scholarly journals Real-Time PCR Determination of IMPDH1 and IMPDH2 Expression in Blood Cells

2007 ◽  
Vol 53 (6) ◽  
pp. 1023-1029 ◽  
Author(s):  
Sara Bremer ◽  
Helge Rootwelt ◽  
Stein Bergan
Keyword(s):  
Real Time ◽  
Cultured Cells ◽  
De Novo ◽  
Geometric Mean ◽  
Calcineurin Inhibitors ◽  
Housekeeping Genes ◽  
Immunosuppressive Drugs ◽  
Inosine Monophosphate Dehydrogenase ◽  
Gene Expressions ◽  
Nucleotide Synthesis

Abstract Background: Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide synthesis and is implicated in cell cycle control. Inhibition of this enzyme is associated with immunosuppressive, antiviral, and antitumor activity. IMPDH basal activity increases after initiation of immunosuppressive therapy. Methods: A real-time reverse-transcription PCR assay was developed and validated for mRNA quantification of the 2 human IMPDH isoforms. Target gene expressions were normalized to the geometric mean of 3 housekeeping genes. Assay utility was tested by analyzing patient samples and cultured cells exposed to immunosuppressive drugs such as the IMPDH inhibitor mycophenolic acid. Results: The assay was linear over 6 logs of cDNA input and demonstrated specific quantification of IMPDH1 and IMPDH2 expression in cultured cells and patient samples. Limits of detection and quantification were 10 and 103 copies of cDNA per reaction, respectively. Within-run and total between-day CVs were <15% for normalized expression. Changes in IMPDH1 and 2 expression were observed in patient samples after initiation of an immunosuppressive regimen that included calcineurin inhibitors, mycophenolate mofetil, and steroids. Conclusions: This assay can be used to study the regulation of IMPDH expression and the involvement of the enzymes in immunological and malignant proliferative conditions. This may contribute to the processes of drug development and to the establishment of monitoring strategies for treatment effect and disease activity.

scholarly journals Cellular pyrimidine imbalance triggers mitochondrial DNA–dependent innate immunity

2021 ◽  
Author(s):  
Hans-Georg Sprenger ◽  
Thomas MacVicar ◽  
Amir Bahat ◽  
Kai Uwe Fiedler ◽  
Steffen Hermans ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Cultured Cells ◽  
De Novo ◽  
Metabolic Response ◽  
Interferon Response ◽  
Type I ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
De Novo Pyrimidine Synthesis

AbstractCytosolic mitochondrial DNA (mtDNA) elicits a type I interferon response, but signals triggering the release of mtDNA from mitochondria remain enigmatic. Here, we show that mtDNA-dependent immune signalling via the cyclic GMP–AMP synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS–STING–TBK1) pathway is under metabolic control and is induced by cellular pyrimidine deficiency. The mitochondrial protease YME1L preserves pyrimidine pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33. Deficiency of YME1L causes inflammation in mouse retinas and in cultured cells. It drives the release of mtDNA and a cGAS–STING–TBK1-dependent inflammatory response, which requires SLC25A33 and is suppressed upon replenishment of cellular pyrimidine pools. Overexpression of SLC25A33 is sufficient to induce immune signalling by mtDNA. Similarly, depletion of cytosolic nucleotides upon inhibition of de novo pyrimidine synthesis triggers mtDNA-dependent immune responses in wild-type cells. Our results thus identify mtDNA release and innate immune signalling as a metabolic response to cellular pyrimidine deficiencies.

scholarly journals A Review of the Potential Utility of Mycophenolate Mofetil as a Cancer Therapeutic

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Nazanin Majd ◽  
Kazutaka Sumita ◽  
Hirofumi Yoshino ◽  
Dillon Chen ◽  
Jumpei Terakawa ◽  
...  
Keyword(s):  
Mycophenolate Mofetil ◽  
Signaling Pathways ◽  
De Novo ◽  
Cancer Therapeutics ◽  
Inosine Monophosphate Dehydrogenase ◽  
Purine Nucleotide ◽  
Antitumor Effects ◽  
Nucleotide Synthesis ◽  
Potential Target

Tumor cells adapt to their high metabolic state by increasing energy production. To this end, current efforts in molecular cancer therapeutics have been focused on signaling pathways that modulate cellular metabolism. However, targeting such signaling pathways is challenging due to heterogeneity of tumors and recurrent oncogenic mutations. A critical need remains to develop antitumor drugs that target tumor specific pathways. Here, we discuss an energy metabolic pathway that is preferentially activated in several cancers as a potential target for molecular cancer therapy. In vitro studies have revealed that many cancer cells synthesize guanosine triphosphate (GTP), via the de novo purine nucleotide synthesis pathway by upregulating the rate limiting enzyme of this pathway, inosine monophosphate dehydrogenase (IMPDH). Non-proliferating cells use an alternative purine nucleotide synthesis pathway, the salvage pathway, to synthesize GTP. These observations pose IMPDH as a potential target to suppress tumor cell growth. The IMPDH inhibitor, mycophenolate mofetil (MMF), is an FDA-approved immunosuppressive drug. Accumulating evidence shows that, in addition to its immunosuppressive effects, MMF also has antitumor effects via IMPDH inhibition in vitro and in vivo. Here, we review the literature on IMPDH as related to tumorigenesis and the use of MMF as a potential antitumor drug.

