scholarly journals Genome location dictates the transcriptional response to PolC-inhibition inClostridium difficile

2018 ◽  
Author(s):  
Erika van Eijk ◽  
Ilse M. Boekhoud ◽  
Ed J. Kuijper ◽  
Ingrid M.J.G. Bos-Sanders ◽  
George Wright ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Gene Dosage ◽  
Response Regulator ◽  
Treatment Options ◽  
Gene Copy Number ◽  
Transcriptional Response ◽  
Current Treatment ◽  
Gene Copy ◽  
Growth Defect

AbstractClostridium difficileis a potentially lethal gut pathogen that causes nosocomial and community acquired infections. Limited treatment options and reports of reduced susceptibility to current treatment emphasize the necessity for novel antimicrobials. The DNA-polymerase of gram-positive organisms is an attractive target for the development of antimicrobials. ACX-362E (N2-(3<,4-Dichlorobenzyl)-7-(2-[1-morpholinyl]ethyl)guanine; MorE-DCBG) is a DNA polymerase inhibitor in pre-clinical development as a novel therapeutic againstC. difficileinfection. This synthetic purine shows preferential activity againstC. difficilePolC over those of other organismsin vitroand is effective in an animal model ofC. difficileinfection. In this study we have determined its efficacy against a large collection of clinical isolates. At concentrations below the minimal inhibitory concentration, the presumed slowing (or stalling) of replication forks due to ACX-362E leads to a growth defect. We have determined the transcriptional response ofC. difficileto replication inhibition and observed an overrepresentation of up-regulated genes near the origin of replication in the presence of PolC-inhibitors, but not when cells were subjected to sub-inhibitory concentrations of other antibiotics. This phenomenon can be explained by a gene dosage shift, as we observed a concomitant increase in the ratio between origin-proximal versus terminus-proximal gene copy number upon exposure to PolC-inhibitors. Moreover, we show that certain genes differentially regulated under PolC-inhibition are controlled by the origin-proximal general stress response regulator sigma factor B. Together, these data suggest that genome location both directly and indirectly determines the transcriptional response to replication inhibition inC. difficile.

scholarly journals Genome Location Dictates the Transcriptional Response to PolC Inhibition inClostridium difficile

2018 ◽  
Vol 63 (2) ◽  
pp. e01363-18 ◽  
Author(s):  
Erika van Eijk ◽  
Ilse M. Boekhoud ◽  
Ed J. Kuijper ◽  
Ingrid M. J. G. Bos-Sanders ◽  
George Wright ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Gene Dosage ◽  
Treatment Options ◽  
Gene Copy Number ◽  
Transcriptional Response ◽  
Gene Copy ◽  
Growth Defect ◽  
Preclinical Development ◽  
Content Type

ABSTRACTClostridium difficileis a potentially lethal gut pathogen that causes nosocomial and community-acquired infections. Limited treatment options and reports of reduced susceptibility to current treatment emphasize the necessity for novel antimicrobials. The DNA polymerase of Gram-positive organisms is an attractive target for the development of antimicrobials. ACX-362E [N2-(3,4-dichlorobenzyl)-7-(2-[1-morpholinyl]ethyl)guanine; MorE-DCBG] is a DNA polymerase inhibitor in preclinical development as a novel therapeutic againstC. difficileinfection. This synthetic purine shows preferential activity againstC. difficilePolC over those of other organismsin vitroand is effective in an animal model ofC. difficileinfection. In this study, we have determined its efficacy against a large collection of clinical isolates. At concentrations below the MIC, the presumed slowing (or stalling) of replication forks due to ACX-362E leads to a growth defect. We have determined the transcriptional response ofC. difficileto replication inhibition and observed an overrepresentation of upregulated genes near the origin of replication in the presence of PolC inhibitors, but not when cells were subjected to subinhibitory concentrations of other antibiotics. This phenomenon can be explained by a gene dosage shift, as we observed a concomitant increase in the ratio between origin-proximal and terminus-proximal gene copy number upon exposure to PolC inhibitors. Moreover, we show that certain genes differentially regulated under PolC inhibition are controlled by the origin-proximal general stress response regulator sigma factor B. Together, these data suggest that genome location both directly and indirectly determines the transcriptional response to replication inhibition inC. difficile.

scholarly journals Different Microfluidic Environments for In Vitro Testing of Lipid Nanoparticles against Osteosarcoma

2021 ◽  
Vol 8 (6) ◽  
pp. 77
Author(s):  
Oihane Mitxelena-Iribarren ◽  
Sara Lizarbe-Sancha ◽  
Jay Campisi ◽  
Sergio Arana ◽  
Maite Mujika
Keyword(s):  
Target Therapy ◽  
Treatment Options ◽  
Lipid Nanoparticles ◽  
Cell Number ◽  
Current Treatment ◽  
Drug Dose ◽  
Free Drug ◽  
Tumor Treatment ◽  
Equal Dose

