriage. Finally, we obtained 186 pregnant women who completed all requirements and attended follow-ups from T1 to delivery. After excluding three samples due to low DNA yield, a total of 183 mother and infant pairs were used for the present study. Participant’s recruitment process is shown in detail in Fig. Maternal sociodemographic factors were assessed using a standardized questionnaire administered by trained field data collector . The questionnaire included information on demographics, maternal occupation, education, and pregnancy profile. These data were prospectively collected from medical records or interviews. Maternal sociodemographic characteristics included age, education level , maternal working status (working and not working), and geographical status . Maternal health status included pre-pregnancy BMI, and mid-upper arm circumference (MUAC). Maternal lifestyle included the outdoor activity to measure the sun exposure status during pregnancy and maternal vitamin D and calcium supplementation during pregnancy.2; normal, 18.5–23.49 kg/m2; overweight, 23.5–24.99 kg/m2; Pre-obese, 25–29.99 kg/m2; Obese, ≥30 kg/m2)  in the model and adjusting for age, pre-pregnancy BMI, gestational age at birth, and infant’s gender. The study objectives are shown in Fig. P value for association analysis was ≤0.006 [3 GRS * 3 maternal 25(OH)D level outcomes D during pregnancy) = 9 tests]. For the interaction analysis, corrected P value was ≤0.003 [3 GRS * 2 maternal 25(OH)D outcomes (T1 and T3) * 3 newborn anthropometry outcomes  = 18 tests].Correction for multiple testing was performed using Bonferroni correction. Corrected . There was a significant difference in diastolic blood pressure (DBP), and body weight during the third trimester and there was a significant difference in outdoor activity (hours/day) during the first trimester between those who were vitamin D deficient (VDD) and those with normal vitamin D status (NVD) (p < 0.05). In Table p > 0.05, for all comparisons). The study participants were enrolled at an average age of 29.7 ± 5.68 years. The average of pre-pregnancy Body Mass Index (BMI) was 23.45 ± 4.56 kg/m2. The average gestational duration was 38.88 ± 1.91 weeks and 73.30% of deliveries were normal. Mean birth weight, birth length, and head circumference were 3204.87 ± 494.99 g, 48.56 ± 2.87 cm, and 33.89 ± 2.52 cm, respectively. Approximately 6.80% (n = 12) of newborn babies had low birth weight (LBW) status, while 5.40% (n = 10) were diagnosed with macrosomia. There were < 10% of cases who had adverse pregnancy outcomes such as LBW, SGA, and preterm birth (PTB). However, a higher number of women had babies with a small head circumference (<35 cm) and short birth length (<50 cm) .The characteristics of the study participants stratified based on maternal vitamin D status at T1 and T3 are shown in Table 1Vitamin D status during pregnancyn = 154) of women were deficient  and insufficient  for vitamin D. The serum 25(OH)D levels increased significantly during pregnancy . In the T3, average maternal serum 25(OH)D level was 21.21 ± 10.16 ng/mL. A total of 46.80% (n = 87) of women were vitamin D sufficient, 34.40% (n = 64) were insufficient and 18.80% (n = 35) were deficient. The prevalence of vitamin D deficiency and insufficiency in the T1 lowered from 82.80% (n = 154) to 53.20% (n = 99) in the T3.Average maternal serum 25(OH)D level in T1 was 14.00 ± 6.97 ng/mL. Approximately 82.80% . There was also no significant association between changes in vitamin D status during pregnancy and newborn anthropometry  (Table 2).We found no significant association between 25(OH)D level during T1 and T3 and newborn anthropometric measurements (P = 0.018), but not with T1 25(OH)D levels (P = 0.157). The synthesis-GRS and metabolism-GRS had no effect on 25(OH)D levels and changes in 25(OH)D levels during pregnancy . The association between GRSs and serum 25(OH)D levels during pregnancy are shown in Table There was a significant association between vitamin D-GRS and 25(OH)D levels in T3 (P = 0.004) and changes in 25(OH)D levels during pregnancy . Similar finding was observed even after classifying newborn anthropometry measurements into categorical variables  . Further stratification of study participants based on head circumference cut-off points  [. However, after correction for multiple testing, this interaction was not considered statistically significant (Table ).None of the interactions were statistically significant except for the interaction between vitamin D-GRS and 25(OH)D concentrations in T3 on newborn head circumference measurement ( ≥35 cm)  showed tm) [Fig. . Howevernt Table .Fig. 4InApaI (rs7975232) SNP showed a significant association with 25(OH)D levels in both T1 (0.047) and T3 (p = 0.043), where A allele carriers had significantly lower 25(OH)D concentrations. In addition, A allele carriers of the CYP2R1 (rs12794714) SNP had significantly lower levels of 25(OH)D in both T1 (p = 0.001) and T3 (p < =0.0001). There was also a significant association between GC (rs22282679) SNP and 25(OH)D concentrations in T3 and changes in 25(OH)D levels during pregnancy (P < 0.001), but not in T1 (P = 081). None of the other associations were statistically significant (Supplementary Table Besides exploring the impact of GRS on 25(OH)D levels during pregnancy, the individual effect of the SNPs on 25(OH)D levels was also examined. Under a dominant genetic model, th T1 0.07 and T3 To our knowledge, this is the first study of its kind to investigate whether maternal vitamin D status was associated with newborn anthropometry measurements using a genetic approach. Our study demonstrated a high prevalence (82.80%) of vitamin D deficiency among Indonesian pregnant mothers. Women who had ≥4 vitamin D-decreasing risk alleles had significantly lower levels of serum 25(OH)D during pregnancy. Even though there was no direct association between GRS and newborn anthropometric measurements, mothers of neonates with head circumference < 35 cm had significantly lower 25(OH)D levels if they carried ≥4 risk alleles suggesting that vitamin D deficiency during pregnancy can increase the genetic risk of adverse newborn anthropometry outcomes. Considering that more than half of the study participants were vitamin D deficient (83%), establishing a vitamin D prevention program for pregnant women may be considered to maintain optimal foetal growth and development. Our findings, if replicated in future studies, may have a significant public health impact on initiating strategy to raise the awareness on the importance of vitamin D during pregnancy to prevent vitamin D deficiency and its adverse pregnancy outcomes.Recent studies have shown a significant phenotypic association between serum 25(OH)D levels during pregnancy and adverse pregnancy outcomes such as gestational diabetes mellitus, pre-eclampsia, SGA, LBW and PTB , 24–26. P = 0.004) and changes in serum 25(OH)D levels during pregnancy. Our finding was similar to a study in 759 Chinese Han pregnant women from Zhoushan Pregnant Women Cohort (ZPWC) which also showed that individuals with >3 risk alleles had significantly lower 25(OH)D levels compared to those with 1 risk allele [One of the main findings of our study was the significant association between GRS  and lower serum 25(OH)D levels in the third trimester D and newborn anthropometry . A few recent studies have shown that te women , 27. A rh weight . Conversh weight ; but theVDR gene variants (rs2228570 and rs7975232) have been shown to be associated with the risk of adverse pregnancy outcomes such as PTB, LBW, and SGA status [VDR) and PTB risk [VDR variants and PTB risk in the present study as the PTB variable was not available for all study participants; however, we examined other newborn anthropometry measurements such as birth weight, birth length and head circumference. VDR is required for the vitamin D metabolic pathway where its activation regulates the expression of genes involved in cell proliferation and differentiation [VDR in placental tissues suggesting the role of vitamin D in reproduction and maternal to foetal nutrient transfer mechanism [VDR expression. Furthermore, it is possible that VDR might be a key factor in maternal to foetal nutrient transfer mechanism and adverse pregnancy outcomes and therefore serves as a strong candidate gene for our study.While most of the genetic variants chosen for our study have not been studied previously in relation to the risk of adverse pregnancy outcomes, A status , 38–42. PTB risk . We werentiation . Studiesechanism , 45. HenThe current study has some limitations. Firstly, the sample size was relatively modest; however, we were still able to identify significant associations and interactions in 183 mother and infant pairs after correction for multiple testing. Secondly, sunlight exposure variable was a self-reported outdoor activity and hence the bias involved in assessing sun exposure status cannot be ruled out. Thirdly, we have controlled for known major confounders, but we cannot completely exclude the possibility of other confounders such as the impact of vitamin D-fortified foods as this information was not collected in the present study. Compared to previous studies , our stuIn conclusion, we provide an evidence for an impact of vitamin D-related genetic variations on newborn anthropometry measurements through the influence of serum 25(OH)D levels among Indonesian pregnant Minangkabau women. Before initiating strategies for the implementation of vitamin D supplementation programs in Indonesia to prevent adverse pregnancy outcomes, further large studies are required to confirm our findings.ESM 1(DOCX 214 kb)


