Relative Quantification Method Research Articles

Expression of NR3C1, INSR and SLC2A4 genes in skeletal muscles and CBG in liver depends on age and breed of pigs

The genes encoding glucose transporter 4 (SLC2A4, GLUT4), insulin receptor (INSR) and glucocorticoid receptor (NR3C1) are considered regulators of energy metabolism that may influence fat content in skeletal muscle at different ages and breeds of pigs. In the study we performed analysis of expression of NR3C1, INSR and SLC2A4 genes in two skeletal muscle tissues: semimembranosus and longissimus dorsi muscle in gilts from three breeds of pigs that differed in intramuscular fat content: Duroc (DU), Pulawska (PUL) and Polish Large White (PLW) at 60, 90, 120, 150, 180 and 210 days after weaning. We also analyzed the expression of cortisol binding globulin (CBG) in the liver. Expression was analyzed with real time PCR (qPCR) using a relative quantification method. In blood, the concentration of cortisol, insulin and leptin were evaluated with radioimmunoassay. The concentration of metabolites (triglycerides and glucose) related to the changes of lipids content was also measured. We observed a significant relationship between the breed and the age of pigs and the expression of analyzed genes in muscle tissues (P < 0.01 for all analyzed genes) as well as with two hormones (for insulin P = 0.001; for cortisol P < 0.0001). Leptin level and metabolites in serum were not significant in the model (P > 0.05). The expression of analyzed genes (SLC2A4, INSR and NR3C1 in muscles and CBG in liver) strongly correlates with intramuscular fat content in pigs and is associated with age and breed.

Analysis of Protein Synthesis in Cucumber Leaves after Inoculation with Corynespora cassiicola: A Proteomic Approach.

Cucumber target leaf spot (TLS) disease caused by Corynespora cassiicola has become one of the most important fungal foliar diseases of cultivated cucumbers. However, the defense mechanisms of cucumber plants (Cucumis sativus) against C. cassiicola are still poorly understood. Here, proteins from resistant cucumber plants were analyzed using iTRAQ (isobaric tags for relative and absolute quantification) method. A total of 286 differentially expressed proteins were identified (p < 0.05, ratio > 1.2 or < 0.83) 6 and 24 h after pathogen inoculation in the resistant cultivar Jinyou 38 (the data are available via ProteomeXchange; identifier, PXD012903). Some of the early responses to C. cassiicola infection were revealed, and four factors related to the resistance of cucumber plants to TLS were discovered. First, the proteomic approach revealed modulation of signaling pathways in resistant cucumber plants in response to C. cassiicola infection. Second, the plant immune system recognizes the pathogen and initiates expression of immune response proteins, including those related to plant defense, stress response, signal transduction, cell metabolism, and redox regulation. Third, C. cassiicola activates common stress response pathways; in particular, mildew resistance locus O (MLO) proteins were found to play a crucial role in the TLS prevention. Fourth, rapid activation of the carbohydrate and secondary metabolic pathways, modification and reinforcement of cell walls, and adjustment of the apoplastic environment to the highly stressful conditions were crucial in the cucumber resistance to TLS. Overall, our data contribute to the understanding of interactions between plants and their pathogens and provide new insight into molecular processes involved in the resistance of cucumber plants to disease.

High-sensitivity quantification of glycosphingolipid glycans by ESI-MS utilizing ozonolysis-based release and isotopic Girard's reagent labeling

Quantitative analysis of glycosphingolipids (GSLs) has been hindered by the lack of chromogenic groups for spectral detection or active functional groups for specific derivatization. In this study, a highly sensitive method based on ozonolysis-induced release and isotopic Girard's reagent P labeling of GSL glycans coupled with mass spectrometric detection for the quantification of animal tissue GSLs is developed. First, different approaches for the release of glycans from GSLs were compared with each other and the ozonolysis-based method was found to have the highest glycan yield under relative mild reaction conditions. Then a relative quantification method of ozonolysis-released GSL glycans based on stable isotope labeling using nondeuterated (d0-) and 2,3,4,5,6-pentadeuterated (d5-) Girard's reagent P (GP) was established, and its good linearity, accuracy and reproducibility were statistically verified. Finally, the new method was successfully applied to revealing the difference between porcine brain and liver as well as between the brain of normal and aging rats in GSL glycome by online hydrophilic interaction liquid chromatography coupling with ultraviolet detection and tandem mass spectrometry (HILIC-UV-MS/MS). This novel method is versatile and sensitive, enabling accurate quantitative analysis of tissue GSLs and showing great significance for glycomic studies.

