scholarly journals Detectability of Pseudomonassyringae pv. aesculi from European Horse Chestnut Using Quantitative PCR Compared with Traditional Isolation

Forests ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1062
Author(s):  
Salome Schneider ◽  
Christopher Schefer ◽  
Joana Beatrice Meyer
Keyword(s):  
Quantitative Pcr ◽  
Bacterial Growth ◽  
Target Gene ◽  
Horse Chestnut ◽  
Inoculation Experiment ◽  
Copy Numbers ◽  
Infection Stage ◽  
Time Point

Bleeding cankers on horse chestnut trees (Aesculushippocastanum and Aesculus × carnea), caused by Pseudomonassyringae pv. aesculi, have been reported across Europe. In the present study, we show the successful detection of P. syringae pv. aesculi on symptomatic horse chestnut trees in Switzerland using quantitative PCR (qPCR). However, P. syringae pv. aesculi was also detected by qPCR on trees from which no isolate was obtained through cultivation. Reduced isolation success and low copy numbers of the target gene were correlated with the increasing age of symptomatic horse chestnut trees. The potential of detecting non-viable P. syringae pv. aesculi by qPCR was evaluated using an inoculation experiment with dead bacteria and detection by qPCR and cultivation. The detectability of DNA from P. syringae pv. aesculi cells dropped by 34.5% one day after inoculation and then decreased only slightly until the end of the experiment (22 days after inoculation). In contrast, no bacterial growth was observed at any time point after the inactivation of the bacteria. To protect horse chestnut trees, evaluating the viability and actual infection stage of the bacterium may play an important role.

THE CONTRIBUTION OF ENDOGENOUS UROKINASE (UK) AND TISSUE PLASMINOGEN ACTIVATOR (t-PA) TO SPONTANEOUS CLOT LYSIS IN PLASMA

1987 ◽  
Author(s):  
V Gurewich ◽  
F Emmons ◽  
R Pannell
Keyword(s):  
Bacterial Growth ◽  
Dextran Sulfate ◽  
Platelet Rich Plasma ◽  
Clot Lysis ◽  
Lag Phase ◽  
Contact Activation ◽  
Human Fibrinogen ◽  
Tissue Plasminogen ◽  
The Uk ◽  
Time Point

Spontaneous lysis of 125I-labeled clots was measured in order to study the relative contributions of pro-UK, t-PA and contact activation. Clots were made up from 0.3 ml plasma, platelet rich plasma (PRP) or 2% human fibrinogen (Kabi) and suspended in plasma (3 ml) prepared from blood freshly collected onto citrate. The clots were preincubated to inactivate any residual thrombin which could inactivate pro-UK. Antibiotics were added to suppress bacterial growth. Lysis was approximately linear and went to completion with plasma clots in 12-16 d. and with fibrinogen clots in 6-10 d. Spontaneous lysis was inhibited by aprotinin at >200 KlU/ml and by antibody to t-PA. When the latter was added at t 2-5 d., clot lysis was arrested at each time point suggesting that t-PA was predominantly responsible for the lysis and not just for its initiation. Addition of antibody to UK or immunodepletion of plasma had little effect on spontaneous lysis of plasma or of fibrinogen clots, but retarded lysis of PRP clots. Only after ≥20 ng/ml of pro-UK were added to plasma, was a dose-responsive acceleration of clot lysis observed. The addition of antibodies to t-PA inhibited clot lysis by the added pro-UK (20-80 ng/ml) and greatly prolonged the lag phase of clot lysis by higher concentrations of pro-UK (100-200 ng/ml). Contact activation by dextran sulfate (1-5 μM) had no effect on spontaneous clot lysis but potentiated the effect of > 20 ng/ml added pro-UK. It was concluded that 1) The UK content of resting plasma (3-5 ng/ml) contributes little to lysis of platelet-free clots. 2) The presence of physiological concentrations of t-PA is essential for lysis by endogenous UK and markedly potentiates fibrinolysis by added pro-UK (20-200 ng/ml). 3) For endogenous UK to contribute to fibrinolysis, some amplification of its effect by a cofactor other than t-PA seems to be required. The results indicate augmentation of endogenous pro-UK induced fibrinolysis by platelets and that contact activation stimulates clot lysis by added pro-UK.