The use of lipid nanoparticles as biodegradable shells for controlled drug delivery shows promise as a more effective and targeted tumor treatment than traditional treatment methods. Although the combination of target therapy with nanotechnology created new hope for cancer treatment, methodological issues during in vitro validation of nanovehicles slowed their application. In the current work, the effect of methotrexate (MTX) encapsulated in different matrices was evaluated in a dynamic microfluidic platform. Effects on the viability of osteosarcoma cells in the presence of recirculation of cell media, free MTX and two types of blank and drug-containing nanoparticles were successfully assessed in different tumor-mimicking microenvironments. Encapsulated MTX was more effective than the equal dose free drug treatment, as cell death significantly increased under the recirculation of both types of drug-loaded nanoparticles in all concentrations. In fact, MTX-nanoparticles reduced cell population 50 times more than the free drug when 150-µM drug dose was recirculated. Moreover, when compared to the equivalent free drug dose recirculation, cell number was reduced 60 and 100 points more under recirculation of each nanoparticle with a 15-µM drug concentration. Thus, the results obtained with the microfluidic model present MTX-lipid nanoparticles as a promising and more effective therapy for pediatric osteosarcoma treatment than current treatment options.

Mesenchymal stem cell-derived extracellular vesicles in the failing heart: past, present, and future

2021 ◽  
Author(s):  
Catherine Karbasiafshar ◽  
Frank W. Sellke ◽  
M. Ruhul Abid
Keyword(s):  
Stem Cell ◽  
Extracellular Vesicles ◽  
Treatment Options ◽  
Current Treatment ◽  
Lifestyle Changes ◽  
Challenges And Opportunities ◽  
Artery Disease ◽  
New Treatment

Cardiovascular disease (CVD) is the leading cause of death globally. Current treatment options include lifestyle changes, medication, and surgical intervention. However, many patients are unsuitable candidates for surgeries due to comorbidities, diffuse coronary artery disease or advanced stages of heart failure. The search for new treatment options has recently transitioned from cell-based therapies to stem-cell derived extracellular vesicles (EVs). A number of challenges remain in the EV field, including the effect of comorbidities, characterization, and delivery, However, recent revolutionary developments and insight into the potential of 'personalizing' EV contents by bioengineering methods to alter specific signaling pathways in the ischemic myocardium hold promise. Here, we discuss the past limitations of cell-based therapies, and recent EV studies involving in vivo, in vitro, and omics, and future challenges and opportunities in EV-based treatments in CVD.

Structure and transcription of the actin gene of Trypanosoma brucei

1988 ◽  
Vol 8 (5) ◽  
pp. 2166-2176 ◽  
Author(s):  
M F Ben Amar ◽  
A Pays ◽  
P Tebabi ◽  
B Dero ◽  
T Seebeck ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Splice Site ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Actin Gene ◽  
S1 Nuclease ◽  
Actin Genes ◽  
Gene Copies ◽  
Actin Mrna

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

scholarly journals Increased gene copy number of DEFA1/DEFA3 worsens sepsis by inducing endothelial pyroptosis

2019 ◽  
Vol 116 (8) ◽  
pp. 3161-3170 ◽  
Author(s):  
QiXing Chen ◽  
Yang Yang ◽  
JinChao Hou ◽  
Qiang Shu ◽  
YiXuan Yin ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Copy Number ◽  
Treatment Options ◽  
Gene Copy Number ◽  
High Copy Number ◽  
Gene Copy ◽  
Dependent Manner ◽  
Barrier Dysfunction ◽  
Specific Expression

Sepsis claims an estimated 30 million episodes and 6 million deaths per year, and treatment options are rather limited. Human neutrophil peptides 1–3 (HNP1–3) are the most abundant neutrophil granule proteins but their neutrophil content varies because of unusually extensive gene copy number polymorphism. A genetic association study found that increased copy number of the HNP-encoding gene DEFA1/DEFA3 is a risk factor for organ dysfunction during sepsis development. However, direct experimental evidence demonstrating that these risk alleles are pathogenic for sepsis is lacking because the genes are present only in some primates and humans. Here, we generate DEFA1/DEFA3 transgenic mice with neutrophil-specific expression of the peptides. We show that mice with high copy number of DEFA1/DEFA3 genes have more severe sepsis-related vital organ damage and mortality than mice with low copy number of DEFA1/DEFA3 or wild-type mice, resulting from more severe endothelial barrier dysfunction and endothelial cell pyroptosis after sepsis challenge. Mechanistically, HNP-1 induces endothelial cell pyroptosis via P2X7 receptor-mediating canonical caspase-1 activation in a NLRP3 inflammasome-dependent manner. Based on these findings, we engineered a monoclonal antibody against HNP-1 to block the interaction with P2X7 and found that the blocking antibody protected mice carrying high copy number of DEFA1/DEFA3 from lethal sepsis. We thus demonstrate that DEFA1/DEFA3 copy number variation strongly modulates sepsis development in vivo and explore a paradigm for the precision treatment of sepsis tailored by individual genetic information.