The role of CXCR3 and its ligands CXCL10 and CXCL11 in the pathogenesis of celiac disease.

The chemokine receptor CXCR3 and its ligands CXCL10 and CXCL11 have been suggested to give rise to the most relevant chemokine axis able to facilitate the entrance of immune cells into inflamed tissues and be activated in different inflammatory disorders, such as celiac disease (CD).The aim of this study was to investigate the expression level of CXCR3, CXCL10, and CXCL11 genes in celiac patients compared to healthy controls. Both cohorts have been recruited from the Iranian population.In this case–control study, biopsy specimens were collected from 71 celiac patients (60.5% female) and 90 control subjects (57% female) during 2016. Total RNA was extracted and mRNA expression levels of CXCR3, CXCL10, and CXCL11 genes were investigated by SYBR green qPCR.Based on qPCR and relative quantification method, the mRNA expression levels of CXCR3, CXCL10, and CXCL11 were significantly higher in duodenal biopsies of celiac patients compared to healthy controls in the study population (P = .038, P = .021, and P = .012 respectively).The result of this study showed that CXCR3/CXCL10/CXCL11 signaling axis is overexpressed in the small intestinal mucosa of CD patients compared to controls. This finding might explain the specific enrollment of the main cell populations that infiltrate the epithelium.

ITRAQ-Based Quantitative Proteomic Profiling of Staphylococcus aureus Under Different Osmotic Stress Conditions.

Staphylococcus aureus (S. aureus) is an extremely halotolerant pathogenic bacterium with high osmotic stress tolerance, and it is frequently encountered in aquatic production and preservation. However, the mechanism underlying the extremely high osmotic stress tolerance of S. aureus remains unclear. In this study, the isobaric tags for relative and absolute quantification (iTRAQ) method was used to identify the differentially expressed proteins (DEPs) under different sodium chloride (NaCl) concentrations. Compared with the control group (0% NaCl), the 10 and 20% NaCl groups had 484 DEPs and 750 DEPs, respectively. Compared with the 10% NaCl group, the 20% NaCl group had 361 DEPs. Among the DEPs, proteins involved in fatty acid synthesis, proline/glycine betaine biosynthesis and transportation, stress tolerance, cell wall biosynthesis and the TCA cycle were upregulated, whereas proteins associated with biofilm formation and pathogenic infections were downregulated. The results obtained in this study indicate that under extremely high osmotic stress, modification of the cell membrane structure, increased biosynthesis and transportation of osmotic protectants, and redistribution of energy metabolism contribute to the osmotic stress tolerance of S. aureus, and the infectious ability of the bacteria may be limited. The aim of this study was to provide new insight into how S. aureus tolerates the high-salt conditions involved in aquatic production and preservation.

Validation of reference genes for the normalization of RT-qPCR expression studies on human laryngeal cancer and hypopharyngeal cancer.

Selecting stably expressed reference genes is crucial for evaluating real-time quantitative polymerase chain reaction (RT-qPCR) data via the relative quantification method. In the present-day study, our aim was to select optimal reference genes (RGs) for the investigation of target gene (TG) expression profiling in cancerous human laryngeal and hypopharyngeal tissues. 12 cancerous laryngeal tissues and 10 cancerous hypopharyngeal tissues were investigated. The expression characteristics of 11 reference genes (18S rRNA, GAPDH, B2M, ACTB, TBP, ALAS1, RPL29, HMBS, HPRT1, GUSB, and PUM1), which were commonly used in RT-qPCR for the analysis of gene expression, were investigated using the geNorm, NormFinder, and BestKeeper algorithm programs. HMBS, ALAS1, and B2M were suggested as optimal RGs for studying human laryngeal and hypopharyngeal cancerous tissues together, laryngeal cancerous tissue by itself, and hypopharyngeal cancerous tissue by itself, respectively. If 2 or more reference genes are needed to achieve better standardization, 3 reference genes can optimally be used in combination to improve the accuracy of relative quantitation normalization. The recommended combinations for studying human laryngeal and hypopharyngeal cancerous tissues together, laryngeal cancerous tissue by itself, and hypopharyngeal cancerous tissue by itself were HMBS + HPRT1 + GUSB, ALAS1 + GUSB + HMBS, and B2M + HPRT1 + TBP, respectively. The recommended reference genes could be used to improve the accuracy of gene expression studies on the molecular mechanisms of cancerous human laryngeal and hypopharyngeal tissues. The selected combination of reference genes can effectively improve the accuracy of the relative quantitative diagnosis of gene expression levels, such as messenger RNA, circular RNA, and long-noncoding RNA.