We propose a study showing a novel role of Gcn5p and Ubp8p in the process of ubiquitylation of the yeast proteome which includes main glycolytic enzymes. Interestingly, in the absence of Gcn5p and Ubp8p glucose consumption and redox balance were altered in yeast. We believe that these results and the role of Gcn5p and Ubp8p in sugar metabolism might open new perspectives of research leading to novel protocols for counteracting the enhanced glycolysis in tumors. Saccharomyces cerevisiae, we analyzed the composition of the ubiquitylated proteomes in strains lacking acetyltransferase Gcn5p, Ub-protease Ubp8p, or both to understand their involvement in the regulation of protein ubiquitylation. We analyzed His6Ub proteins with a proteomic approach coupling micro-liquid chromatography and tandem mass spectrometry (μLC-MS/MS) in gcn5Δ, ubp8Δ and ubp8Δ gcn5Δ strains. The Ub-proteome altered in the absence of Gcn5p, Ubp8p, or both was characterized, showing that 43% of the proteins was shared in all strains, suggesting their functional relationship. Remarkably, all major glycolytic enzymes showed increased ubiquitylation. Phosphofructokinase 1, the key enzyme of glycolytic flux, showed a higher and altered pattern of ubiquitylation in gcn5Δ and ubp8Δ strains. Severe defects of growth in poor sugar and altered glucose consumption confirmed a direct role of Gcn5p and Ubp8p in affecting the REDOX balance of the cell.Protein ubiquitylation regulates not only endocellular trafficking and proteasomal degradation but also the catalytic activity of enzymes. In  Conservation in evolution allows the translation of discoveries obtained in simple models to human cells. Accordingly, simple budding yeast is one of the best models to dissect molecular mechanisms regulating complex networks and circuitries . Protein4–11–GCN5, UBP8, or both, expressing His6-Ub for biochemical purification. Next, we analyzed the composition of Ub proteins with a gel- and label-free proteomic approach based on the coupling of micro-liquid chromatography and tandem mass spectrometry (μLC-MS/MS). We have obtained the proteome composition of His6-Ub proteins and their differential expression in the disrupted strains in comparison with the wild type (WT). By subgrouping the Ub proteins identified as differentially expressed for biological function, we found all the main enzymes involved in glycolysis overrepresented in the absence of Gcn5p and Ubp8p and the key glycolytic enzyme phosphofructokinase 1 (Pfk1pmyc) showed increased and altered ubiquitylation in the disrupted strains. We have also found defective growth in low sugar and altered utilization of glucose with effects on the redox balance of the cell in the absence of Gcn5p and Ubp8p. Collectively, our data indicate that Gcn5p and Ubp8p affect the levels of ubiquitylation of fundamental enzymes and alter the fermentative/respiratory flux balance.In the present study, we wanted to analyze whether the global process of protein ubiquitylation might be dependent on Gcn5p, Ubp8p, or both. We performed a shotgun proteomic study of yeast strains disrupted in ubp8Δ, gcn5Δ, and ubp8Δ gcn5Δ strains . This observation is not surprising based on the intrinsic deubiquitylase activity of Ubp8p, whereas we expect that Gcn5p acts indirectly, possibly through the deposition of a counteracting acetyl group on lysines.The 6×His-Ub proteins were expressed in WT,  strains , purifie strains . Figure  diagram . Of the 10.1128/mBio.01504-20.2TABLE S1ubp8Δ, gcn5Δ, and ubp8Δ gcn5Δ S. cerevisiae strains. Download Table S1, XLS file, 0.1 MB.Complete list of Ub proteins detected in WT, Copyright © 2020 De Palma et al.2020De Palma et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ubp8Δ, gcn5Δ, and ubp8Δ gcn5Δ strains respect to the WT strain. To this end, the two algorithms of the software, the differential average (DAve) and the differential confidence index (DCI), were applied, representing the ratio and the confidence in differential expression, respectively, for each protein between two strains. Using stringent filters for DAve and DCI to maximize the confidence of identification and to consider proteins with a variation greater than a fold change of 1.5, a total of 103 proteins were found differentially expressed and reported in ubp8Δ strain compared to the WT. In addition, 49 proteins were more abundant in the gcn5Δ strain compared to WT, and 11 were down-expressed in the gcn5Δ strain. Finally, 13 and 65 proteins were down- and up-expressed, respectively, in the ubp8Δ gcn5Δ strain compared to the WT. In addition, known genetic (G) or physical (P) interactions of each protein with Ubp8p or Gcn5p are indicated in gcn5Δ and double-mutant strains, which suggests that they are functionally related to Gcn5p. In the same way, elongation factor 3A (Yef3p), pyruvate kinase (Cdc19p), cytochrome c1 (Cyt1p), cytoplasmic chaperone (Hsc82p), and protein involved in clathrin-mediated endocytosis (YHR097Cp) equally change in the ubp8Δ and double-mutant strains, thus demonstrating that they are closely related to Ubp8p. Another interesting aspect concerns proteins, such as elongation factor (Eft1p), phosphoglucose isomerase (Pgi1p), NAD-dependent glycerol-3-phosphate dehydrogenase (Gpd1p), superoxide dismutase (Sod1p), AMP deaminase (Amd1p), and uracil (Ura2p), that show a marked increase or reduction in double-mutant ubp8Δ gcn5Δ strain compared to single-mutant strains, demonstrating the synthetic effect exerted by the double deletion. Finally, in gcn5Δ, ubp8Δ, and ubp8Δ gcn5Δ strains against the WT.Using Multidimensional Algorithm Protein Map (MAProMa) software , we alig10.1128/mBio.01504-20.3TABLE S2ubp8Δ, gcn5Δ, and ubp8Δ gcn5Δ strains versus the WT condition. Download Table S2, XLS file, 0.1 MB.Complete list of differentially expressed Ub proteins resulted from the MAProMa comparison of Copyright © 2020 De Palma et al.2020De Palma et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the PFK1 and PFK2) and pyruvate carboxylase (PYC1) mRNA expression  and low (0.1%) glucose. As shown in gcn5Δ and ubp8Δ strains in high glucose (2%) with respect to the WT, indicating defects in fermentation, that was worse in the ubp8Δ gcn5Δ strain, reflecting its functional interaction. These results also indicated the greater growth reduction at low sugar levels in all strains, thus confirming the strong delay of growth in poor sugar medium. Collectively, these data demonstrate respiratory and fermentative defects. In particular, the results obtained in liquid culture  activities, as markers to monitor the channeling of the glucose glycolytic flux toward respiration or fermentation, respectively. In fact, Adh1p is preferentially expressed in fermentative conditions and directly involved in ethanol production  at 18, 21, and 24 h of growth, and the collected data are shown in a histogram (To determine the impact of  culture  show a coduction . In contoduction  19–21).–21.GCN5 duction – previous for ADH  23). Th. ThGCN5  for ADH . The condium see . Based oistogram . The difistogram .10.1128/mBio.01504-20.1FIG S1FIG S1, PDF file, 0.5 MB.Growth curves (filled circles) of indicated strains in high glucose (2%) are diagrammed with measures (open circles) of glucose (in g/liter) consumed at increasing times (in h) in the same strains. Download Copyright © 2020 De Palma et al.2020De Palma et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the GCN5, UBP8, or both in comparison to the wild type. We used the μLC-MS/MS system for the first characterization of H6-Ub proteins obtained from the four examined strains, and then we obtained quantitative information on specific proteins belonging to cellular metabolic processes subjected to fast changes in response to genetic or environmental stimuli. In this way, we were able to identify the trend of relative abundance of differentially expressed proteins among the three mutated strains compared to the WT. In addition, based on proteomic results, we drafted a proteomic interaction map and a grouping of protein subclasses with respect to their biological function for a better visualization of the major pathways involved. In the first instance, the results obtained clearly indicate a central role for Ubp8p and Gcn5p, affecting specific proteins that contribute to the control of cell growth, stress response, and—above all—energetic metabolism. Most of the Ub proteins detected as differentially expressed in the examined comparisons, although not always with the same levels of abundance, were shared and uprepresented in the absence of GCN5 and UBP8 and in the double-disrupted strain. These evidences can be interpreted as due to modification of lysines that can alternatively be acetylated or ubiquitylated , suggesting the contemporary accumulation of ethanol and its partial oxidation by Adh2p with activation of both fermentative and respiratory metabolism. In fact, while Adh1p allows the glycolytic flux to proceed to ethanol through the reoxidation of NADH in the first part of glycolysis, Adh2p oxidizes part of the ethanol to acetaldehyde. This reaction provides the cytoplasmic NADH redox excess for the transdehydrogenases (Nde1p and Nde2p) localized on the outer mitochondrial membrane that transfer these equivalents to the respiratory transport chain  and spotted.The 600/ml), and pellets were treated with 0.3 M NaOH–140 mM β-mercaptoethanol and then incubated in ice with occasional vortexing. Trichloroacetic acid  was added, followed by incubation in ice. After centrifugation, the cells were lysed in HU buffer  and boiled 15 min at 65°C. Cells transformed with pDJ421 encoding 6×His-Ub (4. Cells (0.8 to 1 OD600) were then lysed in guanidinium buffer ; 1/20th of lysate was used as input and TCA (5.5%) precipitated. The lysate was then incubated on a Ni-NTA agarose resin (Qiagen) at 4°C with rotation, after washing with urea buffer , the samples were eluted in 2× sample buffer . For MudPIT analysis, the resin was washed three times with 8 M urea buffer and with 1 M urea buffer; trypsin was then added in 1:20 (wt/wt) enzyme-substrate. After overnight digestion (37°C), the reaction was stopped by acidification with 1 μl of trifluoroacetic acid.For total protein extraction, yeast cultures were collected at exponential phase (0.8 OD6×His-Ub  were gro4 was added to the cultures (Iglesias). Cells were collected (0.8 OD600/ml), and the eluate and lysate samples were loaded on 7.5% SDS-PAGE, blotted on nitrocellulose membranes (Amersham), and hybridized with anti-6×His-Ub (Abcam), anti-Ada2p, anti-myc, and anti-α-tubulin (Santa Cruz). Proteins were detected by using Long-Lasting Chemilumiscent substrate (EuroClone) and visualized by the ChemiDoc MP imaging system (Bio-Rad).Yeast cultures were grown at 28°C semicomplete medium . For expression of His6-Ub, 0.1 mM CuSOgcn5Δ, and ubp8Δ strains) in YP plus 2% glucose were collected. Total RNA extraction was performed using the phenol method and retrotranscribed with a QuantiTect reverse transcription kit (Qiagen). Standard curves of WT genomic DNA (20/0.05 ng) and cDNA (50 ng) were amplified for actin, phosphofructokinases (PFK1 and PFK2), and pyruvate carboxylase (PYC1); all experiments were performed in triplicate. Quantitative RT-PCR (qRT-PCR) experiments were carried out on a Rotor-gene Q (Qiagen) apparatus. The following oligonucleotides that were used: ACT1f, 5′-GCTGAAAGAGAAATTGTCCG-3′; ACT1r, 5′-ACACTTCATGATGGAGTTGTA-3′; PFK1f, 5′-GCTCAAAGTCAAGGTGCTCT-3′; PFK1r, 5′-CCTGAGAAGGTGATGTTGTTG-3′; PFK2f, 5′-GCAGTTTCAACCAAGCCAAC-3′; PFK2r, 5′-CGTTAGAGTTCATACCTGGAG-3′; PYC1f, 5′-CGTACCGCTCATGAACTGT-3′; and PYC1r, 5′-GGATGGTGAAATCTACCTGG-3′.Exponentially growing cells  and resuspended in 0.1% formic acid. Samples were then analyzed by means of a μLC system coupled with a tandem mass spectrometer through a trap-elute configuration. Briefly, 5 μl (3 μg) of each digested peptide mixture was first loaded onto a peptide trap  for concentration and desalting with a pump running in isocratic mode with 0.1% formic acid in