In vivo and in vitro investigation of bacterial type B RNase P interaction with tRNA 3′-CCA

AbstractPlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

Download Full-text

Synthetic Riboswitches for the Analysis of tRNA Processing by eukaryotic RNase P Enzymes

RNA ◽

10.1261/rna.078814.121 ◽

2022 ◽

pp. rna.078814.121

Author(s):

Anna Ender ◽

Nadine Grafl ◽

Tim Kolberg ◽

Sven Findeiss ◽

Peter F. Stadler ◽

...

Keyword(s):

Homo Sapiens ◽

Rnase P ◽

Single Stranded Region ◽

Domains Of Life ◽

The Individual ◽

The Impact ◽

Individual Enzyme ◽

Trna Precursor

Removal of the 5' leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5' leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes – two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

Download Full-text

Factor XII Activation Promotes Platelet Consumption in the Presence of Bacterial-Type Long-Chain Polyphosphate In Vitro and In Vivo

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.118.311193 ◽

2018 ◽

Vol 38 (8) ◽

pp. 1748-1760 ◽

Cited By ~ 15

Author(s):

Jevgenia Zilberman-Rudenko ◽

Stéphanie E. Reitsma ◽

Cristina Puy ◽

Rachel A. Rigg ◽

Stephanie A. Smith ◽

...

Keyword(s):

Factor Xii ◽

Bacterial Type ◽

Long Chain

Download Full-text

A Novel Insight into the Role of Entry Tears in Type B Aortic Dissection: Pressure Measurements in an in Vitro Model

The International Journal of Artificial Organs ◽

10.5301/ijao.5000627 ◽

2017 ◽

Vol 40 (10) ◽

pp. 563-574 ◽

Cited By ~ 3

Author(s):

Stefania Marconi ◽

Ettore Lanzarone ◽

Hector De Beaufort ◽

Michele Conti ◽

Santi Trimarchi ◽

...

Keyword(s):

False Lumen ◽

Patient Characteristics ◽

Acute Type ◽

Type B ◽

Type B Dissection ◽

Vitro Model ◽

Insight Into

Introduction Predicting aortic growth in acute type B dissection is fundamental in planning interventions. Several factors are considered to be growth predictors in the literature and, among them, size and location of entry tears have been recognized to particularly influence the false lumen pressure. In this study, we develop an in vitro setting to analyze the actual impact of size and location of the entry tears on false lumen pressure, in the absence of other confounding factors such as the deformability of the aortic wall. Methods We formalize some indexes that synthetically describe the false lumen pressure with respect to the true lumen pressure. Then, we experimentally derive their values in several configurations of the in vitro setting, and we look for trends in the indexes with respect to the size and location of entry tears. Results: Results show that the tears have a relevant impact on the false lumen pressure, but that their size and location alone are not enough to explain the phenomena observed in vivo. Conclusions To predict the behavior of acute type B dissection, we therefore recommend not limiting to size and location, as many effects may derive from the interactions between these parameters and other patient characteristics.

Download Full-text

Deciphering the tumor-specific immunopeptidome in vivo with genetically engineered mouse models

10.21203/rs.3.rs-669497/v1 ◽

2021 ◽

Author(s):

Tyler Jacks ◽

Alex Jaeger ◽

Lauren Stopfer ◽

Emma Sanders ◽

Demi Sandel ◽

...

Keyword(s):

Transcript Abundance ◽

Genetically Engineered ◽

Ductal Adenocarcinoma ◽

Affinity Tag ◽

Class I Mhc ◽

Mhc I ◽

Novel Approach ◽

In Vitro Investigation

Abstract Effective immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex Class I (MHC-I). Recent developments in proteomics have improved the identification of peptides that are naturally presented by MHC-I, collectively known as the “immunopeptidome”. Current approaches to profile tumor immunopeptidomes have been limited to in vitro investigation, which fails to capture the in vivo repertoire of MHC-I peptides, or bulk tumor lysates, which are obscured by the lack of tumor-specific MHC-I isolation. To overcome these limitations, we report here the engineering of a Cre recombinase-inducible affinity tag into the endogenous mouse MHC-I gene and targeting of this allele to the KrasLSL-G12D/+; p53fl/fl (KP) mouse model (KP; KbStrep). This novel approach has allowed us to isolate tumor-specific MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD) in vivo. With this powerful analytical tool, we were able to profile the evolution of the LUAD immunopeptidome through tumor progression and show that in vivo MHC-I presentation is shaped by post-translational mechanisms. We also uncovered novel, putative LUAD tumor associated antigens (TAAs). Many peptides that were recurrently presented in vivo exhibited very low expression of the cognate mRNA, provoking reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance. Beyond cancer, the KbStrep allele is compatible with a broad range of Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease, and autoimmunity.