NMR for metabolomics studies of Crataegus rhipidophylla Gand.

Crataegus products for food, nutritional, and pharmaceutical uses are available over the counter in the global market. The aerial parts are enriched with biochemicals and are used to treat cardiovascular, respiratory, and liver disorders and cancer. Detailed study to understand the chemical composition and different levels within medicinally important plant tissues needs hours, such as plant response for metabolite flux and quality control purposes. Therefore, comprehensive metabolite profiling (qualitatively and quantitatively) of different tissues [leaves, flowers, stems, fruits (without seeds), and seeds; n = 5] of Crataegus rhipidophylla Gand. was performed by NMR techniques in one session. Fifty-eight compounds of different classes were unambiguously identified with theuse of 1D and 2D NMR techniques (1H, 13C, distortionless enhancement by polarization transfer with 135° angle, correlation spectroscopy, heteronuclear single quantum coherence, heteronuclear multiple bond correlation) and quantified by a relative quantification method. Chemical compounds such as amygdalin (seeds), rutin (flowers), isovitexin (fruits), shikimic acid (leaves), and epicatechin (stems) were identified as authentication as well as discrimination markers for the respective C. rhipidophylla tissues. The fruits contain a significant mixture of important metabolites, followed by flowers and leaves. Trends of metabolite levels, upregulation, and downregulation in plant tissues were found, which highlights the responses of plant tissues in a specific time. NMR-based comprehensive metabolite information was used to develop the chemical signature, quality control chemical markers, and strategies to understand the chemical diversity of C. rhipidophylla. This chemical information from different aerial tissues will provide guidance for the extensive utilization of Crataegus.

Urinary mitochondrial DNA: A potential early biomarker of diabetic nephropathy.

Mitochondrial dysfunction and chronic sterile inflammation are common features of type 2 diabetes. Therefore, we aimed to investigate whether mitochondrial DNA (mtDNA) could be a biomarker implicated in the progression of type 2 diabetes and diabetic nephropathy and explore the underlying mechanism. We developed a method for relative quantification of mtDNA content in clinical practice. qRT-PCR was used to measure the mtDNA content both in vivo in CD-1 mice with diabetes induction by streptozotocin and in vitro in murine endothelial cells and conditionally immortalized mouse podocytes. By pumping mtDNA into the mouse circulation, the effect of mtDNA on the kidney was assessed in mice. In patients with type 2 diabetes (n = 42; 24 males; mean age 57.9 ± 12.00 years), plasma mtDNA was evaluated. Plasma mtDNA content was significantly decreased in patients with type 2 diabetes, particularly those with significant proteinuria. In vitro, high glucose treatment suppressed intracellular mtDNA content and facilitated the extracellular release of mtDNA, so excessive circulatory mtDNA induced by high glucose might be filtered through the kidney and then into urine. Indeed, urinary mtDNA content was significantly increased in both diabetic patients and mice. Moreover, by pumping excess mtDNA into circulation in mice, filtered mtDNA could trigger inflammation and induce kidney injury. Excessive mtDNA filtered through the kidney under diabetic conditions may be involved in chronic renal inflammation. Reduced plasma mtDNA content and increased urinary mtDNA/creatinine ratio might play a potential role as an early biomarker of diabetic nephropathy.

Antimicrobial effects of the ginsenoside Rh2 on monospecies and multispecies cariogenic biofilms.

To investigate the effects of the ginsenoside Rh2 on monospecies and multispecies cariogenic biofilms and explore the mechanism of the antibiofilm effect of Rh2 invitro. Streptococcus mutans, Streptococcus sobrinus and Streptococcus sanguinis were chosen to form the monospecies or multispecies biofilms. Crystal violet staining and laser scanning confocal microscopy were used to observe the effect of Rh2 on biofilms invitro. Cytotoxicity was examined by the Cell Counting Kit-8. The effects of Rh2 on bacterial membranes were observed via transmission electron microscopy (TEM). The isobaric tags for relative and absolute quantification (iTRAQ) method were used to profile the common differentially expressed proteins. Gene expression was analysed by reverse transcription quantitative polymerase chain reaction. In general, the treatment of cariogenic biofilms with Rh2 significantly decreased biomass accumulation by inhibiting bacterial growth and extracellular polysaccharide synthesis without any cytotoxic effects. TEM imaging showed that Rh2 could disrupt the cell membranes of these bacteria. The iTRAQ results indicated that the levels of mannose-specific IIC/D and acetaldehyde/alcohol dehydrogenase were substantially down-regulated, while the mRNA expression of the corresponding genes were significantly changed. Our data revealed a potential application for Rh2 in the protection against dental caries via the inhibition of cariogenic biofilms. This study describes the first application of a ginsenoside against multispecies cariogenic biofilms. Rh2 may serve as an alternative agent to prevent dental caries by effectively modulating the pathogenic potentials of oral biofilms.