water. Then, the automatic switching of a 10-port valve eluted the trapped mixture on a reversed-phase column  for the separation with an acetonitrile gradient ; the gradient profile was 5% eluent B for 5 min, followed by 5 to 40% eluent B for 45 min, 40 to 80% eluent B for 10 min, 80 to 95% eluent B for 5 min, and 95% eluent B for 10 min. The flow rate was 100 μl/min, which was split to achieve a final flux of 2 μl/min. The peptides that were eluted from the C18 column were analyzed directly with a linear ion trap LTQ mass spectrometer equipped with a nano-ESI source (Thermo Fisher Scientific). The spray capillary voltage was set at 1.6 kV, and the ion transfer capillary temperature was held at 220°C. Full mass spectra were recorded over a 400- to 2,000-m/z range in positive-ion mode, followed by five tandem mass spectrometry (MS/MS) events sequentially generated in a data-dependent manner on the top five most intense ions selected from the full MS spectrum, using a dynamic exclusion for MS/MS analysis. In particular, MS/MS scans were acquired setting a normalized collision energy of 35% on the precursor ion. MS scan functions and LC solvent gradients were controlled by the Xcalibur data system, version 1.4 (Thermo Fisher Scientific). A total of 16 LC-MS/MS runs were performed, representing the four yeast strain conditions examined  as technical and biological replicates.Tryptic digest mixtures were desalted using Pierce CS. cerevisiae proteome database. The following criteria were applied: trypsin as enzyme, three missed cleavages per peptide, mass tolerances of ± 0.8 Da for precursor ions and ± 0.6 Da for fragment ions, protein grouping, and strict parsimony principle. Percolator node was used with a target-decoy strategy to give final false discovery rates at a peptide spectrum match level of 0.01 (strict), considering a maximum delta CN of 0.05 (http://bioinfogp.cnb.csic.es/tools/venny/) using the UniProt accession numbers. Cellular function was assigned to each protein according to the GOA database (http://geneontology.org/) and the UniProt database (http://www.uniprot.org/). We were unable to assign the cellular function for some proteins, which were therefore classified as “unknown,” and additional proteins without a complete characterization at the moment of data analysis were classified as “unclear” or “other.” For label-free differential analysis, the 16 protein lists obtained from the SEQUEST algorithm were aligned with an in-house algorithm, MAProMA  . In depth, to select differentially expressed proteins, the four subgroups were compared pairwise, applying thresholds of 0.4 and 5 on the two MAProMa indexes DAve  and DCI , respectively. DAve, which evaluates changes in protein expression, was defined as (X – Y)/(X + Y)/0.5 with values ranging from a maximum of + 2.00 to a minimum of −2.00, while DCI, that evaluates the confidence of differential expression, was defined as (X + Y) × (X – Y)/2 with values ranging from +∞ to –∞. The X and Y terms represent the aSpCs of a given protein in two compared samples. Most confident up-represented proteins showed a DAve ≥ +0.4 and a DCI ≥ +5; most confident down-represented proteins showed a DAve ≤ −0.4 and DCI ≤ −5.All data generated were searched using the Sequest HT search engine contained in the Thermo Scientific Proteome Discoverer software (v2.1) against the UniProt  of 0.05 . Only peein Map)  and comp600.Cell extracts preparation, native polyacrylamide gel electrophoresis (PAGE), alcohol dehydrogenase (Adhp), and glucose-6-phosphate 1-dehydrogenase (G6PDHp) staining assays were carried out as previously described , 43. Adht test . The error bars shown in Experimental results shown in


Renal cell carcinoma (RCC) is a fatal disease, in which the PI3K/AKT/mTOR signaling pathway serves an important role in the tumorigenesis. Previous studies have reported the prognostic significance of PI3K/AKT/mTOR signaling pathway members in RCC; however, there is insufficient evidence to date to confirm this. Thus, the present study aimed to systematically investigate the prognostic roles of multiple PI3K/AKT/mTOR signaling proteins in clear cell RCC (ccRCC) using online large-scale databases.PTEN, PIK3CA, PIK3CB, PIK3CD, PIK3CG, AKT1, AKT2, AKT3 and mTOR were investigated using the Gene Expression Profiling Interactive Analysis (GEPIA) and Oncomine databases, and the protein expression levels of PI3K, AKT and mTOR were detected using western blotting (WB) analysis. In addition, the correlation between mRNA or protein expression levels and the prognostic significance was analyzed using the Kaplan-Meier (K-M) plotter (n = 530), the Human Protein Atlas  and The Cancer Protein Atlas  databases.The mRNA expression profiles of PI3K/AKT/mTOR signaling pathway proteins PTEN, PIK3CA, PIK3CB, AKT1, AKT2 and AKT3, were negatively correlated with ccRCC stages (P < 0.05), though most of their mRNA and protein expression levels were notsignificantly different between ccRCC and normal tissues using GEPIA, Oncomine and WB analyses (P < 0.05). Meanwhile, using the K-M plotter and HPA prognostic analysis, it was found that the mRNA expression levels of the majority of the PI3K/AKT/mTOR signaling pathway members, including PTEN, PIK3CA, PIK3CB, PIK3CG, AKT3 and mTOR were positively correlated with overall survival (OS), whereas PIK3CD mRNA expression was negatively correlated with OS (P < 0.05). Furthermore, TCPA prognostic analysis observed that several of the key molecules of the PI3K/AKT/mTOR signaling pathway  were also positively correlated with OS in patients with ccRCC (P < 0.05). In conclusion, the present study suggested that several members of the PI3K/AKT/mTOR signaling pathway, especially PTEN, may be favorable prognostic factors in ccRCC, which indicated that the PI3K/AKT/mTOR signaling pathway may be implicated in ccRCC initiation and progression.The GEPIA revealed that the mRNA expression of major PI3K/AKT/mTOR pathway members, including  Kidney cancer is a high-risk cancer that demonstrates a high mortality rate, accounting for an estimated 73,750 newly diagnosed cases and 14,830 cancer-related deaths in the United States in 2020 . Clear cApart from the well-known von Hippel-Lindau (VHL) gene, genetic mutations in members of the PI3K/AKT/mTOR signaling pathway are also frequently observed in RCC, which promotes the hyperactivation of the PI3K/AKT/mTOR signaling cascade . The PI3fatty acid synthase (FASN) and AKT3 were prognostic factors for the survival of patients with ccRCC; and using the Oncomine and K-M plotter databases, our previous study reported that several mRNA biomarkers, such as FASN and ATP citrate lyase (ACLY), may be prognostic factors for ccRCC  , The Canor ccRCC . In the or ccRCC . In addihttp://gepia2.cancer-pku.cn/), which was based on the UCSC Xena project, was used to analyze the mRNA expression status of the following nine members of the PI3K/AKT/mTOR signaling cascade in ccRCC, as described previously , AKT  and mTOR. Briefly, the mRNA expression data of 523 ccRCC and 100 normal kidney specimens from TCGA and Genotype-Tissue Expression (GTEx) were loaded into the server to analyze the differentially expressed genes between the two groups. The RNA-seq results were reported as the number of transcripts per million (TPM). The following filter indexes were set: expression mode was expression DIY, dataset was ccRCC, log-scale was log2 (TPM + 1), and P-value cut-off was 0.05. Then the mRNA expression parameters, including sample size and statistical box plot were displayed. For consistency analysis, the mRNA expression levels of the nine genes in 523 ccRCC and 72 matched normal specimens from TCGA, and their correlationship between the genes and the pathological stages in 523 ccRCC specimens were also analyzed.The GEPIA server  was used to validate the mRNA expression levels of the PI3K/AKT/mTOR pathway members, as previously described . And 3 cases of ccRCC tissues (including cancer and adjacent non-cancerous), which were collected from 3 patients  , were ushttp://www.kmplot.com), which contained the RNA-seq data from Gene Expression Omnibus (GEO), Cancer Biomedical Informatics Grid (caBIG) and TCGA, was used to analyze the correlation between the mRNA expression levels of each PI3K/AKT/mTOR signaling pathway member and the prognosis of patients with ccRCC (n = 530), as described previously  were loaded into the online server, and the K-M survival plots were used to identify their correlation with the OS for discovery analysis. The hazard ratio (HR), with 95% confidence intervals (CI), and the log-rank P-value were calculated automatically on the webpage after ≤150 months follow-up. The relationship between the gene expression levels and clinicopathological parameters, for example, sex, pathological stages and grades, of ccRCC, were also investigated.The K-M plotter , which retrieved the RNA-seq data from the Genomic Data Commons (GDC) Data Portal, was used to validate the prognosis of PI3K/AKT/mTOR signaling pathway genes in patients with ccRCC (n = 528). The K-M plot and log-rank P-value were also calculated accordingly after ≤150 months follow-up.The HPA database  was used to analyze the correlation between the PI3K/AKT/mTOR signaling pathway protein expression levels and the prognosis of patients with ccRCC (n = 445) after ≤150 months follow-up. The log-rank and univariate Cox P-values were calculated automatically. The relationship between the protein expression levels and the clinicopathological parameters, for instance, the pathological stage and Fuhrman grade of ccRCC, were also investigated.TCPA database . For GEPIA analysis, the mRNA expression levels of PI3K/AKT/mTOR signaling pathway members in ccRCC and normal tissues were analyzed using one-way ANOVA. For Oncomine and WB analyses, the PI3K/AKT/mTOR expression levels were analyzed using a two-tailed Student’s PTEN, PIK3CA, PIK3CB, PIK3CD, PIK3CG, AKT1, AKT2, AKT3 and mTOR) between ccRCC and normal kidney tissues. The mean mRNA expression levels of these genes were not significantly different between the 523 ccRCC and 100 normal tissues . Similar results were obtained between 523 ccRCC and 72 matched normal samples using GEPIA analysis , PIK3CA (P < 0.001), PIK3CB (P = 0.010), AKT1 (P < 0.001), AKT2 (P < 0.001) and AKT3 (P < 0.001), were negatively correlated with their stages  except PTEN , PIK3CA  and AKT1 .Then Oncomine database  was used to validate their differential expression , which dP = 0.774, P = 0.585, P = 0.480, respectively; Finally, further WB analysis displayed that the protein expression levels of PI3K, AKT and mTOR were not significantly changed between ccRCC and normal specimens (PTEN  and mTOR  were associated with improved OS in patients with ccRCC. For the multiple AKT genes, AKT3  was observed to be a favorable factor; however AKT1  and AKT2  were