Abstract P015: Factors Altering Luminal Succinate Effect Blood Pressure

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Kathleen S Hering-Smith ◽  
Weitao Huang ◽  
Fred Teran ◽  
Ryo Sato ◽  
Lee Hamm
Keyword(s):  
Target Gene ◽  
Digital Pcr ◽  
Normal Diet ◽  
Kidney Cortex ◽  
Knock Out ◽  
Acid Diet ◽  
Copy Numbers ◽  
Clearance Studies ◽  
Activity Dependent

Luminal succinate (Suc) has been reported to activate its receptor Sucnr1, stimulate renin release and increase BP in certain contexts. NaDC1 (Na + dicarboxylate cotransporter), located only on apical membrane in the proximal tubule of the kidney, reabsorbs filtered citrate and Suc, and is upregulated in acidosis. NaDC1 regulation is key in preventing stones and in maintaining acid-base homeostasis; but its role in BP regulation is not known. We postulate that luminal Suc alters BP. Our purpose was to examine the role of NaDC1, luminal Suc, and acidosis in BP regulation. To address these issues, we used NaDC1 KO (knock out) and WT (wild type) mice on normal diet or 72 hr acid diet (AD). Acidosis should lower luminal Suc due to upregulation of NaDC1 in WT mice. AD was associated with statistically significant BP decreases in both male and female WT but not in NaDC1 KO. Clearance studies compared Suc infused (SI) with non-infused NaDC1 KO and WT. ANOVA showed borderline increases between some groups: WT males on AD, BP increased from 71.48 ± 2.30 to 81.63 ± 2.16 with SI; and NaDC1 KO females on normal diet BP increased from 86.20 ± 3.98 to 96.48 ± 3.39 with SI. KO was only associated with increased BP in males on AD (p=0.05). Recently Khamaysi et al (JASN 2019) found only activity-dependent BP was altered by Suc. So, in additional studies we used telemetry (BP/T) for 24/7 monitoring of NaDC1 KO vs WT. On normal diets, mean arterial pressure (MAP) was significantly higher in KO than in WT. MAP at 9 pm: KO 122.44 ± 1.85 vs WT 110.40 ± 2.59, p <0.002, 5 am: KO 118.81 ± 2.64 vs WT 103.18 ± 4.00, p <0.01 and 8 am: KO 106.86 ± 1.28 vs 99.22 ± 2.64, p <0.02. Acidosis appeared to lessen these BP/T differences. Thus, increased luminal delivery of succinate due to KO of NaDC1 yields significant MAP increases during active periods. We examined levels of Sucnr1 in kidney cortex using droplet digital PCR (data are expressed as copy numbers of target gene in 1 ng PCR reaction). Sucnr1 expression was higher in NaDC1 KO (1343.6 ± 51) vs WT (1052.8 ± 50, p< 0.007) mice on normal diet. When fed acid diet Sucnr1 expression in KO fell (1039.6 ± 33) but increased in WT (1148 ± 50, p <0.006). In sum luminal Suc predominantly via NaDC1 reabsorption, does influence BP but this is modified by a variety of factors such as acidosis and activity.

scholarly journals In-planta Gene Targeting in Barley using Cas9, with and without Geminiviral Replicons

2021 ◽  
Author(s):  
Tom Lawrenson ◽  
Alison Hinchliffe ◽  
Martha Clarke ◽  
Yvie Morgan ◽  
Wendy Harwood
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Target Gene ◽  
In Planta ◽  
Crop Species ◽  
Increase Copy Number ◽  
Independent Gene ◽  
Copy Numbers ◽  
Wide Range ◽  
Repair Template

