The JAK2 617V>F mutation triggers erythropoietin hypersensitivity and terminal erythroid amplification in primary cells from patients with polycythemia vera

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 1013-1021 ◽  
Author(s):  
Sabrina Dupont ◽  
Aline Massé ◽  
Chloé James ◽  
Irène Teyssandier ◽  
Yann Lécluse ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Quantitative Polymerase Chain Reaction ◽  
Selective Advantage ◽  
Erythroid Progenitors ◽  
Molecular Events ◽  
Polymerase Chain ◽  
Late Stages ◽  
Early Phases

Abstract The JAK2 617V>F mutation is frequent in polycythemia vera (PV) and essential thrombocythemia (ET). Using quantitative polymerase chain reaction (PCR), we found that high levels of JAK2 617V>F in PV correlate with increased granulocytes and high levels of hemoglobin and endogenous erythroid colony formation. We detected normal progenitors and those that were heterozygous or homozygous for the mutation by genotyping ET and PV clonal immature and committed progenitors. In PV patients, we distinguished homozygous profiles with normal, heterozygous, and homozygous progenitors from heterozygous profiles with only heterozygous and normal progenitors. PV patients with a heterozygous profile had more mutated, committed progenitors than did other PV and ET patients, suggesting a selective amplification of mutated cells in the early phases of hematopoiesis. We demonstrated that mutated erythroid progenitors were more sensitive to erythropoietin than normal progenitors, and that most homozygous erythroid progenitors were erythropoietin independent. Moreover, we observed a greater in vitro erythroid amplification and a selective advantage in vivo for mutated cells in late stages of hematopoiesis. These results suggest that, for PV, erythrocytosis can occur through two mechanisms: terminal erythroid amplification triggered by JAK2 617V>F homozygosity, and a 2-step process including the upstream amplification of heterozygous cells that may involve additional molecular events.

Breast Milk and Saliva Lactoferrin Levels and Postnatal Cytomegalovirus Infection

2020 ◽  
Author(s):  
Kristin E. D. Weimer ◽  
Hunter Roark ◽  
Kimberley Fisher ◽  
C. Michael Cotten ◽  
David A. Kaufman ◽  
...  
Keyword(s):  
Breast Milk ◽  
Very Low Birth Weight ◽  
Quantitative Polymerase Chain Reaction ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lactating Mothers ◽  
Life Threatening ◽  
Polymerase Chain

Abstract Objective Very low birth weight preterm infants are at risk for life-threatening infections in the NICU. Breast milk protects against infections but carries the risk of infection by cytomegalovirus (CMV) shed in mother's milk. Lactoferrin is a breast milk and saliva protein with potent neutralizing activity against CMV. Study Design VLBW, maternal breast milk fed infants in the NICU and their lactating mothers were enrolled and followed for 3 months/discharge. Breast milk and infant saliva samples were collected biweekly. Maternal CMV status was determined on breast milk. CMV was measured using quantitative polymerase chain reaction and lactoferrin by enzyme-linked immunosorbent assay. Results In an in vitro neutralization assay, the IC90 of purified human lactoferrin against CMV was 2.08 ng/mL. Bovine lactoferrins were more potent, IC90s > 10-fold higher. Lactoferrin was detected in all breast milk (median: 3.3 × 106 ng/mL) and saliva (median: 84.4 ng/swab) samples. Median CMV load in breast milk was 893 copies/mL. There was no correlation between breast milk lactoferrin concentration and CMV load. Five infants acquired postnatal CMV. There was no difference in saliva or breast milk lactoferrin concentration for mother–infant pairs and postnatal CMV acquisition. Conclusion Lactoferrin neutralizes CMV in vitro, but concentrations in breast milk and saliva are likely too low for effective neutralization in vivo.

214 USE OF VIRUS ISOLATION AND QUANTITATIVE POLYMERASE CHAIN-REACTION TECHNIQUES TO DETECT BOVINE VIRAL DIARRHEA VIRUS (BVDV) IN SINGLE OR SMALL GROUPS OF PRE-IMPLANTATION BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 214
Author(s):  
J. Waldrop ◽  
M. Givens ◽  
K. Riddell ◽  
P. Galik ◽  
D. Stringfellow
Keyword(s):  
Small Groups ◽  
Virus Isolation ◽  
Quantitative Polymerase Chain Reaction ◽  
Bovine Embryos ◽  
Chain Reaction ◽  
Diarrhea Virus ◽  
Polymerase Chain ◽  
Viral Diarrhea Virus