scholarly journals Use of Belatacept as Alternative Immunosuppression in Three Renal Transplant Patients withDe NovoDrug-Induced Thrombotic Microangiopathy

2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
Federico Cicora ◽  
Marta Paz ◽  
Fernando Mos ◽  
Javier Roberti
Keyword(s):  
Renal Transplant ◽  
Thrombotic Microangiopathy ◽  
De Novo ◽  
Parvovirus B19 ◽  
Calcineurin Inhibitors ◽  
Immunosuppressive Drugs ◽  
Drug Induced ◽  
Transplant Patients ◽  
Proliferation Signal Inhibitors ◽  
Renal Transplant Patients

Thrombotic microangiopathy (TMA), a severe complication of renal transplantation, is a pathological process involving microvascular occlusion, thrombocytopenia, and microangiopathic hemolytic anemia. It generally appears within the first weeks after transplantation, when immunosuppressive drugs are used at high doses.De novoTMA may also be drug-induced when calcineurin inhibitors or proliferation signal inhibitors are used. We report three cases ofde novodrug-induced TMA in renal transplant patients who were managed by replacing calcineurin inhibitors or proliferation signal inhibitors with belatacept, a primary maintenance immunosuppressive drug, which blocks the CD28 costimulation pathway, preventing the activation of T lymphocytes. To identify the cause of TMA, we ruled out HUS, hepatitis C serology, HIV serology, parvovirus B19, cytomegalovirus, anti-HLA antibodies, and prolonged activated partial thromboplastin time. We suspect that the TMA was caused by the calcineurin inhibitors or proliferation signal inhibitors. Belatacept treatment was initiated at a dose of 10 mg/kg on days 1, 5, 14, 28, 60, and 90; maintenance treatment was 5 mg/kg once a month for 1 year. Belatacept, in combination with other agents, prevented graft rejection in three patients.

scholarly journals Real-Time Quantitative Reverse Transcription-PCR Analysis of Expression Stability of Actinobacillus pleuropneumoniae Housekeeping Genes during In Vitro Growth under Iron-Depleted Conditions

2005 ◽  
Vol 71 (6) ◽  
pp. 2949-2954 ◽  
Author(s):  
K. Klitgaard Nielsen ◽  
M. Boye
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reverse Transcription ◽  
Geometric Mean ◽  
Housekeeping Genes ◽  
Actinobacillus Pleuropneumoniae ◽  
Internal Controls ◽  
Rt Pcr ◽  
Bacterial Gene

ABSTRACT The aims of the present investigation were to develop and test a sensitive and reproducible method for the study of gene expression in the porcine lung pathogen Actinobacillus pleuropneumoniae by real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal controls, as such controls have not been defined yet for this bacterium. Bacterial gene expression was studied during in vitro exponential and early stationary growth in medium with and without sufficient iron, respectively. First, the stability of expression of five genes, the glyA, tpiA, pykA, recF, and rhoAP genes involved in basic housekeeping, was evaluated on the basis of the mean pairwise variation. All the housekeeping genes included were stably expressed under the conditions investigated and consequently were included in the normalization procedure. Next, the geometric mean of the internal control genes was used to correct five genes of interest. These genes were three genes involved in iron acquisition (tbpA, exbB, and fhuD), the heat shock protein gene groEL, and a putative quorum-sensing gene (luxS). The level of tbpA, exbB, and fhuD expression in A. pleuropneumoniae showed significant up-regulation under iron-restricted conditions compared to bacteria grown in medium with sufficient iron. The observed expression patterns of the genes of interest were consistent with previous observations. This study therefore lends further support to the use of real-time quantitative RT-PCR, with the glyA, tpiA, pykA, recF, and rhoAP genes as internal controls, for future similar gene expression studies in A. pleuropneumoniae.

scholarly journals Compositional complexity of rods and rings

2018 ◽  
Vol 29 (19) ◽  
pp. 2303-2316 ◽  
Author(s):  
Cara R. Schiavon ◽  
Maxwell E. Griffin ◽  
Marinella Pirozzi ◽  
Raman Parashuraman ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
De Novo ◽  
Glucose Deprivation ◽  
Cell Types ◽  
Guanine Nucleotide ◽  
Inosine Monophosphate Dehydrogenase ◽  
Nucleotide Synthesis ◽  
Inosine Monophosphate ◽  
Binding Partners ◽  
Rods And Rings

Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH (inosine monophosphate dehydrogenase). They have been observed across a wide variety of cell types and species and can be induced to form by inhibitors of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis; however, the function and regulation of RRs is poorly understood. Here we show that the regulatory GTPase, ARL2, a subset of its binding partners, and several resident proteins at the endoplasmic reticulum (ER) also localize to RRs. We also have identified two new inducers of RR formation: AICAR and glucose deprivation. We demonstrate that RRs can be disassembled if guanine nucleotides can be generated by salvage synthesis regardless of the inducer. Finally, we show that there is an ordered addition of components as RRs mature, with IMPDH first forming aggregates, followed by ARL2, and only later calnexin, a marker of the ER. These findings suggest that RRs are considerably more complex than previously thought and that the function(s) of RRs may include involvement of a regulatory GTPase, its effectors, and potentially contacts with intracellular membranes.

Synthesis and In Vitro Enzymatic Studies of New 3-Aryldiazenyl Indoles as Promising Helicobacter pylori IMPDH Inhibitors

2019 ◽  
Vol 19 (5) ◽  
pp. 376-382 ◽  
Author(s):  
Sachin Jangra ◽  
Gayathri Purushothaman ◽  
Kapil Juvale ◽  
Srimadhavi Ravi ◽  
Aishwarya Menon ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Bacterial Infections ◽  
Immunosuppressive Drugs ◽  
Multi Drug Resistance ◽  
Metabolic Enzyme ◽  
World Population ◽  
Inhibitor Molecule ◽  
Nucleotide Synthesis ◽  
H Pylori

Background & Objective:Helicobacter pylori infection is one of the primary causes of peptic ulcer followed by gastric cancer in the world population. Due to increased occurrences of multi-drug resistance to the currently available antibiotics, there is an urgent need for a new class of drugs against H. pylori. Inosine 5′-monophosphate dehydrogenase (IMPDH), a metabolic enzyme plays a significant role in cell proliferation and cell growth. It catalyses guanine nucleotide synthesis. IMPDH enzyme has been exploited as a target for antiviral, anticancer and immunosuppressive drugs. Recently, bacterial IMPDH has been studied as a potential target for treating bacterial infections. Differences in the structural and kinetic parameters of the eukaryotic and prokaryotic IMPDH make it possible to target bacterial enzyme selectively.Methods:In the current work, we have synthesised and studied the effect of substituted 3-aryldiazenyl indoles on Helicobacter pylori IMPDH (HpIMPDH) activity. The synthesised molecules were examined for their inhibitory potential against recombinant HpIMPDH.Results:In this study, compounds 1 and 2 were found to be the most potent inhibitors amongst the database with IC50 of 0.8 ± 0.02µM and 1 ± 0.03 µM, respectively.Conclusion:When compared to the most potent known HpIMPDH inhibitor molecule C91, 1 was only four-fold less potent and can be a good lead for further development of selective and potent inhibitors of HpIMPDH.

scholarly journals Ever-increasing viral diversity associated with the red imported fire ant Solenopsis invicta (Formicidae: Hymenoptera)

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
César Augusto Diniz Xavier ◽  
Margaret Louise Allen ◽  
Anna Elizabeth Whitfield
Keyword(s):  
Solenopsis Invicta ◽  
De Novo ◽  
Rna Virus ◽  
Housekeeping Genes ◽  
Virus Genome ◽  
Single Strand ◽  
Viral Diversity ◽  
Red Imported Fire Ant ◽  
Sequence Comparisons ◽  
Data Set

Abstract Background Advances in sequencing and analysis tools have facilitated discovery of many new viruses from invertebrates, including ants. Solenopsis invicta is an invasive ant that has quickly spread worldwide causing significant ecological and economic impacts. Its virome has begun to be characterized pertaining to potential use of viruses as natural enemies. Although the S. invicta virome is the best characterized among ants, most studies have been performed in its native range, with less information from invaded areas. Methods Using a metatranscriptome approach, we further identified and molecularly characterized virus sequences associated with S. invicta, in two introduced areas, U.S and Taiwan. The data set used here was obtained from different stages (larvae, pupa, and adults) of S. invicta life cycle. Publicly available RNA sequences from GenBank’s Sequence Read Archive were downloaded and de novo assembled using CLC Genomics Workbench 20.0.1. Contigs were compared against the non-redundant protein sequences and those showing similarity to viral sequences were further analyzed. Results We characterized five putative new viruses associated with S. invicta transcriptomes. Sequence comparisons revealed extensive divergence across ORFs and genomic regions with most of them sharing less than 40% amino acid identity with those closest homologous sequences previously characterized. The first negative-sense single-stranded RNA virus genomic sequences included in the orders Bunyavirales and Mononegavirales are reported. In addition, two positive single-strand virus genome sequences and one single strand DNA virus genome sequence were also identified. While the presence of a putative tenuivirus associated with S. invicta was previously suggested to be a contamination, here we characterized and present strong evidence that Solenopsis invicta virus 14 (SINV-14) is a tenui-like virus that has a long-term association with the ant. Furthermore, based on virus sequence abundance compared to housekeeping genes, phylogenetic relationships, and completeness of viral coding sequences, our results suggest that four of five virus sequences reported, those being SINV-14, SINV-15, SINV-16 and SINV-17, may be associated to viruses actively replicating in the ant S. invicta. Conclusions The present study expands our knowledge about viral diversity associated with S. invicta in introduced areas with potential to be used as biological control agents, which will require further biological characterization.