not. As for the PI3K genes, the majority were identified as protective factors, including PIK3CA , PIK3CB  and PIK3CG , whereas PIK3CD demonstrated the opposite effect . These results suggested that different members of AKT or PI3K may serve distinct roles in the prognosis of ccRCC.For survival analysis, the public online resource K-M plotter was used to calculate the clinical significance of individual genes compared with OS in 530 patients with ccRCC ; Table 1PTEN, PIK3CA and PIK3CB were positively correlated with a favorable OS in stage I, III and IV patients with ccRCC . Furthermore, increased PIK3CG expression level was correlated with an improved OS in stage I, II and III patients with ccRCC . Similar results were achieved for AKT3 and mTOR; AKT3 was found to be positively correlated with a favorable prognosis in stage III and IV patients with ccRCC  and the overexpression of mTOR was correlated with an improved OS in stage I and II patients with ccRCC . AKT1 was discovered to be a favorable factor in stage I [HR 95% CI = 0.44 (0.24–0.80)], whereas it found to be a poor factor in stage II and III patients with ccRCC . PIK3CD mRNA expression levels were associated with a favorable OS in stage III [HR 95% CI = 0.55 (0.31–0.96)], but a worse OS in stage IV [HR 95% CI = 2.42 (1.25–4.68)] patients with ccRCC. AKT2 mRNA expression was not correlated with the tumor stages of patients with ccRCC (P > 0.05). These results suggested that the prognostic role of PI3K/AKT/mTOR signaling pathway members may be markedly influenced by TNM stages.In addition to investigating OS, cancer staging, sex and grading were further investigated to determine the correlation between mRNA expression levels and OS in ccRCC patients. According to the TNM staging system , the ccRPTEN, PIK3CA, PIK3CB, PIK3CG and AKT3 were positively correlated with a favorable OS in both females  and males . mTOR was positively correlated with an improved OS in female patients with ccRCC [HR 95% CI = 0.27 (0.14–0.52)], whereas AKT2 was positively correlated with poor OS in males [HR 95% CI = 1.71 (1.15–2.55)]. Moreover, PIK3CD was positively correlated with a favorable OS in females [HR 95% CI = 0.57 (0.34–0.97)], but poor OS in male patients with ccRCC [HR 95% CI = 1.81 (1.22–2.68)]. These results suggested that sex may affect the prognostic role of PI3K/AKT/mTOR signaling pathway members in patients with ccRCC.Regarding the association of these genes with the sex of patients, the prognostic values of individual genes were further investigated in patients with ccRCC. As illustrated in Similarly, the prognostic value of the genes in different grades of ccRCC was also evaluated. As illustrated in PTEN (P < 0.001), PIK3CA (P < 0.001), PIK3CB (P < 0.001), PIK3CG (P = 0.004), AKT3 (P < 0.001) and mTOR (P = 0.001) were correlated with a significantly improved OS in 528 patients with ccRCC, whereas the overexpression of PIK3CD mRNA (P = 0.048) was significantly correlated with poor OS. AKT1 mRNA expression levels demonstrated no significant correlation with the prognosis of patients with ccRCC (P = 0.082), with the K-M plotter results providing similar findings. In addition, HPA analysis identified AKT2 as a hazard factor (P = 0.004), but it was not correlated with patients’ prognosis (P = 0.055), which was assessed using K-M plotter analysis.For HPA validation analysis, the prognostic significance of the nine genes was examined, which found that the majority of genes demonstrated identical prognosis (OS) following the same follow-up period in another 528 patients with ccRCC . SpecifiPTEN , p-AKT  and p-mTOR  were associated with an improved OS in patients with ccRCC, which is consistent with previous findings , they were identified as favorable prognostic factors by univariate Cox Proportional Hazards model analysis . However, PIK3CA and p-AKT (T308) expression levels were not correlated with tumor prognosis in patients with ccRCC (P > 0.05).Since the activity of the PI3K/AKT/mTOR signaling cascade is largely regulated through phosphorylation events, the prognostic roles of core executors of the PI3K/AKT/mTOR signaling pathway, with or without phosphorylation, were investigated in patients with ccRCC. For this purpose, 445 cases of ccRCC from TCPA database were used for the statistical analysis ; Table 4findings . AlthougPTEN, AKT and p-AKT (S473) were positively correlated with both tumor stages  and tumor grades  in patients with ccRCC, which is consistent with previous studies  expression and tumor stages or grades of patients with ccRCC (P > 0.05).As tumor staging and grading could markedly influence the statistical results of prognosis, correlation analysis between protein expression levels and ccRCC stages or grades were further performed . The exp studies . HoweverP > 0.05), suggesting that the activation of the PI3K/AKT/mTOR signaling pathway in patients with ccRCC may not be dependent on their transcriptional regulation. Although WB displayed an upward trend of AKT protein expression in ccRCC, more samples were needed to clarify this issue. When striatified by clinical stage of the 523 ccRCC cases using GEPIA, the mRNA expression levels of six genes, i.e., PTEN, PIK3CA, PIK3CB, AKT1, AKT2 and AKT3, were negatively correlated with their stages (P < 0.05). Further prognostic analysis using the K-M plotter and HPA demonstrated that the mRNA expression levels of the major PI3K/AKT/mTOR signaling pathway members  were positively correlated with a favorable OS in patients with ccRCC, whereas PIK3CD mRNA expression levels were positively correlated with a poor OS (P < 0.05). Accordingly, TCPA analysis demonstrated that the protein expression levels of PTEN, p-AKT (S473) and p-mTOR (S2448) were correlated with an improved OS (P < 0.05). The relationship between their mRNA or protein expression levels and several clinicopathological features, such as sex, pathological stage and Fuhrman grade, were also analyzed. The results demonstrated that the majority of the stratified prognosis was consistent with the overall prognosis, which confirmed the validity of our conclusion. In general, the prognosis between the mRNA and protein expression levels of members of the PI3K/AKT/mTOR signaling pathway was consistent, with the majority being recognized as favorable prognostic factors in patients with ccRCC. The mRNA expression levels of the six genes, i.e., PTEN, PIK3CA, PIK3CB, AKT1, AKT2 and AKT3, were negatively correlated with their stages, which was also consistent with their favorable prognosis in ccRCC. This suggested that the function of PI3K/AKT/mTOR in ccRCC was not only dependent on the mRNA and protein expression levels, but also reliant on protein activation through phosphorylation, such as p-AKT and p-mTOR. What’s more, it also indicated that the activation of PI3K/AKT/mTOR was a complicated process, and further molecular biological, pathological and biochemical experiments were needed to support the prognosis of PI3K/AKT/mTOR pathway members in ccRCC. To the best of our knowledge, this was the first report studying the prognostic role of mRNA and protein expression levels of multiple PI3K/AKT/mTOR pathway members, such as PTEN, AKT1 and mTOR, in ccRCC simultaneously. Our findings indicated that they may be potential prognostic markers for ccRCC, in addition to suggesting that PI3K/AKT/mTOR pathway signaling abnormalities may be involved in ccRCC tumorigenesis and progression.ccRCC is the most prevalent kidney cancer; however, its molecular mechanism remains to be fully elucidated . The PI3PTEN. PTEN has been identified as one of the most commonly lost or mutated tumor suppressor genes in human cancers  marker, whereas PTEN was not an independent prognostic marker of RCC. However, two meta-analyses indicated that PTEN was a favorable prognostic factor for patients with ccRCC  were associated with favorable disease-specific survival (DSS) in 375 patients with RCC; and p-AKT (S473) expression levels were correlated with a poorer OS for 440 patients with RCC. In addition, mTOR and p-mTOR (S2448) in 10 ccRCC and normal kidney tissues, and found that p-mTOR (S2448), but not mTOR expression levels, were increased in ccRCC. A subsequent study by the same authors comprehensively investigated the differential expression levels of mTOR and p-mTOR in 342 primary and 90 metastatic ccRCC tissues, and revealed that high p-mTOR expression levels, but not mTOR levels, were associated with impaired OS  and several proteins [such as PTEN and p-AKT (S473) ] were favorable prognostic factors for ccRCC, which indicated that the PI3K/AKT/mTOR signaling pathway may be implicated in ccRCC progression, which may also provide a potential therapeutic target for patients with ccRCC. These findings are contradictory to the common knowledge that the PI3K/AKT/mTOR signaling pathway promotes the initiation and progression of RCC. However, mTOR signaling is necessary for multiple biological effects, including energy metabolism homeostasis, autophagy, cell survival and apoptosis. Thus, the prognostic roles of PI3K/AKT/mTOR signaling pathway members in patients with RCC may be different under specific physiological or pathological conditions, although this hypothesis requires to be further investigated.Integrated bioinformatics analysis confirmed that the PI3K/AKT/mTOR pathway was activated in ccRCC, but the prognostic significance was not fully elucidated . ThroughPTEN, PIK3CA, PIK3CB, PIK3CD, PIK3CG, AKT1, AKT2, AKT2 and mTOR, in ccRCC and normal tissues using GEPIA and Oncomine analyses, and detected PI3K, AKT and mTOR protein expression using WB analysis. The prognostic significance of these genes and proteins in ccRCC patients was evaluated using K-M plotter, HPA and TCPA analysis, which suggested that several members of the PI3K/AKT/mTOR signaling pathway, especially PTEN, may be favorable prognostic factors in ccRCC. To the best of our knowledge, our study was the first to reveal the detailed OS of nine PI3K/AKT/mTOR signaling pathway members simultaneously. These results further suggested that the PI3K/AKT/mTOR signaling pathway may be implicated in ccRCC initiation and progression, and that components could be used as potential therapeutic targets in ccRCC.The present study investigated the mRNA expression profiles of multiple PI3K/AKT/mTOR signaling pathway components, including 10.7717/peerj.9261/supp-1Table S1Click here for additional data file.10.7717/peerj.9261/supp-2Table S2Click here for additional data file.10.7717/peerj.9261/supp-3Table S3Click here for additional data file.10.7717/peerj.9261/supp-4Dataset S1Click here for additional data file.10.7717/peerj.9261/supp-5Dataset S2Click here for additional data file.10.7717/peerj.9261/supp-6Dataset S3Click here for additional data file.10.7717/peerj.9261/supp-7Dataset S4Click here for additional data file.