AbstractAdvances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications, however this remains inefficient especially in key crop species. Here we describe successful, heritable gene targeting in barley using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase copy number of the repair template. Without the replicon, we were able to delete 150bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared to the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1 and subsequently the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

scholarly journals Impact of the HLA-DRB1 shared epitope on responses to treatment with tofacitinib or abatacept in patients with rheumatoid arthritis

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Wataru Hirose ◽  
Masayoshi Harigai ◽  
Koichi Amano ◽  
Toshihiko Hidaka ◽  
Kenji Itoh ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Disease Activity Score ◽  
Shared Epitope ◽  
Clinical Effectiveness ◽  
Regression Analyses ◽  
Erythrocyte Sedimentation ◽  
Copy Numbers ◽  
The Mean ◽  
The Impact ◽  
Time Point

Abstract Objectives The aim of this study was to compare the clinical effectiveness of tofacitinib and abatacept and clarify the impact of the HLA-DRB1 shared epitope (SE) on responses to these treatments in patients with rheumatoid arthritis (RA). Methods After adjustments by propensity score matching, 70 out of 161 patients receiving tofacitinib and 70 out of 131 receiving abatacept were extracted. The clinical effectiveness of both drugs over 24 weeks and the impact of the copy numbers of SE on effectiveness outcomes were investigated. Results The percentage of patients in remission in the 28-joint count disease activity score using the erythrocyte sedimentation rate (DAS28-ESR) did not significantly differ between patients receiving tofacitinib and abatacept at week 24 (32% vs 37%, p = 0.359). The mean change at week 4 in DAS28-ESR from baseline was significantly greater in patients receiving tofacitinib than in those receiving abatacept (− 1.516 vs − 0.827, p = 0.0003). The percentage of patients in remission at week 4 was 30% with tofacitinib and 15% with abatacept (p = 0.016). When patients were stratified by the copy numbers of SE alleles, differences in these numbers did not affect DAS28-ESR scores of patients receiving tofacitinib. However, among patients receiving abatacept, DAS28-ESR scores were significantly lower in patients carrying 2 copies of SE alleles than in those carrying 0 copies at each time point throughout the 24-week period. Furthermore, the percentage of patients in remission with DAS28-ESR at week 24 was not affected by the copy numbers of SE alleles in patients receiving tofacitinib (p = 0.947), whereas it significantly increased as the copy numbers became higher in patients receiving abatacept (p = 0.00309). Multivariable logistic regression analyses showed a correlation between the presence of SE and DAS28-ESR remission in patients receiving abatacept (OR = 25.881, 95% CI = 3.140–213.351, p = 0.0025), but not in those receiving tofacitinib (OR = 1.473, 95% CI = 0.291–7.446, p = 0.639). Conclusions Although the clinical effectiveness of tofacitinib and abatacept was similar at week 24, tofacitinib was superior to abatacept for changes from baseline in DAS28-ESR and the achievement of remission at week 4. SE positivity was associated with the achievement of DAS28-ESR remission by week 24 in patients receiving abatacept, but not in those receiving tofacitinib.

scholarly journals Detailed temporal dissection of an enhancer cluster reveals two distinct roles for individual elements

2020 ◽  
Author(s):  
Henry Thomas ◽  
Elena Kotova ◽  
Axel Pilz ◽  
Merrit Romeike ◽  
Andreas Lackner ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Target Gene ◽  
Enhancer Element ◽  
Expression Levels ◽  
Genomic Position ◽  
First Intron ◽  
Super Enhancer ◽  
Time Point

AbstractMany genes are regulated by multiple enhancers that often simultaneously activate their target gene. Yet, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that form a previously identified enhancer cluster and activate the Fgf5 locus during exit from naïve murine pluripotency. Four elements are located downstream of the Fgf5 gene and form a super-enhancer. Each of these elements contributes to Fgf5 induction at a distinct time point of differentiation. The fifth element is located in the first intron of the Fgf5 gene and contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. This amplifier element strongly accumulates paused RNA Polymerase II but does not give rise to a mature Fgf5 mRNA. By transplanting the amplifier to a different genomic position, we demonstrate that it enriches for high levels of paused RNA Polymerase II autonomously. Based on our data, we propose a model for a mechanism by which RNA Polymerase II accumulation at a novel type of enhancer element, the amplifier, contributes to enhancer collaboration.