Because of its broad distribution among populations of cattle and its association with materials of animal origin used in embryo production, bovine viral diarrhea virus (BVDV) is a potential problem in applications of embryo technologies. While some isolates of BVDV are known to associate with both in vivo-derived and in vitro-produced bovine embryos, it has yet to be determined if the quantity of virus associated with exposed zona pellucida-intact embryos is sufficient to infect susceptible recipient cows via the intrauterine route. Techniques to detect and quantify BVDV associated with single transferable embryos are important to determine the risk of transmitting BVDV via embryo transfer. The objectives of this study were to define reproducible techniques to detect and quantify BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using virus isolation (VI) or real time quantitative polymerase chain reaction (Q-PCR) assays. In vivo-derived and in vitro-produced embryos were exposed for 2 h to approximately 106-cell culture infective doses (50% endpoint) per mililiter of a high affinity strain of BVDV, SD-1, and then washed according to IETS guidelines. Embryos were assayed in groups of five or two embryos, or single. There were 5 replicates of the group of five embryos, 4 of the group of two embryos, and 3 of the single embryos for the in vivo-derived embryos undergoing VI; 5, 4, and 2 replicates, respectively, undergoing Q-PCR, and 2, 5, and 2 replicates, respectively, for the in vitro-produced embryo groups undergoing VI and Q-PCR. Those to be assayed by VI were sonicated and the sonicate fluids were layered onto Madin Darby Bovine Kidney (MDBK) cells and passaged to allow for viral replication; an immunoperoxidase monolayer assay was then used for viral detection. A Roche� RNA/DNA extraction kit (Roche Diagnostic Systems, Inc., Somerville, NJ, USA) was used to extract RNA from virally exposed embryos, and extracted samples were assayed in duplicate Q-PCR reactions consisting of 100 �L. The primers used were L1 and U3 which are specific for conserved areas of the 5 prime nontranslated regions of the viral genome of BVDV. The PCR product was detected using hybridization probes s1 and s2 as in Struder et al. 2002 Biologicals 40, 289-296. In vivo-derived groups of five or two embryos, or single embryos, were positive for BVDV 100, 50, and 30% of the time, respectively, when VI was used and 100, 75 and 100%, respectively, when Q-PCR was used. The virus was detected in all of the in vitro-produced embryo groups of five, or two embryos, or single embryos, 100% of the time using VI, and in 100, 80, and 100% respectively, using Q-PCR. The virus isolation technique is highly sensitive but the need to destroy embryos by sonication to identify any embryo-associated virus precludes its use for embryos intended for transfer. Techniques for Q-PCR were sufficiently sensitive to detect and quantify 10 copies of RNA in a sample and to detect BVDV associated with single embryos.

Pure Heterozygous and Mixed JAK2 V617F Genotypes Result in Distinct Mechanisms of Erythrocytosis in Polycythemia Vera.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 662-662
Author(s):  
Sabrina Dupont ◽  
Aline Masse ◽  
Chloe James ◽  
Nicole Casadevall ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Jak2 V617f ◽  
Molecular Event ◽  
Erythroid Progenitors ◽  
Low Frequencies