A Combined Chemical Derivatization/Mass Spectrometric Method for theEnhanced Detection and Relative Quantification of Protein Ubiquitination.

Mass spectrometry (MS) is a sensitive analytical technique with wide application across the sciences including for the detection of peptides and proteins in biological analysis. Ubiquitinated (Ub) proteins are typically analyzed by proteolytic digestion and subsequent chromatographic separation followed by MS detection of the resulting isopeptides. Here we describe a novel method which enables enhanced detection of this important posttranslational modification (PTM) by use of a simple chemical labeling strategy prior to Data-Independent Acquisition (DIA) using a SWATH-based acquisition approach on a suitable Quadrupole-Time-Of-Flight (Q-TOF) mass spectrometer.

Evaluation of potential gene expression as early markers of insulin resistance and non-alcoholic fatty liver disease in the Indonesian population

Early detection of insulin resistance (IR) or non-alcoholic fatty liver disease (NAFLD) is crucial to preventing future risks of developing chronic diseases. The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), Liver Fat Score (LFS), and Fatty Liver Index (FLI) are generally employed to measure severity stages of IR and NAFLD. The study of gene expressions could explain the molecular mechanisms that occur early on in IR and NAFLD; thus providing potential early markers for both diseases. This study was conducted to evaluate the gene expressions that could potentially be early markers of IR and NAFLD. All participants (n = 21) had normal blood glucose and were categorized as without hepatosteatosis (n = 10), at higher risk of hepatosteatosis (n = 6), and hepatosteatosis (n = 5). Gene expression analysis was performed using the 2-∆∆CT relative quantification method. There were significant differences in galnt2 (p &lt; 0.002) and sirt1 (p &lt; 0.010) expression between the first and the third tertiles of HOMA-IR; and in ptpn1 (p &lt; 0.012) expression between the first and the second tertiles of LFS. In conclusion, the expressions of galnt2 and sirt1 could be used as early markers of IR, while the expression of ptpn1 could be employed as an early marker of NAFLD.

ITRAQ-based phosphoproteomic analysis reveals host cell's specific responses to Toxoplasma gondii at the phases of invasion and prior to egress

Protein phosphorylation plays a key role in host cell-T. gondii interaction. However, the phosphoproteome data of host cell at various phases of T. gondii infection has not been thoroughly described. In this study, we assessed the host phosphoproteome data with isobaric tags for relative and absolute quantification (iTRAQ) method during the phases of T. gondii invasion (30 min post infection, PI) and prior to egress (28 h PI). Our iTRAQ analysis revealed a total of 665 phosphoproteins, among which the significantly regulated phosphoproteins in different between-group comparisons were further analyzed. Functional analysis of these significantly regulated phosphoproteins suggested that T. gondii modulated host cell processes through phosphorylation including cell cycle regulation, inducing apoptosis, blocking the synthesis of some inflammatory factors, mediating metabolism to support its proliferation at the infection phase prior to egress, and utilizing membrane and energy from host cell, reorganizing cytoskeleton to favor its invasion and PV formation at the phase of invasion. The phosphorylation level of Smad2, CTNNA1, and HSPB1 identified with western blot revealed a consistent trend of change with iTRAQ result. These newly identified and significantly regulated phosphoproteins from our phosphoproteome data may provide new clues to unravel the host cell's complex reaction against T. gondii infection and the interaction between the host cell and T. gondii.