scholarly journals Correlation of Expression of CD25 in Hematopoietic Stem/Progenitor Cell Fraction of Bone Marrow Cells with Response to Tyrosine Kinase Inhibitors in Chronic Myelogenous Leukemia Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3061-3061 ◽  
Author(s):  
Daiki Karigane ◽  
Hidenori Kasahara ◽  
Masatoshi Sakurai ◽  
Eri Matsuki ◽  
Keiichi Tozawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Quantitative Pcr ◽  
Research Funding ◽  
Progenitor Cell ◽  
Kinase Inhibitors ◽  
Bone Marrow Cells ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Copy Numbers ◽  
Tki Discontinuation

Abstract Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of chronic myelogenous leukemia (CML). The treatment with TKIs continues to improve the depth of response and overall survival of CML patients, but the life-long use of TKI is known to be associated with late complications such as cardiovascular events as well as heavy financial burden, and thus impairs the quality of life. To overcome these issues, many studies evaluating the possibility of TKI discontinuation have been ongoing worldwide. In order to achieve durable treatment-free remission, it is crucial to understand the dynamics of CML-leukemia initiating cells (LICs). We previously reported that CD25 was highly expressed in murine and human CML-LICs (Kobayashi CI et al., Blood, 2014). The aim of this study was to assess whether the proportion of CD25 positive cells in hematopoietic stem/progenitor cell fraction of bone marrow cells in CML patients treated with TKIs is associated with their molecular response and could serve as a novel surrogate marker to stop TKI therapy. Methods: Bone marrow samples were obtained from patients with CML in chronic phase who were diagnosed and have been treated solely with TKIs at Keio University Hospital (Tokyo, Japan). This study was approved by the institutional ethical committee and informed consent was obtained from each patient. Both quantitative and qualitative PCR of BCR-ABL was performed using bone marrow mononuclear cells (BMMNCs). The proportion of CD25 positive cells in bone marrow hematopoietic stem/progenitor cell (HSPC; CD34+CD38-) fraction was evaluated by flow cytometry using FITC-labeled anti-CD34, PE- labeled anti-CD38 and APC-labeled anti-CD25 antibodies. The response to TKIs at the time of evaluation was determined according to the previous report (Yoshida C et al., Int J Clin Oncol, 2012): complete cytogenetic remission (CCyR) defined as Philadelphia chromosome undetectable and quantitative PCR copy numbers >731 among BMMNCs; major molecular remission (MMR) as quantitative PCR copy numbers ≤731, and complete molecular remission (CMR) as undetectable BCR-ABL by quantitative and qualitative PCR. Results: Bone marrow samples obtained from 95 patients were evaluated (median age 53 years old; male/female, 67/28). Analysis was performed prior to TKI exposure in nine patients and under TKI therapy including 2nd generation TKI in 64 patients (imatinib, 22; dasatinib, 33; nilotinib, 9). Remaining 22 patients were treatment free because they enrolled in a clinical trial of TKI discontinuation. The proportion of CD25 positive cells in HSPC fraction significantly decreased in patients with prior TKI exposure relative to patients at diagnosis (n=86; Mean 4.2%, SD 7.0% vs n=9; Mean 22.4%, SD 11.3%, P<0.01). In addition, the proportion of CD25 positive cells in HSPC fraction significantly correlated the level of quantitative PCR (Figure, P<0.0001), and MMR was also dividable from CMR by the proportion of CD25 positive cells (MMR; n=29, Mean 4.6%, SD 4.8% vs CMR; n=49, Mean 2.6%, SD 2.5%, P<0.05). The expression of CD25 was still detected in the majority of patients who achieved CMR, including those who sustained CMR after the discontinuation of TKIs. Conclusion: We confirmed that the expression of CD25 in HSPC fraction of CML patients was significantly correlated with the response to TKI therapy, and may serve as an asset to select patients who are likely to achieve durable treatment-free survival. Figure Figure. Disclosures Karigane: Celgene: Honoraria. Sakurai:Celgene: Honoraria. Matsuki:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Nippon Shinyaku: Honoraria. Kikuchi:Celgene: Honoraria; Takeda Pharmaceutical Company: Honoraria; Kyowa Hakko Kirin: Honoraria. Mitsuhashi:LSI Medience: Consultancy. Okamoto:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Astellas Pharma Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Teijin Pharma Limited: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