Abstract The JAK2 V617F mutation is present in most patients with polycythemia vera (PV) and half with essential thrombocythemia (ET). Using real-time quantitative PCR, we analyzed the levels of JAK2 V617F in granulocytes and/or bone marrow mononuclear cells from 159 PV and 149 ET patients. High JAK2 V617F levels were correlated with higher leukocyte, granulocyte, hemoglobin values and higher endogenous erythroid colony formation. Thus, the phenotype of PV and ET may be closely linked to the JAK2 V617F level, which may reflect the clonal genotypic pattern of hematopoietic progenitor cells. It is thought that the occurrence of the mitotic recombination, which generates homozygous JAK2 V671F clones, is a key molecular event for the onset of PV. In this work, we aimed to study the consequences of the clonal JAK2 V617F genotype on the amplification properties and erythropoietin (EPO) hypersensitivity of PV (n=14) and ET (n=6) progenitors. Analysis of clonal genotypic patterns shows that ET patients harbor a mix of heterozygous and normal progenitors. Interestingly, we distinguish pure heterozygous PV profiles (3/14 patients) with no homozygous progenitors from homozygous PV profiles (11/14 patients) with normal, heterozygous and homozygous progenitors. Similar low frequencies of mutated immature progenitors, comprising long-term culture-initiating cells and lympho-myeloid progenitors, are found in ET and PV. In contrast, PV patients with pure heterozygous PV profiles have striking higher proportions (>90%) of mutated committed progenitors than other PV and ET patients. This result suggests a selective amplification of heterozygous cells in the early phases of hematopoiesis. Furthermore, by using increasing concentrations of EPO, homozygous mutated erythroid progenitors are demonstrated to be more sensitive to EPO than heterozygous ones, a majority of the former (69,5%) being EPO independent. Moreover, we demonstrate a two to three fold increase in in vitro amplification of ET and PV progenitor cells when compared to normal ones in serum free liquid culture containing IL3, Stem Cell Factor, Dexamethasone and 1 IU/mL EPO. In addition, the quantification of the mutated allele in immature CD34+CD38- cells, in CD34+CD38+ committed progenitor cells, in mature erythroblasts (GPA+) and in granulocytes shows a marked in vivo selective advantage of mutated cells in late stages of hematopoiesis. These results suggest that in PV, erythrocytosis results from two distinct mechanisms: a terminal erythroid amplification advantage triggered by homozygosity or a two-step process including the upstream amplification of heterozygous cells that may involve additional molecular event(s).

scholarly journals In Vivo Association of Ku with Mammalian Origins of DNA Replication

2001 ◽  
Vol 12 (11) ◽  
pp. 3386-3401 ◽  
Author(s):  
Olivia Novac ◽  
Diamanto Matheos ◽  
Felipe D. Araujo ◽  
Gerald B. Price ◽  
Maria Zannis-Hadjopoulos
Keyword(s):  
Dna Replication ◽  
Quantitative Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Analysis ◽  
Origin Firing ◽  
Origin Binding Protein ◽  
M Phase ◽  
Polymerase Chain ◽  
In Cells

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association withors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80+/+ and Ku80−/− mouse embryonic fibroblasts. A decrease of ∼70% in in vitro DNA replication was observed when the Ku80−/− extracts were used, compared with the Ku80+/+ extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.

scholarly journals Inhibiting SLC26A4 reverses cardiac hypertrophy in H9C2 cells and in rats

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8253
Author(s):  
Liqun Tang ◽  
Xiaoqin Yu ◽  
Yangyang Zheng ◽  
Ning Zhou
Keyword(s):  
Cardiac Hypertrophy ◽  
Natriuretic Peptide ◽  
Quantitative Polymerase Chain Reaction ◽  
Pendred Syndrome ◽  
Abdominal Aortic ◽  
Polymerase Chain ◽  
Gsk 3Β

Background It has been confirmed that mutations in solute carrier family 26 member 4 (SLC26A4) contribute to pendred syndrome. However, the role of SLC26A4 in cardiac hypertrophy and the signaling pathways remain unclear. Methods Cardiomyocytes were treated by 200 µM phenylephrine (PE) to induce cardiac hypertrophy. Also, the expression of SLC26A4, GSK3, cardiac hypertrophy markers including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was detected through real-time quantitative polymerase chain reaction (RT-qPCR). Flow cytometry assay was used to test the apoptosis of PE-induced cardiomyocytes transfected by small interfere RNA (siRNA)-SLC26A4. Furthermore, we detected the expression of autophagy-related markers including light chain 3 (LC3) and P62. Finally, we established a rat model of abdominal aortic constriction (AAC)-induced cardiac hypertrophy in vivo. Results RT-qPCR results showed that the mRNA expression of SLC26A4 was significantly up-regulated in PE-induced cardiac hypertrophy. After inhibiting SLC26A4, the release of ANP and BNP was significantly decreased and GSK3β was elevated in vivo and in vitro. Furthermore, inhibiting SLC26A4 promoted apoptosis of cardiac hypertrophy cells. In addition, LC3 was down-regulated and P62 was enhanced after transfection of siRNA-SLC26A4. Conclusion Our findings revealed that SLC26A4 increases cardiac hypertrophy, and inhibiting SLC26A4 could decrease the release of ANP/BNP and promote the expression of GSK-3β in vitro and in vivo. Moreover, SLC26A4 silencing inhibits autophagy of cardiomyocytes and induces apoptosis of cardiomyocytes. Therefore, SLC26A4 possesses potential value to be a therapeutic target of cardiac hypertrophy, and our study provides new insights into the mechanisms of cardiac hypertrophy.