Gene Expression Profiling of Hyperdiploid Multiple Reveal Complex Gene Dosage Effects and an mRNA Translation/Protein Synthesis Signature.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1538-1538
Author(s):  
Wee-Joo Chng ◽  
Scott Van Wier ◽  
Gregory Ahmann ◽  
Tammy Price-Troska ◽  
Kim Henderson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Differentially Expressed Genes ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Mrna Translation ◽  
Differentially Expressed ◽  
Gene Copy ◽  
Gene Dosage Effect ◽  
Expression Of Genes

Abstract Hyperdiploid MM (H-MM), characterized by recurrent trisomies constitute about 50% of MM, yet very little is known about its pathogenesis and oncogenic mechanisms. Studies in leukemia and solid tumors have shown gene dosage effect of aneuploidy on gene expression. To determine the possible gene dosage effect and deregulated cellular program in H-MM we undertook a gene expression study of CD138-enriched plasma-cell RNA from 53 hyperdiploid and 37 non-hyperdiploid MM (NH-MM) patients using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). Gene expression data was analyzed using GeneSpring 7 (Agilent Technologies, Palo Alto, CA). Genes differentially expressed between H-MM and NH-MM were obtained by t-test (p&lt;0.01). The majority of the differentially expressed genes (57%) were under-expressed in H-MM. Genes located on the commonly trisomic chromosomes were mostly (but not always) over-expressed in H-MM and constitute 76% of over-expressed genes. Chromosome 1 contained the most differentially expressed genes (17%) followed by chromosome 12 (9%), and 19 (8%). To examine the relationship of gene copy number to gene expression, we examined the expression of genes on chromosomes 9 and 15 in subjects with 2 copies (15 normal control and 20 NH-MM) and 3 copies (12 H-MM) of each chromosome as detected by interphase FISH. We then derived a ratio of the mean expression of each gene on these chromosomes between patients with 3 copies and 2 copies of the chromosome. If a simple relationship exists between gene expression and gene copy number, one would expect the ratio of expression of most genes on these two chromosomes to be about 1.5 in H-MM compared to NH-MM. However, many genes have ratios either higher than 2 or lower than 0.5. Furthermore, when the heterogeneity of cells with underlying trisomies is taken into consideration by correcting the ratio for the number of cells with trisomies, the actual ratio is always lower than the expected ratio. When the expression of genes on a chromosome was compressed to a median value, this value was always higher in the trisomic chromosomes for H-MM compared to NH-MM. The data suggests that although gene dosage influence gene expression, the relationship is complex and some genes are more gene dosage dependent than others. Amongst the differentially expressed genes with known function, 33% are involved in mRNA translation/protein synthesis. Of note, 37 of the top 100 differentially expressed genes are involved in these processes. In particular, 60 ribosomal protein (RP) genes are significantly (p&lt;0.05) upregulated in H-MM. This signature in H-MM is not associated with increase proliferation as measured by PCLI. This predominant signature suggests that deregulated protein synthesis may be important for the biology of H-MM. Many of these RPs are involved in the synthesis of product of oncogenic pathways (e.g. MYC, NF-KB pathways) and may mediate the growth and survival of tumor cells. It is therefore possible that these tumor cells may be sensitive to the disruption of mRNA translation/protein synthesis. Targeting the mTOR pathway with rapamycin may therefore be useful for treatment of H-MM.

scholarly journals Influence of Promoter, Gene Copy Number, and Preexisting Immunity on Humoral and Cellular Responses to a Vectored Antigen Delivered by a Salmonella enterica Vaccine

2008 ◽  
Vol 16 (1) ◽  
pp. 78-87 ◽  
Author(s):  
Manvendra Saxena ◽  
Peter J. Coloe ◽  
Peter M. Smooker
Keyword(s):  
Salmonella Enterica ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Heterologous Antigen ◽  
Cell Responses ◽  
Vaccine Trials ◽  
Serovar Typhimurium ◽  
Promoter Gene ◽  
Dna Plasmid

ABSTRACT Attenuated Salmonella strains are currently in production as vaccines for protection of animals against salmonellosis. Such commercial strains offer the potential to deliver heterologous antigen to protect animals against other diseases. One vaccine strain, attenuated Salmonella enterica serovar Typhimurium (STM-1), was tested for the ability to deliver ovalbumin and to induce immune responses in mice. Two vaccine trials were performed testing the influence of promoter choice, the location of the encoding DNA (plasmid or chromosome), and the effect of preexisting homologous or heterologous immunity. The results demonstrated that humoral and T-cell responses were induced from either of two promoters, from either the plasmid or the chromosome, and that preexposure to the empty homologous vector, STM-1, or the heterologous vector, S. enterica serovar Enteritidis, had no detrimental effect on subsequent antigen-specific responses. In the case of homologous preexposure, responses were generally greater, and this was correlated with an increased uptake of Salmonella by macrophages in vitro after opsonization with immune sera.