Sustainable and green synthesis of nanocomposites for degradation of pharmaceuticals was developed via immobilization and stabilization of the biological strong oxidizing agents, peroxidase enzymes, on a solid support. Sol–gel encapsulated enzyme composites were characterized using electron microscopy , atomic force microscopy, FTIR spectroscopy, and thermogravimetric analysis. Horseradish peroxidase (HRP) and lignin peroxidase (LiP) were adsorbed onto magnetite nanoparticles and sol–gel encapsulated in a surface silica layer. Encapsulation enhanced the stability of the biocatalysts over time and thermal stability. The biocatalysts showed appreciable selectivity in oxidation of the organic drinking water pollutants diclofenac, carbamazepine, and paracetamol with improved activity being pharmaceutical specific for each enzyme. In particular, sol–gel encapsulated LiP- and HRP-based nanocomposites were active over 20 consecutive cycles for 20 days at 55 °C (24 h/cycle). The stability of the sol–gel encapsulated catalysts in acidic medium was also improved compared to native enzymes. Carbamazepine and diclofenac were degraded to 68% and 64% by sol–gel LiP composites respectively at pH 5 under elevated temperature. Total destruction of carbamazepine and diclofenac was achieved at pH 3 (55 °C) within 3 days, in the case of both immobilized HRP and LiP. Using NMR spectroscopy, characterization of the drug decomposition products, and decomposition pathways by the peroxidase enzymes suggested. Apart from the widespread problem of heavy metal pollution aggravated by warmer weather and increased acidity , there itzerland , Spain [tzerland , Sweden tzerland , and in tzerland . Even ottzerland .What is surprising, efficiency of conventional WWTPs against micropollutants was close to 0%, but with high variability for individual compounds . Better 3, H2O2 and UV [Besides adsorbents, other technologies for pharmaceutical removal, for example by ozone and UV, are already available for public. For instance, pilot-scale tests were run with ozonation for reduction of 24 pharmaceuticals at 10 full-scale wastewater treatment plants in southern Sweden with reasonable level reduction of pharmaceuticals reaching on average 65–88% . UV-trea2 and UV . Neverth2 and UV .In this respect, due to its high selectivity and performance, enzymatic water treatment was proposed as an attractive alternative solution ,17,18. Phanerochaete chrysosporium was studied for in vitro degradation of CBZ and diclofenac. LiP was found to completely degrade diclofenac at pH 3.0–4.5 with 3–24 ppm H2O2, while the degradation efficiency of CBZ was mostly below 10% [For example, crude lignin peroxidase (LiP), from the white rot fungus elow 10% . In anotelow 10% ,21. Horseradish peroxidase has also been evaluated for removal of drugs such as diclofenac  4, Sigma-Aldrich Sweden AB, cat. no. 8.00658 98%, Stockholm, Sweden); iron (II) chloride tetrahydrate ; iron (III) chloride hexahydrate ; sodium acetate tri-hydrate ; glacial acetic acid ; hydrogen peroxide ; acetaminophen ; diclofenac sodium salt ; ethanol (96%); 0.1 M HCl; 0.1 M NaOH; 0.06 M phosphate buffer (pH 7.0); horseradish peroxidase, ; lignin peroxidase ; ABTS , Sigma-Aldrich Sweden AB, Stockholm, Sweden). The following reagents were used in the applied synthetic procedures: tetraethoxysilane  and 48 g of Ferric chloride (FeCl3) were dissolved in 1l of deionized water. This solution was added dropwise to 250 mL of ammonia solution . Black precipitate was collected and washed several times by distilled water to pH = 7. The obtained Fe3O4 particles were spherical with an average diameter of about 14 ± 3 nm, and a specific surface area of about 96 m2/g. Magnetite was prepared by co-precipitation of iron (II) and iron (III) chlorides with ammonia solution under a nitrogen atmosphere . For tha3O4 was suspended in 50 mL of water/ethanol  mixture and then 5 mg of the enzyme dissolved in 5 mL of phosphate buffer was added to the magnetite suspension. The enzyme was left to absorb for 1.5 h, then 0.2 mL of 1% NH4F in water was added. Finally, 4 mL TEOS in 5 mL EtOH was added over 30 min. After 5 h, the solution became viscous and transformed into a gel. The mature gel was washed by EtOH and water. The composites were freeze-dried overnight. 200 mg Fe−1. Triplicate measurements were made for each assay of enzyme activity. ABTS was prepared as a 0.2 mM solution in acetate buffer at pH 5.0. With each cycle of oxidation, 3 µL of 3.6% H2O2 and 0.997 mL of ABTS was added to interact for 24 h with the composite powder at defined temperature. Composites were separated by magnet within 1 min. After separation, the ABTS solutions were filtered through 0.2 µm cellulose membranes in order to separate composite particles. ABTS concentration was measured at 420 nm in a quartz cuvette with a light path length of 1 cm.One unit (U) of enzyme activity was defined as the amount of enzyme required to oxidize 1 µmol of ABTS per minute with free enzyme activities expressed in ULAll drugs solutions were prepared in water or acetate buffer with corresponding pH. Initial concentrations were 17.6 µg/mL for CBZ, 100 µg/mL for paracetamol and 50 µg/mL for diclofenac.2O2 (3.6%) + 1 mL ABTS in sodium acetate buffer) after reacting for 10 min. Then, 1 mL of crude enzyme solutions in phosphate buffer, (HRP and LiP) with C = 0.06 mg/mL (corresponding to the mass of enzyme in composite), were mixed with 1 mL of 0.01 mg/mL of paracetamol solution, or diclofenac solution or with 1 mL of 17 mkg/mL of carbamazepine solutions, following with 3 µL of H2O2 (3.6%) addition. Solutions were reacting for 5 days, then filtered through 0.2 µm cellulose membrane filters.Initial enzyme activity was measured after by ABTS test  mixture and then 5 mg of the enzyme dissolved in 5 mL of phosphate buffer was added to the magnetite suspension; the enzyme was left to absorb for 1.5 h) under vigorous shaking with following 3 µL of H2O2 (3.6%) addition.Experiments were done with and without enzymes; experiments were multiplicated and ran in a few cycles. 1 mL of drug solution in corresponding buffer was added to 30 mg of magnetite-enzyme sample (200 mg Fe2O2 (3.6%). Samples were thermostated at a certain temperature and time. 1 mL of drug solution in corresponding buffer was added to 30 mg of sol–gel sample under vigorous shaking, followed by the addition of 3 µL of H30 mg of sample has been shaking at 55 °C for 24 h in deionized water. After composite decantation by magnet, solutions were filtered through 200 µm membrane filter and measured by UV–Vis at λ = 280 nm. Then, ABTS was added and solution was kept for 24 h at 55 °C to react. Activity was measured at λ = 420 nm.2O. 600 MHz Bruker Avance Cryoprobe instrument was used.500 µL of a sample solution after removal of the nanocomposite was filtered through 0.2 µm cellulose membranes and mixed with 55 µL of DConcentrations of drugs were measured using UV–Vis spectroscopy at λ = 243 nm for the paracetamol, λ = 273 nm for the diclofenac, and at λ = 280 for CBZ. All solutions were filtered through 0.2 µm cellulose membrane filters in order to separate composite particles. Perkin Elmer Lambda 2 instrument was used.Micrographs of samples were obtained on an Hitachi TM-1000 environmental scanning electron microscope. Samples were mounted on specimen stubs coated by carbon tabs and viewed at 15 kV. −1 to 4000 cm−1, in transmittance mode. All spectra were smoothed and their baselines corrected.FTIR spectra of all samples were recorded as KBr pellets on a Perkin-Elmer Spectrum 100 instrument. A total of eight scans were carried out on wavenumbers from 400 cmSurface structures of samples were studied using a Bruker FastScan Bio Atomic Force Microscope (AFM) in ScanAsyst mode. Wet samples were air-dried for 2 days prior to investigation. For native enzymes, samples were prepared by depositing 2 µL of 0.06 mg/mL solution on a mica plate. Composite samples were prepared by deposition of 2 µL of 0.001 mg/mL suspension on mica plate.TEM observations were made using a Philips CM12 instrument  operated at 100 kV.−1, using a Perkin-Elmer Pyris 1 instrument. Thermogravimetric analysis was carried out in air with a heating rate of 10 °C minDSC was performed with Mettler-Toledo DSC 820instrument. The method consisted in heating of the sample in 40 µL Al crucible at a rate 8 °C/min and airflow rate 50 mL/min up to 600 °C.Statistical analysis was performed using Origin 2017 software. Error bars in the graphs were expressed as mean square deviation of the data. Trametes Versicolor laccase revealed higher enzyme performance when its immobilized on positive surfaces [Some materials, due to their surface charge and adsorbed counterions, provide special ‘microenvironment’ for enzymes, thus increasing their activity. For instance, modeling for surfaces . Surfacesurfaces .3O4) surface in a first step, followed then by formation of a SiO2 shell using a sol–gel method. Magnetite was used as an active surface for freezing enzyme conformation, whereas silica shell formation was used to prevent enzyme leakage by desorption, with a potential to provide increased pH and temperature resistance for the developed composites. In the present work, we applied a combined approach, consisting of adsorption of the enzyme on magnetite  appears at 800 cm−1 and 1000–1230 cm−1 and can be attributed to Si-O-Si vibrations in silica. Absorption band at 1630 cm−1 belongs to water molecule vibrations together with the broad absorption band 3000 cm−1 to 3600 cm−1.From the literature, it was reported that above pH 7.5 and below 4.0, LiP enzymes lose all activity in less than 5 h . The natActivity was evaluated using a model ABTS oxidation test. Each cycle was conducted for 24 h at 55 °C. As observed in Enzyme leakage tests, were performed using both UV–Vis at λ = 280 nm to detect enzyme, and to evaluate possible enzymatic activity of leachate . Apparently, enzyme is gradually releasing from the composite . That isAfter testing ability of the composites in oxidation of model compounds, they were used for oxidation of several common water drug pollutants. It worth to notice that in our studies, at pH 7, at room and elevated temperatures (55 °C), the activity against selected drugs was negligible for both LiP and HRP composites. 