Thin Film Chemical Deposition Techniques as a Tool for Fingerprinting of Free Fatty Acids by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Metabolic fingerprinting is a powerful analytical technique, giving access to high-throughput identification and relative quantification of multiple metabolites. Because of short analysis times, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is the preferred instrumental platform for fingerprinting, although its power in analysis of free fatty acids (FFAs) is limited. However, these metabolites are the biomarkers of human pathologies and indicators of food quality. Hence, a high-throughput method for their fingerprinting is required. Therefore, here we propose a MALDI-TOF-MS method for identification and relative quantification of FFAs in biological samples of different origins. Our approach relies on formation of monomolecular Langmuir films (LFs) at the interphase of aqueous barium acetate solution, supplemented with low amounts of 2,5-dihydroxybenzoic acid, and hexane extracts of biological samples. This resulted in detection limits of 10-13-10-14 mol and overall method linear dynamic range of at least 4 orders of magnitude with accuracy and precision within 2 and 17%, respectively. The method precision was verified with eight sample series of different taxonomies, which indicates a universal applicability of our approach. Thereby, 31 and 22 FFA signals were annotated by exact mass and identified by tandem MS, respectively. Among 20 FFAs identified in Fucus algae, 14 could be confirmed by gas chromatography-mass spectrometry.

Profiling of carboxyl-containing metabolites in smokers and non-smokers by stable isotope labeling combined with LC-MS/MS

Profiling of carboxyl-containing metabolites in smokers and non-smokers provides insight into the smoking-related biological events and causal relationships between exposure and adverse events. However, more comprehensive analysis of carboxyl-containing metabolites in bio-matrices with high sensitivity and accuracy is challenging. In this work, stable isotope labeling in combination with liquid chromatography-tandem mass spectrometry was used for untargeted profiling and relative quantification of carboxyl-containing metabolites in plasma of smokers and non-smokers. A pair of isotope labeling reagents, N, N-dimethylethylenediamine (DMED) and d4-DMED was used to label carboxyl-containing metabolites. Since the isotope labeled dimethylamino moieties of DMED and d4-DMED are easily fragmented and lost as characteristic neutral fragments of 45 and 49 Da, respectively, double neutral loss scans can be used to profiling of carboxyl-containing metabolites. Subsequently, based on the ion pair parameters obtained from double neutral loss scans, relative quantification method was developed. As a result, 269 carboxyl-containing metabolite candidates were discovered, and 88 metabolite candidates were found to have significant alterations between smokers and non-smokers. 7Z, 10Z-hexadecadienoic acid, myristic acid and 3β-hydroxy-5-cholestenoic acid with significant differences confirmed by standard comparison are linked to smoking related inflammation, abnormal bile acid synthesis and cholesterol metabolism.

Transcriptomic Profile Identified a Specific Signature in Children with Acute Myeloid Leukemia (AML) and Primary Induction Failure (PIF): Preliminary Data and Future Perspectives