scholarly journals Rapid Real-Time Fluorescent PCR Gene Dosage Test for the Diagnosis of DNA Duplications and Deletions

2000 ◽  
Vol 46 (10) ◽  
pp. 1574-1582 ◽  
Author(s):  
Clara Ruiz-Ponte ◽  
Lourdes Loidi ◽  
Ana Vega ◽  
Angel Carracedo ◽  
Francisco Barros
Keyword(s):  
Real Time ◽  
Capillary Tube ◽  
Gene Dosage ◽  
Melting Curve ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Target Sequence ◽  
Hereditary Neuropathy ◽  
Target Dna ◽  
Fluorescent Pcr

Abstract Background: Current methods to determine gene dosage are time-consuming and labor-intensive. We describe a new and rapid method to assess gene copy number for identification of DNA duplications or deletions occurring in Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), respectively. Methods: We studied 16 patients with HNPP, 4 with CMT1A, and 49 control subjects. We used real-time PCR on the LightCycler system with use of a single capillary tube and no post-PCR handling. A polymorphic fragment of the PMP22 gene was amplified to determine gene dosage for heterozygous samples. The presence of two alleles was used to indicate that no deletion was present in HNPP samples. The ratio obtained between the areas under each allele melting curve of heterozygous CMT1A samples was used to determine whether the sequence was duplicated or normal. Homozygous samples required a competitive gene dosage test, where the ratio between the areas under the melting curves of the target DNA of samples and of the competitor molecule was used to determine whether the target sequence was duplicated, deleted, or normal. Samples from HNPP, CMT1A, and controls were analyzed. Results: Area ratios were ∼0.6, 1.0, and 2.0 for HNPP, control, and CMT1A samples, respectively. The results agreed with those obtained by Southern blotting and microsatellite analysis in the same samples. Conclusions: Direct and competitive real-time fluorescent PCR can differentiate one, two, or three copies of the target DNA. The method described is sensitive and accurate for detection of CMT1A duplications and HNPP deletions and is faster and easier than current methods.

scholarly journals Differential in vitro activity of individual drugs and bedaquiline-rifabutin combinations against actively multiplying and nutrient-starved Mycobacterium abscessus

2020 ◽  
Author(s):  
Jin Lee ◽  
Nicole Ammerman ◽  
Anusha Agarwal ◽  
Maram Naji ◽  
Si-Yang Li ◽  
...  
Keyword(s):  
Bactericidal Activity ◽  
Treatment Options ◽  
Current Treatment ◽  
Mycobacterium Abscessus ◽  
Bacterial Populations ◽  
Treatment Regimens ◽  
Novel Treatment ◽  
Limited Effectiveness ◽  
Dependent Activity

AbstractCurrent treatment options for lung disease caused by Mycobacterium abscessus complex infections have limited effectiveness. To maximize the use of existing antibacterials and to help inform regimen design for treatment, we assessed the in vitro bactericidal activity of single drugs against actively multiplying and net non-replicating M. abscessus populations in nutrient-rich and nutrient starvation conditions, respectively. As single drugs, bedaquiline and rifabutin exerted bactericidal activity only against nutrient-starved and actively growing M. abscessus, respectively. However, when combined, both bedaquiline and rifabutin were able to specifically contribute bactericidal activity at relatively low, clinically relevant concentrations against both replicating and non-replicating bacterial populations. The addition of a third drug, amikacin, further enhanced the bactericidal activity of the bedaquiline-rifabutin combination against nutrient-starved M. abscessus. Overall, these in vitro data suggest that bedaquiline-rifabutin may be a potent backbone combination to support novel treatment regimens for M. abscessus infections. This rich dataset of differential time-and concentration-dependent activity of drugs, alone and together, against M. abscessus also highlights several issues affecting interpretation and translation of in vitro findings.

scholarly journals Genome instability drives epistatic adaptation in the human pathogen Leishmania

2021 ◽  
Vol 118 (51) ◽  
pp. e2113744118
Author(s):  
Giovanni Bussotti ◽  
Laura Piel ◽  
Pascale Pescher ◽  
Malgorzata A. Domagalska ◽  
K. Shanmugha Rajan ◽  
...  
Keyword(s):  
Translational Control ◽  
Copy Number ◽  
Biomarker Discovery ◽  
Genome Instability ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Copy Number Variations ◽  
Gene Copy ◽  
Snorna Gene ◽  
Ideal System

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2′-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

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