Will Imitanib Be Useful for Patients with Polycythemia Vera?.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2601-2601 ◽  
Author(s):  
Amos S. Gaikwad ◽  
Srdan Verstovsek ◽  
Ko-Tung Chang ◽  
Donghoon Yoon ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Glucose Uptake ◽  
Polycythemia Vera ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Jak2 V617f ◽  
Erythroid Progenitors ◽  
Jak2 Mutation

Abstract Polycythemia vera (PV) is an acquired myeloproliferative, clonal stem cell disorder characterized by cytokine hypersensitivity. Several groups reported a clonal mutation in the pseudo kinase domain of the Janus kinase 2 (Jak2) protein, substituting phenylalanine at position 617 with valine (V617F) that causes constitutive activation of the JAK/STAT pathway in PV patients. Chronic myelogenous leukemia is another myeloproliferative disorder associated with aberrant tyrosine kinase (bcr-abl) that arises from t(9:22) translocation. Constitutive bcr-abl tyrosine kinase activity suppresses apoptosis leading to survival and proliferation of cells at low cytokine concentrations. Imitanib has been developed to inhibit the bcr-abl kinase activity and has had impressive therapeutic efficacy. However, imitanib also inhibits other tyrosine kinases. Clinical benefits of imitanib in PV have been reported. Mouse FDCP cells over-expressing either the wild-type Jak2 (JAK/W) or two cell lines with the V617F mutation (V617F), one with cytokine hypersensitivity and the other cytokine independent, were created. We examined the effect of imitanib by MTT proliferation assay and propidium iodide staining analysis. No appreciable changes in the proliferation and DNA content were observed in all three cell-lines after imitanib treatment at ~1μM (the concentration effective for the bcr-abl expressing cells and achievable in vivo in imitanib treated patients). However, after 72h of exposure, the cells expressing JAK2 V617F mutant showed 50% inhibition of growth at ~6μM imitanib with no significant effect on the growth of cells expressing JAK/W. To further understand the mechanism of growth inhibition of V617F cells by imitanib, we examined the metabolism of these cells since the constitutively active tyrosine kinases has been demonstrated to change the glucose metabolism. Imitanib treatment (5μM) for 72h caused about 30% decrease in the glucose uptake in V617F cells with only a marginal (~5%) decrease in glucose uptake was observed in the JAK/W cells. We then examined the effect of imitanib on in vitro expanded native human erythroid progenitors (CD71 and CD235a positive cells) from three normal and four PV individuals who expressed variable proportion of mutant JAK2 V617F alleles. In this small study, between 18–30% decrease in the proliferation of the cells from PV patients was seen with 1–2μM of imitanib compared to 8% seen with normal erythroid progenitors; however, the in vitro expansion was accompanied by a decrease of proportion of cells with JAK2 mutation (see abstract Prchal et. Al. at this meeting). Further, in ongoing clinical studies, one of 5 tested PV patients who showed an excellent clinical response to imitanib therapy had decrease of V617F mutation from 58 to 19% in circulating granulocytes. We conclude that high concentration of imitanib is required to achieve the cytotoxic effects in reporter cells transfected with JAK2 mutation that are not readily achievable in vivo; however, lower doses (but still significantly higher than those needed for bcr-abl expressing cells) are effective in native PV progenitors. These data do not exclude possible useful therapeutic effect of imitanib in PV either alone or in combination with other drugs. Amos Gaikwad, Ph.D. and Srdan Verstovsek, M.D. contributed equally to this study.

An integrated approach to bovine oocyte quality: from phenotype to genes

2016 ◽  
Vol 28 (9) ◽  
pp. 1276 ◽  
Author(s):  
Leslie Angulo ◽  
Catherine Guyader-Joly ◽  
Sylvain Auclair ◽  
Christelle Hennequet-Antier ◽  
Pascal Papillier ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Quantitative Polymerase Chain Reaction ◽  
Integrated Approach ◽  
Oocyte Quality ◽  
Developmental Rates ◽  
Polymerase Chain ◽  
The Difference ◽  
Differential Genes