2, 3, 4, Diclofenac is well known for its recalcitrance and difficulty for biodegradation ,44,45 inJust adsorbed enzymes were evaluated in diclofenac and paracetamol degradation tests. Non encapsulated composites were active only in one cycle, after that enzyme was deactivated and washed out from the surface. Data presented on 1H NMR were: 7.24 (d), 7.09 (t), 6.93 (t), 6.43 (d), 3.77 (s). The NMR spectra of initial diclofenac and diclofenac after interaction with native HRP and LiP enzymes are shown in NMR spectra for diclofenac and its metabolites after interaction with sol–gel encapsulated HRP and LiP enzyme composites at pH 5 are shown in From NMR spectra we can conclude that diclofenac removal by sol–gel encapsulated HRP and LiP enzymes at pH 3 lead to total degradation (see NMR on The summarized pathways during enzymatic degradation of diclofenac is given on scheme below. 1H NMR are the following: 9.5 (s), 6.9 (d), 7.2 (d), 2.1 (s). As can be observed form NMR spectra of paracetamol after interaction with native enzymes, aromatic protons, inherent initial paracetamol molecules, disappear  transformation by laccase is given in [b) and 10,11-dihydro-10,11-epoxy-CBZ (EP-CBZ) (compound c). At the same time, major metabolites of CBZ formed by myeloperoxidase [d and e). Mechanisms for aromatic compound hydroxylation by enzymes are thoroughly discussed in [The products after CBZ interaction with native HRP- and LiP enzymes are presented in given in . The tworoxidase  were 2- ussed in .Apparently, in case of native HRP and LiP enzymes interaction with CBZ, aromatic ring cleavage occurs, which reflects in disappearance of aromatic protons signal around 7.5 ppm.1H NMR were: 7.4–7.27 (m), 6.90 (s), 4.95 (s).In case of carbamazepine, only a decrease in concentration, but no metabolites were observed during decomposition by sol–gel encapsulated enzymes at pH 5 see . ChemicaAt pH 3, total destruction of CBZ was observed. Possible products for CBZ during enzymatic degradation are not certain. Since the aromatic protons in NMR spectra of CBZ products disappear a–c, it iThe reason for increased enzymatic activity may be explained by literature data. For example, it was suggested  that theResults of decomposition for selected drugs by LiP and HRP composites under different conditions are summarized in To summarize, a decrease in pH appreciably increased the enzymes activities. Changing pH to 5.0 increased the activity of the composites allowing them to decompose more than 50% of diclofenac a and CBZAn explanation for poor paracetamol degradation may lie in a different mechanism of oxidation. In contrast to laccases, peroxidases are heme-containing enzymes, which utilize hydrogen peroxide when oxidized releasing two electrons. Then the peroxidase is reduced via a two-step one-electron oxidation process. Lignin peroxidase  oxidizes phenolic compounds and reduces molecular oxygen to water, generating intermediate radicals. These radicals are different from those generated by laccase, resulting in different pollutant degradation mechanisms for the two enzymes. Transformation of polyphenols, such as lignin, to low-molecular compounds is the most frequent reaction of LiP in nature . For exaLower oxidation efficiencies for immobilized enzymes can be attributed to an increased mass transfer requirement and limited accessibility of active sites after enzyme immobilization ,56. At pPeroxidase enzymes HRP and LiP, efficiently immobilized on iron oxide nanoparticles by adsorption from solution, maintain their active conformation. Adsorption is especially visible in quantitative measurements of adhesion by AFM. The advantage of the proposed approach was in potential preservation of advantageous configuration of enzyme molecules when adsorbed on magnetite nanoparticles, which was confirmed by further tests. Further sol–gel coating by silica results in permanent immobilization with resistance to ‘washing off’ and enhanced stability in time and against elevated temperatures and acidity. Synthesized composites were active in diclofenac, carbamazepine, and paracetamol removal. Immobilized LiP was more active in diclofenac removal than HRP and even laccase, as was anticipated based on earlier studies. The silica-coated enzyme composites revealed enhanced stability at elevated temperatures. Nevertheless, the silica encapsulated HRP- and LiP enzyme composites showed limited ability for paracetamol degradation. In addition, cation-radicals apparently formed by peroxidase enzymes, were more effective for CBZ and diclofenac removal than in case of paracetamol.


In part A (n = 8), patients received a single 1-h intravenous infusion of [14C]-pevonedistat 25 mg/m2. In part B (n = 7), patients received pevonedistat 25 or 20 mg/m2 on days 1, 3, and 5 in combination with, respectively, docetaxel 75 mg/m2 or carboplatin AUC5 plus paclitaxel 175 mg/m2 on day 1 every 3 weeks. Following the single dose of [14C]-pevonedistat 25 mg/m2 in part A, there was a parallel log-linear decline in plasma and whole blood pevonedistat concentration, with systemic exposure of unchanged pevonedistat representing 41% of drug-related material . The mean terminal half-life of pevonedistat and drug-related material in plasma was 8.4 and 15.6 h, respectively. Pevonedistat distributed preferentially in whole blood with a mean whole-blood-to-plasma ratio for pevonedistat AUC∞ of 40.8. By 1 week post dose, the mean recovery of administered radioactivity was 94% (41% in urine and 53% in feces). The pevonedistat safety profile during both study parts was consistent with previous clinical experience, with no new safety signals observed. In part B, pevonedistat in combination with docetaxel or carboplatin plus paclitaxel was generally well tolerated. ClinicalTrials.gov identifier: NCT03057366.Pevonedistat (TAK-924/MLN4924) is an investigational small-molecule inhibitor of the NEDD8-activating enzyme that has demonstrated preclinical and clinical activity across solid tumors and hematological malignancies. Here we report the results of a phase I trial characterizing the mass balance, pharmacokinetics, and clearance pathways of [The online version of this article (10.1007/s10637-020-01017-x) contains supplementary material, which is available to authorized users. Pevonedistat TAK-924/MLN4924) is an investigational small-molecule inhibitor of the neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) , 2. NAE 4/MLN4924Pevonedistat has demonstrated potent antitumor activity in preclinical studies, demonstrating growth inhibition across a variety of tumor cell lines including solid tumors  and hematological malignancies  , 8, 10. 2 in combination with docetaxel and 20 mg/m2 in combination with carboplatin plus paclitaxel. Pevonedistat was well tolerated in both combinations, with the most common treatment-emergent adverse events (TEAEs) including fatigue, nausea, constipation, diarrhea, vomiting, and anemia. Sustained clinical responses were observed in patients receiving pevonedistat in combination with carboplatin plus paclitaxel , and the ORR in combination with docetaxel was 16%.Numerous phase I/Ib studies have examined the activity of pevonedistat alone or in combination with other agents in multiple tumor types, including advanced solid tumors, melanoma, AML, myelodysplastic syndromes, multiple myeloma, and lymphoma , 11–16. 2). Pevonedistat population PK was assessed using data from six studies of pevonedistat alone or in combination with current standard-of-care agents -pevonedistat in patients with advanced solid tumors. The study comprised two parts: part A assessed mass balance, PK, metabolite profiles, safety, and tolerability in patients who received a single IV infusion of [14C]-pevonedistat, while in part B, safety, tolerability, and disease response were assessed in patients who received treatment with pevonedistat in combination with either docetaxel or carboplatin plus paclitaxel.In vitro metabolism studies have suggested a major role for CYP3A in pevonedistat elimination pathways; however, strong and moderate CYP3A inhibitors did not result in clinically meaningful increases in pevonedistat exposure . Here we14C]-pevonedistat 25 mg/m2  at a single center . Patients were required to stay at the part A study site for approximately 8 days or until the discharge criteria were met, with a maximum estimated confinement period of 9–14 days. Upon completion of part A, patients had the option to participate in part B at Medical Center Hungarian Defense Forces, Budapest, Hungary. In part B, patients received treatment in 3-week cycles with either: pevonedistat 20 mg/m2 on days 1, 3, and 5 plus carboplatin AUC5 and paclitaxel 175 mg/m2 on day 1; or pevonedistat 25 mg/m2 on days 1, 3, and 5 plus docetaxel 75 mg/m2 on day 1. Treatment was administered for 12 cycles or until discontinuation criteria were met.This was a two-part (A and B), open-label, multicenter, mass balance, distribution, metabolism, excretion (DME) study in adult patients with advanced solid tumors. In part A, patients received a single IV infusion of [14C]-pevonedistat 25 mg/m2 (part A).The primary objectives of the study were to assess the mass balance  and to characterize the PK of pevonedistat  and total radioactivity  following a single infusion of -pevonedistat 25 mg/m2 without modification or interruption. The actual administered dose ranged from 40 mg to 50 mg based on patient BSA. In part B, patients received a median of 5.0 treatment cycles of pevonedistat/carboplatin/paclitaxel and 3.5 cycles of pevonedistat/docetaxel. All patients received at least 2 cycles of treatment, and 2 patients in the pevonedistat/carboplatin/paclitaxel arm received 11 cycles.All patients in part A received the planned dose of [14C]-pevonedistat 25 mg/m2, the mean t1/2 of pevonedistat and drug-related material  in plasma was 8.4 and 15.6 h, respectively, and the mean t1/2 of pevonedistat and drug-related material in whole blood was 13.9 and 36.6 h, respectively. Figure 14C]-pevonedistat. The longer t1/2 of drug-related material compared with pevonedistat that is observed in both plasma  ratio of pevonedistat versus total drug-related material in plasma and whole blood was 0.41 and 0.44, respectively, indicating that > 50% of the total radioactivity in circulation was accounted for by pevonedistat metabolites  administered radioactivity was recovered in excreta (combined urine and feces), with 41% (± 7.9%) and 53% (± 9.5%) of administered radioactivity recovered in urine and feces, respectively, by 1 week post dose. The majority of the radioactivity was recovered by 96 h post dose.The geometric mean pevonedistat plasma CL was 32.2 L/h and geometric mean CL/h Table . Figure 14C]-pevonedistat (part A) and after multiple doses of pevonedistat plus chemotherapy (part B) is presented in Table An overall summary of the safety profile following a single dose of 1C-pevonedIn part A, 4 patients (50%) experienced at least one TEAE (Table 4), among whom 1 patient (12.5%) had a TEAE that was determined by the investigator to be drug-related (Grade 1 dizziness). There were no serious