Background. Acute Myeloid Leukemia is a heterogeneous disease, characterized by the uncontrolled proliferation of hematopoietic precursor cells. AML accounts for 15% of acute leukemias in children, with a cure rate of 55-60% as overall survival (Pession A Blood 2013). Complete remission (CR) is achieved in 80-85% of children with AML. Current protocol defines CR by morphological evaluation (blasts <5%) and cytofluorimetric analyses (blasts <0,1%), after two cycles of high-dose chemotherapy. Around 10-15% of children and 30% of adults do not achieve remission and are defined as Primary Induction Failure (PIF). The majority of these cases are addressed to other form of high-dose chemotherapy blocks, experimental therapies or directly to hematopoietic stem cell transplantation (HSCT) as salvage treatment, showing a very poor prognosis (Quarello P et al. British Journal of Haematology 2015). Strong efforts to generate biological data in order to define this subtype of AML are needed for early identification and for target therapy design.Aim. Our study aims to characterize a specific gene-expression profile in children with PIF-AML in order to early detect peculiar genetic aberrations and to identify potential targets to new compounds.Materials and Methods. We analyzed diagnostic samples from 4 children with PIF-AML, diagnosed and treated in our Center, from 2013 to 2016. All cases were female and 3 had more than 10 years of age. One presented with a monosomy of chromosome 11; two with complex karyotype (one with NUP98-NSD1 rearrangement) and one with no cytogenetic and molecular markers. After two cycles of Induction phase, one case showed complete blast regeneration (>60%); in the other three cases we detected, by flow-cytometry, a blast rate between 10-20%. In the attempt to compare these cases with those presenting with very high-risk (VHR) features but achieving CR after induction (Primary Induction Responder, PIR), we included in our analyses 8 children with VHR-PIR-AML, diagnosed and treated in our Center: 2 with FLT3-ITD and NUP98-NSD1 fusion genes; 2 with FLT3-ITD and complex karyotype; 2 with DEK-CAN fusion genes (1 with FLT3-ITD); 2 with NUP98 rearrangements and complex karyotype. We performed whole transcriptome analyses using Human Transcriptome Array 2.0 (Affymetrix). Then we compared our analyses with a recently published data set, including a large population of children with AML (Bisio V Leukemia 2017). Based on transcriptomic findings, we determined the expression of specific genes, by Real-time PCR using gene-specific probes (UPL probes by Roche Merck, Germany), applying a QuantStudio7 Flex System technology (Applied Biosystem, California, USA) and the relative quantification method (the comparative 2-ΔΔCt method). We applied a t-student test, for statistical analyses among different subgroups.Results. A direct comparison of PIF-AML vs PIR-AML subgroups revealed 919 protein-coding and 8 pri-miRNA overexpressed genes (fold-change >3, FDR adjusted p value <0.05) and 18 protein coding and 43 pri-miRNA down-regulated genes (fold-change >-3, FDR adjusted p value <0.05). Marked differences were observed in cancer-related genes such as KIT, PTEN, FLT3 and POU4F1, and in cell-cycle related genes such as cyclin transcripts (CNNG1, CCNA1, CCND2, CCNJ, CCNC, CCNY). Moreover some genes that discriminate PIF from PIR subgroups are also differentially expressed between cases with NUP98-negative and NUP98-positive AML (PDGFD, CCNA1). The latter group is considered as a VHR-AML subtype. Based on these findings, we selected several genes including HOX-B3, HOX-B5, CEBP-D, CCNA1, PDGF-D and performed a Real-Time PCR with specific-gene probes. Comparing gene expression data of our 4 PIF-AML cases with 8 VHR-PIR-AML cases, we observed a strong statistically significant different expression in CCNA1 (p<0.0001) and PDGF-D (p<0.0001).Conclusions. Our findings defined a characteristic profile of PIF-AML, highlighting the overexpression of cell-cycle related genes (CCNG1, CCNA1) and several potential targets (KIT, FLT3, PDGF-D) to be considered for an alternative treatment in this very poor-prognostic subgroup of patients. In conclusion our results suggest that PIF-AML samples bear a specific gene expression profile, not overlapping with those of known molecular and karyotype AML subgroups. However, a larger population study is needed to confirm our preliminary data. DisclosuresNo relevant conflicts of interest to declare.

Post-Therapy Measurable Residual Disease Monitoring in Peripheral Blood Using Overexpressed Genes in Childhood Acute Myeloid Leukemia