In cattle, early embryonic failure plays a major role in the limitation of reproductive performance and is influenced by genetic effects. Suboptimal oocyte quality, including an inadequate store of maternal factors, is suspected to contribute to this phenomenon. In the present study, 13 Montbeliarde cows were phenotyped on oocyte quality, based on their ability to produce viable embryos after in vitro maturation, fertilisation and culture for 7 days. This discriminated two groups of animals, exhibiting developmental rates below 18.8% or above 40.9% (relative to cleaved embryos). Using microarrays, transcriptomic profiles were compared between oocytes collected in vivo from these two groups of animals. The difference in oocyte development potential was associated with changes in transcripts from 60 genes in immature oocytes and 135 genes in mature oocytes (following Bonferroni 5% correction). Of these, 16 and 32 genes were located in previously identified fertility quantitative trait loci. A subset of differential genes was investigated on distinct samples by reverse transcription–quantitative polymerase chain reaction. For SLC25A16, PPP1R14C, ROBO1, AMDHD1 and MEAF6 transcripts, differential expression was confirmed between high and low oocyte potential animals. Further sequencing and searches for polymorphisms will pave the way for implementing their use in genomic selection.

Analysis of the Dynamics of the Electric Capacitance of a Cell Monolayer at the Late Stages of Caco-2 cell Differentiation

2019 ◽  
Vol 35 (6) ◽  
pp. 87-90
Author(s):  
S.V. Nikulin ◽  
V.A. Petrov ◽  
D.A. Sakharov
Keyword(s):  
Impedance Spectroscopy ◽  
Cell Monolayer ◽  
Microfluidic Chips ◽  
Russian Science ◽  
Electric Capacitance ◽  
A Cell ◽  
Static Conditions ◽  
Late Stages

The real-time monitoring of electric capacitance (impedance spectroscopy) allowed obtaining evidence that structures which look like intestinal villi can be formed during the cultivation under static conditions as well as during the cultivation in microfluidic chips. It was shown in this work via transcriptome analysis that the Hh signaling pathway is involved in the formation of villus-like structures in vitro, which was previously shown for their formation in vivo. impedance spectroscopy, intestine, villi, electric capacitance, Hh The study was funded by the Russian Science Foundation (Project 16-19-10597).

scholarly journals Akt kinase LANCL2 functions as a key driver in EGFR-mutant lung adenocarcinoma tumorigenesis

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Yuqing Lou ◽  
Jianlin Xu ◽  
Yanwei Zhang ◽  
Wei Zhang ◽  
Xueyan Zhang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Line ◽  
Quantitative Polymerase Chain Reaction ◽  
Mutant Cell ◽  
A549 Cells ◽  
The Cancer Genome Atlas ◽  
Akt Kinase ◽  
Egfr Expression

AbstractEpidermal growth factor receptor (EGFR) is a key oncogene in lung adenocarcinoma (LUAD). Resistance to EGFR tyrosine kinase inhibitors is a major obstacle for EGFR-mutant LUAD patients. Our gene chip array, quantitative polymerase chain reaction validation, and shRNA-based high-content screening identified the Akt kinase lanthionine synthetase C-like protein 2 (LANCL2) as a pro-proliferative gene in the EGFR-mutant LUAD cell line PC9. Therefore, we investigated whether LANCL2 plays a role in promoting cell proliferation and drug resistance in EGFR-mutant LUAD. In silico clinical correlation analysis using the Cancer Genome Atlas Lung Adenocarcinoma dataset revealed a positive correlation between LANCL2 and EGFR expression and an inverse relationship between LANCL2 gain-of-function and survival in LUAD patients. The EGFR-mutant LUAD cell lines PC9 and HCC827 displayed higher LANCL2 expression than the non-EGFR-mutant cell line A549. In addition, LANCL2 was downregulated following gefitinib+pemetrexed combination therapy in PC9 cells. LANCL2 knockdown reduced proliferation and enhanced apoptosis in PC9, HCC827, and A549 cells in vitro and suppressed murine PC9 xenograft tumor growth in vivo. Notably, LANCL2 overexpression rescued these effects and promoted gefitinib + pemetrexed resistance in PC9 and HCC827 cells. Pathway analysis and co-immunoprecipitation followed by mass spectrometry of differentially-expressed genes in LANCL2 knockdown cells revealed enrichment of several cancer signaling pathways. In addition, Filamin A and glutathione S-transferase Mu 3 were identified as two novel protein interactors of LANCL2. In conclusion, LANCL2 promotes tumorigenic proliferation, suppresses apoptosis, and promotes gefitinib+pemetrexed resistance in EGFR-mutant LUAD cells. Based on the positive association between LANCL2, EGFR, and downstream Akt signaling, LANCL2 may be a promising new therapeutic target for EGFR-mutant LUAD.

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