TEAEs (SAEs). One patient with sarcoma died during the study after completing part A; the patient experienced an SAE of Grade 4 sarcoma , which resulted in death 1 day after onset and 25 days after the part A pevonedistat dose.All 7 patients in part B experienced at least one TEAE; the most common were vomiting, which occurred in 4 patients (57.1%) overall, and nausea, diarrhea and asthenia in 3 patients (42.9%) overall (Table 4). At least one drug-related TEAE was experienced by 6 patients (85.7%) overall . The most common drug-related TEAE was vomiting, which occurred in 3 patients (42.9%) overall (2 patients in the pevonedistat/carboplatin/paclitaxel arm and 1 patient in the pevonedistat/docetaxel arm), and 2 patients overall experienced increased alanine aminotransferase (ALT), increased aspartate aminotransferase (AST), nausea , and asthenia (1 patient in each arm). Four patients (57.1%) in part B had Grade ≥ 3 TEAEs, which were considered to be drug-related in 2 patients . One patient in the pevonedistat/carboplatin/paclitaxel arm had a TEAE of Grade 3 pneumonia resulting in study drug discontinuation. Three patients (42.9%) had at least one SAE, none of which were considered related to treatment; 2 were receiving pevonedistat/carboplatin/paclitaxel and 1 was being treated with pevonedistat/docetaxel. Two on-study deaths occurred in part B, neither of which were attributed to any of the study drugs. The deaths were due to general physical health deterioration in a patient receiving pevonedistat/carboplatin/paclitaxel, and disease progression 9 days after the last dose in a patient receiving pevonedistat/docetaxel.n = 6) or an SAE .In part B, all 7 patients were evaluable for response. One patient achieved a best response of partial response (per RECIST version 1.1), and 5 patients achieved a best response of stable disease. The remaining patient experienced progressive disease. All 7 patients subsequently discontinued treatment due to progressive disease  and extrinsic  factors on PK is a crucial component of the clinical pharmacology characterization of anticancer agents to inform appropriate dosing and administration across patient populations and clinical contexts of use , 19. Radgnancies . In patignancies . In this∞ was 40.8, suggesting a preferential distribution of pevonedistat in whole blood. The preferential distribution to red blood cells is considered to be due to binding to carbonic anhydrase II (CAII) (Takeda data on file), an intracellular enzyme abundant in human erythrocytes -pevonedistat 25 mg/m2 in cancer patients confirm a predominant role for metabolic clearance and a minor role of renal clearance in the disposition of this agent. Recovery of radioactivity was nearly complete (94%) in 1 week following single-dose IV administration. PK data support low systemic clearance, presence of circulating metabolite(s), and extensive whole blood partitioning of pevonedistat and total drug-related material. Further analyses of metabolite profiles in plasma and excreta collected in this study will provide greater molecular resolution of the biotransformation pathways of pevonedistat in humans. These findings, taken together, provide important information to enable rational assessment of the impact of intrinsic and extrinsic factors on pevonedistat exposure as part of the ongoing clinical pharmacology evaluations in the development of this investigational anticancer agent.In conclusion, the results of this radiolabeled mass balance study of the NAE inhibitor pevonedistat following a single 1-h infusion of [ESM 1(PDF 574 kb)


Streptomyces sp. strain ventii, an environmental isolate recovered from deep-sea hydrothermal vents in the Pacific Ocean, is presented along with the resequenced draft genomes of the type strains Streptomyces bohaiensis 11A07 and Streptomyces lonarensis NCL 716.The draft genome of  Streptomyces sp. strain ventii, an environmental isolate recovered from deep-sea hydrothermal vents in the Pacific Ocean, is presented along with the resequenced draft genomes of the type strains Streptomyces bohaiensis 11A07 and Streptomyces lonarensis NCL 716.The draft genome of  Streptomyces are Gram-positive, spore-forming, filamentous bacteria that often synthesize desirable antimicrobials, cytotoxins, and other lead compounds . The sample was diluted 1:1,000 in sterile Instant Ocean, spread onto 1/10 Zobell marine agar 2216 with sterile swabs, and incubated at 28°C for 2 weeks. Strain maintenance was performed on International Streptomyces Project 2 (ISP2) medium supplemented with 0.1 M sodium phosphate buffer to a pH of 8.0 (buffered ISP2) at 28°C ; adapter sequence removal, read quality trimming , and analysis were performed using BBDuk in the BBMap package (http://sourceforge.net/projects/bbmap/), and genomes were assembled with SPAdes v. 3.14.0 using the “–careful” option and specifying kmers of 21, 33, 55, 77, 99, and 121  Vents Program at Oregon State University and the NOAA Pacific Marine Environmental Laboratory. A microbial mat near hydrothermal vents on the Axial Seamount 46.06°N, 130°W) at a depth of 2,190 m was collected with a custom syringe-based sampler on the remotely operated vehicle (ROV) Jason II  and display higher mean coverage in fewer contigs with more of the genomes in scaffolds greater than 50 kb (The relevant genome characteristics are presented in an 50 kb . Removalan 50 kb .The whole-genome shotgun projects, BioSample material, and raw reads have been deposited in DDBJ/ENA/GenBank under the accession numbers listed in


Galleria mellonella, have recently been established as a non-mammalian infection model for the Mycobacterium tuberculosis complex (MTBC). To gain further insight into the potential of this model, we applied proteomic (label-free quantification) and transcriptomic (gene expression) approaches to characterise the innate immune response of G. mellonella to infection with Mycobacterium bovis BCG lux over a 168 h time course. Proteomic analysis of the haemolymph from infected larvae revealed distinct changes in the proteome at all time points . Reverse transcriptase quantitative PCR confirmed induction of five genes , which encoded proteins found to be differentially abundant from the proteomic analysis. However, the trend between gene expression and protein abundance were largely inconsistent (20%). Overall, the data are in agreement with previous phenotypic observations such as haemocyte internalization of mycobacterial bacilli (hemolin/β-actin), formation of granuloma-like structures (Hdd11), and melanization (phenoloxidase activating enzyme 3 and serpins). Furthermore, similarities in immune expression in G. mellonella, mouse, zebrafish and in vitro cell-line models of tuberculosis infection were also identified for the mechanism of phagocytosis (β-actin). Cecropins , which share the same α-helical motif as a highly potent peptide expressed in humans (h-CAP-18), were induced in G. mellonella in response to infection, giving insight into a potential starting point for novel antimycobacterial agents. We believe that these novel insights into the innate immune response further contribute to the validation of this cost-effective and ethically acceptable insect model to study members of the MTBC.The larvae of the insect  Mycobacterium tuberculosis (MTB) (Tuberculosis (TB) is the leading cause of global infectious disease mortality, with a quarter of the world’s population believed to be infected with the causative agent, is (MTB) . A largeis (MTB) . WHO is is (MTB) . In ordeGalleria mellonella (Greater wax moth) have become increasingly popular as an infection model to study bacterial  and funalbicans , Aspergiumigatus  and Maduetomatis ] pathogeentation . This inentation . Furtherntenance .G. mellonella lacks adaptive immunity, its innate immune system is complex and shares functional and anatomical similarities to those found in mammals , including MTB  with MTB  to study the immune mechanisms of mycobacterial control, proving to be a valuable organism in mycobacterial research  supplemented with 0.2% glycerol , 0.05% polysorbate-80  and 10% albumin dextrose catalase , 50 µg/ml of hygromycin , and incubated at 37°C in an orbital shaker. For enumeration of colony forming units (CFU), 10-fold serial dilutions of BCG lux were plated onto Middlebrook 7H11 agar  supplemented with 0.5% glycerol, 10% oleic acid albumin dextrose catalase , 50 µg/ml of hygromycin, and statically incubated at 37°C. The bioluminescence, relative light unit (RLU)/ml of BCG lux, was measured using a luminometer  and 1% decanal (Sigma-Aldrich) as the substrate. RLU was used as a relative and rapid estimation of CFU at a ratio of 3:1/4:1 in vitro and in vivo, respectively, as previously determined , weight (250 mg), motility and colour were picked for experimentation and acclimatised to room temperature before experimentation.Last instar lux was prepared and injection of healthy G. mellonella larvae were carried out as previously described  via the last left proleg of the larva. Injection is the preferred method over feeding, as dosing of BCG lux can be more accurately controlled. Infected larvae were transferred to a Petri dish lined with filter paper and incubated in a vented box at 37°C in the dark. N = 30 larvae were infected for each experimental group, unless otherwise stated, and each experiment consisted of three biological replicates (N = 90). The negative infection controls were naïve larvae, mock infected with phosphate-buffered saline (PBS), with tween . Survival of the infected larvae were recorded for up to 1-week post infection (pi). Larvae were considered dead when they failed to respond to touch. Where appropriate, a t-test was carried out to determine statistical significance.Mid-log phase BCG escribed . G. melllux in vivo, over a one week-time course was determined at 0, 24, 48, 72, 96, and 168 h pi. Five larvae were individually homogenized and the RLU of the homogenate was measured using a luminometer as previously described , correlating the data against the six-frame translation of the expressed sequence tags (EST) contigs for G. mellonella, with the addition of the proteomes of insect species Hyalophora cecropia, Manduca sexta, Bombyx mori (M. bovis BCG (via PRIDE partner repository with the dataset identifier PXD015250.Cell free haemolymph proteins (75 µg) were processed for proteomics using previously described and established protocols . Proteinbyx mori , and a povis BCG . The MS 2 transformed and one-way analysis of variance (ANOVA) and t-tests were carried out between the naïve reference and 4, 48, and 168 h BCG lux pi larval haemolymph samples. Significance was determined using p-value of 0.05 as a cut-off