Background:Even though children with acute myeloid leukemia (AML) receive a very intensive chemotherapy and most achieve a complete remission (CR) ~30% of patients suffer from relapse. Post-treatment monitoring of measurable residual disease (MRD) can allow detection of a re-emerging leukemic clone several months before clinical relapse, and studies are in progress that aim at treating children with a molecular relapse.Specific genetic aberrations can be used for disease monitoring after therapy completion, but even though oncogenic fusion transcripts are more common in childhood than adult AML, NPM1 mutation is much rarer and consequently MRD measurements based on these aberrations are clinically applicable in only ~40% of childhood AML patients. Thus, a considerable fraction of patients do not have a suitable leukemia-specific molecular MRD target, but genes with an abnormally high expression in the leukemic cells might be candidate MRD targets in those patients. WT1 overexpression in childhood AML is well described, and if distinctly overexpressed at diagnosis, serves as a suitable MRD target in a large proportion of patients.Gene expression profiling has identified several other genes with an abnormally high expression in the leukemic blasts compared to normal hematopoietic cells.We investigated the applicability of 4 leukemia-associated genes (PRAME, GAGED2, ST18, SPAG6) as targets for early detection of relapse in peripheral blood (PB) in a Danish cohort of childhood AML patients, defined child-specific reference values of gene expression based on a large material of PB and BM samples from hematologically healthy children, and investigated gene expression levels under the presence of infection.Methods:We investigated the expression of 4 leukemia-associated genes (PRAME, GAGED2, ST18, SPAG6) in hematologically healthy children (n=53) and during suspected infection in febrile but otherwise healthy children (n=90). Gene expression in de novo AML at diagnosis (n=50) and during follow-up (n=20) was compared with child-specific reference values. We defined the 95th percentile of expression levels in hematologically healthy children as the upper limit of normal expression.RT-qPCR analyses were performed in compliance with EAC protocols and due to concordant qPCR efficiencies the ΔΔCq method for relative quantification could be applied.Results:At AML diagnosis, 64% had high expression of at least 1 of the 4 genes defined as >20-fold overexpression compared to hematologically healthy children. Nine out of 10 patients (90%) without established molecular MRD targets or high WT1 expression had high expression of at least 1 of the 4 genes. All 7 children with t(9;11) had GAGED2>1000-fold overexpressed. Gene expression was quantified in 99 PB samples (163 RT-qPCR analyses) during follow-up in 20 patients with distinct overexpression at diagnosis. All 10 patients with PB sampling performed within 100 days of disease recurrence displayed expression above normal by a median of 1.6 months (range 0.5-6 months) before hematological relapse. Patients with CBF-AML had a significantly longer interval between molecular relapse and hematological relapse than patients with non-CBF-AML (2.5 months (range 0.8-6 months) vs. 0.9 months (range 0.5-2 months), p=0.047). One patient with PB sampling performed only once at 119 days prior to hematological relapse did not show any molecular evidence of disease recurrence before hematological relapse. Only 1 of 96 (1%) post-therapy follow-up analyses performed in 9 patients in continuous CR for >5 years after diagnosis had expression above normal. In this case, a 9-year-old girl in continuous CR had an increase in ST18 expression, however the increase was transient and returned to normal level in the following samples.We found no clinically relevant influence of fever on gene expression levels, except for GAGED2, where 21% of febrile children had expression above normal.Conclusions:Sequential post-therapy monitoring of overexpressed genes in PB can predict relapse in childhood AML patients and facilitates molecular MRD monitoring in 90% of patients without a leukemia-specific target or WT1 overexpression. Frequent PB sampling (every 4-6 weeks) is necessary to detect an upcoming relapse, however in the post-treatment follow-up setting PB serves as an attractive and easily accessible source of preference compared to BM aspiration. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Living apart-together: Microhabitat differentiation of cryptic nematode species in a saltmarsh habitat.

Coexistence of highly similar species is at odds with ecological theory of competition; coexistence, then, requires stabilizing mechanisms such as differences in ecological niche. In the bacterivore nematode Litoditis marina species complex, which occurs associated with macro-algae, four cryptic lineages (Pm I-IV) co-occur in the field along the south-western coast and estuaries of The Netherlands. Here we investigate the temporal and/or spatial niche differentiation in their natural environment using a qPCR-based detection and relative quantification method. We collected different algal species (i.e. two Fucus species and Ulva sp.) and separated algal structures (i.e. receptacula, thalli, non-fertile tips and bladders) at different sampling months and times (i.e. twice per sampling month), to examine differences in microhabitat use between coexisting L. marina species. Results demonstrate that the cryptic species composition varied among different algal species and algal structures, which was also subject to temporal shifts. Pm I dominated on Fucus spp., Pm II showed dominance on Ulva sp., while Pm III overall had the lowest frequencies. Microhabitat partitioning was most pronounced between the two cryptic species which had similar microbiomes (Pm I and Pm II), and less so between the two species which had significantly different microbiomes (Pm I and Pm III), suggesting that species which share the same microhabitats may avoid competition through resource partitioning. The interplay of microhabitat differentiation and temporal dynamics among the cryptic species of L. marina implies that there is a complex interaction between biotic components and abiotic factors which contributes to their coexistence in the field.

Hsa-microRNA-101-3p expression in human urinary bladder cancer: Correlation with tumor pathology