scholarly journals Regulation of virulence and β-lactamase gene expression in Staphylococcus aureus isolates: cooperation of two-component systems in bloodstream superbugs

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sanaz Dehbashi ◽  
Hamed Tahmasebi ◽  
Behrouz Zeyni ◽  
Mohammad Reza Arabestani
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Gene Expressions ◽  
Resistance Development ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Lactamase Gene

Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA)-bloodstream infections (BSI) are predominantly seen in the hospital or healthcare-associated host. Nevertheless, the interactions of virulence factor (VFs) regulators and β-lactam resistance in MRSA-BSI are unclear. This study aims to characterize the molecular relationship of two-component systems of VFs and the expression of the β-lactamase gene in MRSA-BSI isolates. In this study, 639 samples were collected from BSI and identified by phenotypic methods. We performed extensive molecular characterization, including SCCmec type, agr type, VFs gene profiles determinations, and MLST on isolates. Also, a quantitative real-time PCR (q-RT PCR) assay was developed for identifying the gene expressions. Results Ninety-one (91) S. aureus and 61 MRSA (67.0%) strains were detected in BSI samples. The presence of VFs and SCCmec genes in MRSA isolates were as follows: tst (31.4%), etA (18.0%), etB (8.19%), lukS-PVL (31.4%), lukF-PV (18.0%), lukE-lukD (16.3%), edin (3.2%), hla (16.3%), hlb (18.0%), hld (14.7%), hlg (22.9%), SCCmecI (16.3%), SCCmecII (22.9%), SCCmecIII (36.0%), SCCmecIV (21.3%), and SCCmecV (16.3%). Quantitative real-time PCR showed overexpression of mecRI and mecI in the toxigenic isolates. Moreover, RNAIII and sarA genes were the highest expressions of MRSA strains. The multi-locus sequence typing data confirmed a high prevalence of CC5, CC8, and CC30. However, ST30, ST22, and ST5 were the most prevalent in the resistant and toxigenic strains. Conclusion We demonstrated that although regulation of β-lactamase gene expressions is a significant contributor to resistance development, two-component systems also influence antibiotic resistance development in MRSA-BSI isolates. This indicates that resistant strains might have pathogenic potential. We also confirmed that some MLST types are more successful colonizers with a potential for MRSA-BSI.

scholarly journals Extended haplotype-phasing of long-read de novo genome assemblies using Hi-C

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zev N. Kronenberg ◽  
Arang Rhie ◽  
Sergey Koren ◽  
Gregory T. Concepcion ◽  
Paul Peluso ◽  
...  
Keyword(s):  
Zebra Finch ◽  
Cultured Cells ◽  
De Novo ◽  
Single Cells ◽  
Variant Calling ◽  
Chromatin Interaction ◽  
Extended Haplotype ◽  
Benchmark Datasets ◽  
And Performance ◽  
Genome Assemblies

AbstractHaplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. To date, these assemblies have been best created with complex protocols, such as cultured cells that contain a single-haplotype (haploid) genome, single cells where haplotypes are separated, or co-sequencing of parental genomes in a trio-based approach. These approaches are impractical in most situations. To address this issue, we present FALCON-Phase, a phasing tool that uses ultra-long-range Hi-C chromatin interaction data to extend phase blocks of partially-phased diploid assembles to chromosome or scaffold scale. FALCON-Phase uses the inherent phasing information in Hi-C reads, skipping variant calling, and reduces the computational complexity of phasing. Our method is validated on three benchmark datasets generated as part of the Vertebrate Genomes Project (VGP), including human, cow, and zebra finch, for which high-quality, fully haplotype-resolved assemblies are available using the trio-based approach. FALCON-Phase is accurate without having parental data and performance is better in samples with higher heterozygosity. For cow and zebra finch the accuracy is 97% compared to 80–91% for human. FALCON-Phase is applicable to any draft assembly that contains long primary contigs and phased associate contigs.

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