and Benjamini-Hochberg correction . LFQ intensities were logrrection  was applG. mellonella larvae infected with BCG lux, five larvae were individually homogenized using a Ribolyser in a 2 ml lysing tube containing 900 µl of TRIzol  and six 1/8-inch metal beads  at 4, 48 and 168 h pi. The lysate was processed to extract RNA using an RNeasy Mini Kit  following the manufacturer’s instructions. RNA of five larvae were pooled and cDNA was synthesised using the QuantiTect Reverse Transcription kit , using 1 µg gDNA treated RNA as recommended by the manufacturer’s protocol. Real-time quantitative PCR (RT-qPCR) was carried out using the QuantiFast SYBR® Green RT-PCR kit  on a StepOne Plus real-time PCR system  targeting genes using primers in gloverin, cecropin, hemolin, insect metalloproteinase inhibitor (IMPI) and ubiquitin genes were acquired from prior publications , which returned expected matches . Fold changes for each gene, relative to the naïve control at t = 0 h, are shown using the comparative CT (ΔΔCt) method using ubiquitin as the house keeping gene. Gene expression was determined using three independent experiments, and each sample was run in triplicate. The Mann-Whitney U test was used for statistical analyses.To quantify gene expression in ications . The prilux survival over a time course of 96 h  compared to those non-immunized  within G. mellonella was monitored over a 168 h time course, where bioluminescence of the larval homogenate was used as a rapid and validated method of quantifying changes in mycobacterial load in vivo  was monitored similarly; at this lower dose RLU continued to decline until 96 h, where a slight increase in RLU was observed at 168 h pi  infected larvae at 4, 48, and 168 h pi, and the proteomic profiles were compared to that of 0 h control (naïve larvae). A total of 2013 peptides were identified representing 185 proteins with two or more peptide matches, of which 104 proteins were found at all time points. A number of differentially abundant proteins , with a fold change of > ± 1.5 were found, i.e., 6 at 4 h pi, 54 at 48 h pi, and 86 at 168 h pi, and a total 11 proteins were deemed exclusive . These exclusive proteins were used in statistical analysis following imputation of the zero value as described in the methodology. Principal component analysis (PCA) of filtered proteins clearly distinguished the proteome of each time point, and clustered independent replicates of each time point together , hemolin (+2.2 fold) and arginine kinase (+1.6 fold) were found differentially more abundant in larval haemolymph compared to 0 h. Tubulin alpha chain (-2.4 fold), and heat shock protein 25.4 (-1.7 fold) were differentially less abundant in the larval haemolymph compared to 0 h , gloverin (+56.3 fold), hemolin (+55.7 fold), cecropin-A (+47.5 fold), putative defence protein Hdd11 (+18.3 fold), serpin-4B (+14 fold), prophenoloxidase activating enzyme 3 (+14 fold), insect metalloproteinase inhibitor (+11.5 fold), serpin-11 (+5.9 fold), transgelin (+4.6 fold), Hdd-1 like protein (+2.9 fold), serpin-3a (+2.3 fold). Proteins which decreased in relative abundance 48 h pi were GAPDH (-14.7 fold), serine proteinase (-9.4 fold), lysozyme-like protein 1 (-4.74 fold), scolexin (-2.1 fold), and 27 kDa haemolymph protein (-1.9 fold) , hemolin (+87.8 fold), cecropin-D-like peptide (+54.42 fold), chemosensory protein 7 (+42.2 fold), transgelin (+26.6 fold), prophenoloxidase activating enzyme 3 (+24.6 fold), serpin-4B (+20.9 fold), cecropin-A (+16.3 fold), serpin-11 (+15.1 fold), hdd1 (+10.8 fold), serpin-2 (+10.3 fold), gloverin (+7.61 fold), serpin-3a (+2.6 fold). Proteins which decreased in relative abundance 169 h pi were 27 kDa haemolymph protein (-9.6 fold), serine proteinase (-9.1 fold), GAPDH (-7.7 fold), scolexin (-1.9 fold), and apolipophorin (-1.7 fold) , and an uncharacterised protein containing an undecaprenyl diphosphate synthase domain were detected. However, no proteins with a known function associated with virulence, intracellular metabolism or survival, were detected at 168 h pi. A list of detected BCG lux proteins is available in LFQ proteomics analysis additionally identified 15 BCG G. mellonella larvae were challenged with high (1 x 107 CFU) and low (1x105 CFU) doses of BCG lux. Over a 168 h time course, the expression of five genes of the G. mellonella innate immune system were measured at 4, 48, and 168 h pi, via RT-qPCR using the ΔΔCt method, ubiquitin as the housekeeping gene, and naïve non-infected larvae as the reference. Expression of the house keeping gene ubiquitin and four others encoding proteins that were differentially expressed in the proteomic analysis, i.e., hemolin, IMPI, gloverin, and cecropin, were measured. Hemolin, and its encoded protein, was found to be differentially abundant at all time points, IMPI, and its encoded protein, was found in relatively consistent abundance at 48 and 168 h pi, as was Hdd11 and its encoded protein, which is of interest as it is involved in the formation of granuloma-like structures, which are a hallmark of TB infection . However, the level of gene expression was lower but of similar levels between 48 and 168 h pi for both genes , while cecropin mirrored high dose infection but at significantly lower levels of gene expression  at 4 h, 48 h, and 168 h pi using LFQ proteomics. Distinct changes in larval proteome in response to infection at all three time points were found, with the least changes in differentially abundant proteins being at 4 h pi.Previously we described, using survival curves, bacterial counts, and electron microscopy, a BCG  complex . The aim studies . Using tG. mellonella hemolin may detect lipoglycans  found in abundance on the mycobacterial cell wall , may reflect the cytoskeletal redistribution and remodelling which takes place during phagocytosis . In linelux. Cecropin A, cecropin-D-like protein, and gloverin were differentially more abundant than at 0 h at both 48 h and 168 h pi, but not at 4 h pi. Cecropins are amphipathic α-helical AMPs, reported to be effective against both Gram-positive and Gram-negative bacteria by permeating the bacterial cell wall (6 CFU of S. aureus 24 h pi (lux infection led to increased expression of cecropin-D-like peptide (+145.2 fold), gloverin (+56.3 fold), and cecropin-A (+47.5 fold) at 48 h pi. Although variance in the infectious dose and time point complicates comparisons, the higher abundance of gloverin in response to a lower infectious dose of S. aureus may indicate that gloverin is preferentially expressed in the presence of Gram-positive bacteria, or alternatively the higher abundance of cecropin-D-like peptide may indicate BCG lux is a more potent inducer. In mammals, cathelicidins, cationic α-helical AMPs, are released by leukocytes in response to infection (S. aureus) and negative (Pseudomonas aeruginosa and E. coli) bacteria through permeabilization and disruption of the cell membrane  and tissue inhibitors of MMPs (TIMPs) (G. mellonella (G. mellonella is yet to be fully annotated (Hdd11 (homologous to Noduler) is associated with the formation of nodules or granuloma-like structures; facilitated by crosslinking pathogens and haemocytes by Hdd11 . In linea larvae , where nd to 0 h . Althoug (TIMPs) . IMPI, f (TIMPs) . IMPI do (TIMPs) . MMPs anllonella . The channotated .S. aureus (2 x 106 CFU) induced more abundant levels of phenoloxidase activating enzyme 3 and serpin-4b by 6 h pi compared to the 0 h control , 48 h prior to infection with a lethal dose of BCG lux (2x107 CFU), resulted in improved survival outcome when compared to the non-immunized control. This observation, commonly referred to as immune priming, has widely been reported in invertebrate infection models  and low (1x105 CFU) doses, led to differential gene expression of innate immune genes, with higher dose infection inducing more substantial changes. Changes in gene expression were detected in gloverin, cecropin, IMPI, hemolin, and Hdd11 regardless of infectious dose when compared to the 0 h control. Induction of expression was highest at 4 h pi, and thereafter decreased over the time course despite the presence of a persistent infection. Similar observations were made in an RT-qPCR study of G. mellonella innate immune responses to infection with Riemerella anatipestifer, a Gram-negative bacterium, where genes involved in innate immunity , were highest at 4 h pi, and declined over time    reported beetle) . Studies beetle) , with co beetle) . Further beetle) . It is g beetle) . The preG. mellonella to mycobacterial infection using molecular transcriptomic and proteomic approaches. The proteomic profiles were unique over the course of infection. Responses were similar to those in G. mellonella infected with fungal granulomatous infectious disease. BCG lux proteins were detected, associated with intracellular survival and metabolism in vertebrate hosts, indicating that G. mellonella induces similar stress responses. RT-qPCR confirmed the proteomic analysis. However, gene expression and protein abundance was not always in agreement, and careful comparison of data should be made in future studies. Several targets for further studies, such hemolin as an opsonising agent and cecropin as an antimycobacterial agent, were identified and will be further investigated. While this study focused on the host response to infection, future studies will evaluate the mycobacterial response to humoral innate immunity of G. mellonella. The data presented here provide a vital insight into the G. mellonella – mycobacterial infection model and highlights its value for accelerating in TB research.In conclusion, to our knowledge, this is the first report of the innate immune response of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/MA, GS, YL, BR, KK, PL, and SN conceptualized the study. MA drafted the manuscript. MA, GS, and YL carried out the experimental procedures. MA and GS carried out experimental analysis. MA, GS, YL, BR, KK, PR, and SN reviewed, edited, and approved the manuscript for submission. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Biotechnology and Biological Sciences Research Centre (BBSRC) [BB/P001262/1] to PL and YL, and the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) [NC/R001596/1] to PL, SN, BR, and YL. GS is the recipient of a Maynooth University doctoral studentship. The Q-exactive mass spectrometer was funded under the Science Foundation Ireland (SFI) Research Infrastructure Call 2012, [12/RI/2346] (3). This publication emanated from research supported in part by a research grant from the SFI and is co-funded under the European Regional Development Fund [12/RC/2275_P2].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.