scholarly journals Rapid screening mutations of first-line-drug-resistant genes in Mycobacterium tuberculosis strains by allele-specific real-time quantitative PCR

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6696 ◽  
Author(s):  
Pengpeng Yang ◽  
Yuzhu Song ◽  
Xueshan Xia ◽  
A-Mei Zhang
Keyword(s):  
Quantitative Pcr ◽  
Pcr Primers ◽  
Drug Resistant ◽  
First Line ◽  
Real Time Quantitative Pcr ◽  
Resistant Genes ◽  
Copy Numbers ◽  
Specificity And Sensitivity ◽  
Allele Specific ◽  
Line Drug

Tuberculosis (TB) is a worldwide health, economic, and social burden, especially in developing countries. Drug-resistant TB is the most serious type of this burden. Thus, it is necessary to screen drug-resistant mutations by using a simple and rapid detection method. A total of 32 pairs of allele-specific PCR (AS-PCR) primers were designed to screen mutation and/or wild-type alleles of 16 variations in four first-line drug-resistant genes (katG, rpoB, rpsL, and embB) of TB strains. A pair of primers was designed to amplify 16S rRNA gene and to verify successful amplification. Subsequently, we tested the specificity and sensitivity of these AS-PCR primers. The optimized condition of these AS-PCR primers was first confirmed. All mutations could be screened in general AS-PCR, but only 13 of 16 variations were intuitively investigated by using real-time quantitative PCR (qPCR) and AS-PCR primers. The results of specificity assay suggested that the AS-PCR primers with mutation and/or wildtype alleles could successfully amplify the corresponding allele under optimized PCR conditions. The sensitivity of nine pairs of primers was 500 copy numbers, and the other seven pairs of primers could successfully amplify correct fragments with a template comprising 103 or 104 copy numbers template. An optimized AS-qPCR was established to screen drug-resistant mutations in TB strains with high specificity and sensitivity.

scholarly journals Comparison of Different Approaches To QuantifyStaphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese

2001 ◽  
Vol 67 (7) ◽  
pp. 3122-3126 ◽  
Author(s):  
Ingeborg Hein ◽  
Angelika Lehner ◽  
Petra Rieck ◽  
Kurt Klein ◽  
Ernst Brandl ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Taqman Probe ◽  
Gene Copy ◽  
Real Time Quantitative Pcr ◽  
Copy Numbers ◽  
Gene Copies ◽  
Different Types ◽  
Pure Cultures ◽  
Gene Copy Numbers

ABSTRACT Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures ofS. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60nuc gene copies/μl) than using a fluorigenic TaqMan probe (6 nuc gene copies/μl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 � 102 to 6.4 � 102 copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.

scholarly journals In-planta Gene Targeting in Barley Using Cas9 With and Without Geminiviral Replicons

2021 ◽  
Vol 3 ◽  
Author(s):  
Tom Lawrenson ◽  
Alison Hinchliffe ◽  
Martha Clarke ◽  
Yvie Morgan ◽  
Wendy Harwood
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Target Gene ◽  
Wheat Dwarf Virus ◽  
In Planta ◽  
Crop Species ◽  
Independent Gene ◽  
Copy Numbers ◽  
Wide Range ◽  
Repair Template

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications; however, this remains inefficient especially in key crop species. Here, we describe successful, heritable gene targeting in barley at the target site of Cas9 using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase the copy number of the repair template. Without the replicon, we were able to delete 150 bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared with the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false-positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1, and subsequently, the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

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