IntroductionMicroRNAs (miRs) are a class of small, conserved, non-protein-coding RNAs that regulate gene expression by controlling translation of target messenger RNA. In cancer, miRs can act either as tumor suppressors or oncogenes. Reduced expression of hsa-miR-101-3p has been observed in different types of cancers, and was thus considered to be a tumor suppressor. In urinary bladder cancer (BC) cell lines, hsa-miR-101-3p was found to suppress cancer cells motility, however studies in human BC patients, correlating hsa-miR-101-3p expression to pathological findings are very limited. This study aims at assessing hsa-miR-101-3p expression in human BC in correlation to tumor pathology in transurethral resection biopsies. Material and methodsTransuretheral bladder tissue specimens were collected from 100 bladder cancer patients. Only bladder transitional cell carcinoma (BTCC) patients (n = 80) were considered. Total RNA was extracted and following reverse transcription, expression of hsa-miR-101-3p was assessed by quantitative real time PCR using Taqman assay. The fold change for hsa-miR-101-3p expression between malignant and surrounding non-malignant tissues was calculated using relative quantification method. Expression of hsa-miR-101-3p was correlated to tumor stage, grade, and lymphatic/ vascular involvement. ROC curve was done to calculate the discriminative ability of hsa-miR-101-3p expression between malignant and non-malignant tissues. ResultsHsa-miR-101-3p was significantly under-expressed in all malignant versus non-malignant tissue samples (median 13.6 (9.8–19.4) vs. median 10.9 (7.4–16.2), p < 0.001) respectively. Mann-Whitney test showed statistically significant hsa-miR-101-3p underexpression in high vs. low tumor grades (p < 0.001), and T2 vs. T1 tumor stages (p < 0.001). There was no significant association between hsa-miR-101-3p underexpression and lymphatic/vascular invasion. ROC curve demonstrated hsa-miR-101-3p to highly discriminate between malignant and non-malignant tissue (p < 0.001).At cut-off value >10.85, hsa-miR-101-3p showed sensitivity of 83% and specificity of 56%. ConclusionsHsa-miR-101-3p can highly discriminate between malignant and non-malignant samples in BTCC, and seems to associate with tumor progression and aggressiveness. It might be a promising diagnostic and perhaps prognostic marker for BTCC.

Phenotyping and relative quantification of vitamin D binding protein in a paediatric population by using liquid chromatography-tandem mass spectrometry.

Adequate concentrations of vitamin D are required to ensure bone health and minimize the incidence of multiple extraskeletal diseases. Although total 25-hydroxyvitamin D (25OHD) remains the recommended biomarker for assessing vitamin D status, it has been speculated that free 25OHD correlates better with clinical outcomes. The calculation of free 25OHD depends on the concentrations of vitamin D binding protein (DBP), the determination of which involves different immunoassays and has led to varying results and conclusions. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and relative quantification of DBP isoforms. We used serum samples from healthy children ( n = 79), mainly Caucasian (88%). Proteins were denatured, reduced, alkylated and digested with trypsin. Purified peptides were analysed by LC-MS/MS. The DBP phenotype was established by using the combinations of tryptic peptides associated with each of the three isoforms and one peptide common to all of them to perform relative quantification. The genotyping of volunteers ( n = 7) facilitated verification of the ability of our method to correctly identify the DBP phenotype. The DBP phenotype was correctly established in all samples from volunteers, based on the 100% correlation observed with the genotype. The most common DBP phenotype in Caucasian children was 2/1S (34%) and the rarest 1F/1F (2%). The relative quantification of DBP concentrations did not show statistically significant differences between phenotypes ( P = 0.11). LC-MS/MS enabled simultaneous phenotyping and relative quantification of DBP, while avoiding the analytical limitations of immunoassays and confirming similar concentrations of DBP in all phenotypes.

Human kallikrein-related peptidase 12 (KLK12) splice variants discriminate benign from cancerous breast tumors

ObjectivesAs kallikrein-related peptidase 12 (KLK12) has been implicated in the cancer progression and alternative splicing plays significant role in this disease, the aim of this study was to examine the expression profile and the clinical impact of the KLK12 splice variants in breast cancer. Design and methodsTotal RNA was isolated and reverse transcripted from 141 tissues. Afterwards, quantitative real-time PCR were conducted, followed by the performance of the comparative CT (2-ΔΔCT) method for relative quantification, whilst their correlation with the clinicopathological features of breast malignancies were assessed by statistical analysis. ResultsBoth KLK12sv1/2 and KLK12sv3 showed higher expression in non-cancerous than in cancerous samples. KLKsv1/2 (P = 0.001) upregulated and KLK12sv3 (P < 0.001) downregulated in the malignant compared to the benign tumors and their discriminative ability was verified by ROC curve analysis. Moreover, KLK12sv3 was associated with grade (P = 0.012) and hormonal receptor status (P = 0.001). Furthermore, Kaplan-Meier and Cox regression analyses showed that patients with positive KLK12sv1/2 and KLK12sv3 levels presented a significantly longer disease-free survival (P = 0.014 and P = 0.013, respectively) and overall survival (P = 0.062 and P = 0.004, respectively). ConclusionsOur results demonstrate the discriminative value of KLK12sv1/2 and KLK12sv3 between benign and malignant breast tumors as well as their potential favorable prognostic significance in breast adenocarcinoma.