Download Full-text

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

10.1101/452920 ◽

2018 ◽

Author(s):

Agnes Karasik ◽

Carol A. Fierke ◽

Markos Koutmos

Keyword(s):

Mitochondrial Diseases ◽

Protein S ◽

Rnase P ◽

Ribonuclease P ◽

Mitochondrial Rna ◽

P Protein ◽

Adenosyl Methionine ◽

Methylation Activity

ABSTRACTHuman mitochondrial ribonuclease P (mtRNase P) is an essential three protein complex that catalyzes the 5’ end maturation of mitochondrial precursor tRNAs (pre-tRNAs). MRPP3 (Mitochondrial RNase P Protein 3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl methionine (SAM)-dependent methyltransferase MRPP1/2 sub-complex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the MRPP1 co-factor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process and methylate human mitochondrial pre-tRNAs.

Download Full-text

Comparative in vitro and in vivo activity of temocillin (BRL 17421) and ampicillin against Haemophilus influenzae type b.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.23.1.182 ◽

1983 ◽

Vol 23 (1) ◽

pp. 182-184 ◽

Cited By ~ 2

Author(s):

R Yogev ◽

W Glogowski ◽

E Connor

Keyword(s):

Haemophilus Influenzae ◽

Haemophilus Influenzae Type ◽

Haemophilus Influenzae Type B ◽

Type B

Download Full-text

Infrared Spectroscopy with a Fiber-Coupled Quantum Cascade Laser for Attenuated Total Reflection Measurements Towards Biomedical Applications

Sensors ◽

10.3390/s19235130 ◽

2019 ◽

Vol 19 (23) ◽

pp. 5130 ◽

Cited By ~ 3

Author(s):

Ine L. Jernelv ◽

Karina Strøm ◽

Dag Roar Hjelme ◽

Astrid Aksnes

Keyword(s):

Infrared Spectroscopy ◽

Quantum Cascade Lasers ◽

Spectral Power ◽

Total Reflection ◽

Attenuated Total Reflection ◽

Mid Infrared ◽

Quantum Cascade ◽

In Vitro Investigation

The development of rapid and accurate biomedical laser spectroscopy systems in the mid-infrared has been enabled by the commercial availability of external-cavity quantum cascade lasers (EC-QCLs). EC-QCLs are a preferable alternative to benchtop instruments such as Fourier transform infrared spectrometers for sensor development as they are small and have high spectral power density. They also allow for the investigation of multiple analytes due to their broad tuneability and through the use of multivariate analysis. This article presents an in vitro investigation with two fiber-coupled measurement setups based on attenuated total reflection spectroscopy and direct transmission spectroscopy for sensing. A pulsed EC-QCL (1200–900 cm − 1 ) was used for measurements of glucose and albumin in aqueous solutions, with lactate and urea as interferents. This analyte composition was chosen as an example of a complex aqueous solution with relevance for biomedical sensors. Glucose concentrations were determined in both setup types with root-mean-square error of cross-validation (RMSECV) of less than 20 mg/dL using partial least-squares (PLS) regression. These results demonstrate accurate analyte measurements, and are promising for further development of fiber-coupled, miniaturised in vivo sensors based on mid-infrared spectroscopy.

Download Full-text

In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: a novel approach for cancer treatment

Blood ◽

10.1182/blood.v95.3.731.003k28_731_737 ◽

2000 ◽

Vol 95 (3) ◽

pp. 731-737 ◽

Cited By ~ 58

Author(s):

C. Cobaleda ◽

I. Sánchez-Garcı́a

Keyword(s):

Cancer Treatment ◽

Chromosomal Abnormalities ◽

Human Cancer ◽

Rnase P ◽

Catalytic Rna ◽

Fusion Genes ◽

Novel Approach ◽

Chimeric Molecules

One major obstacle to the effective treatment of cancer is to distinguish between tumor cells and normal cells. The chimeric molecules created by cancer-associated chromosomal abnormalities are ideal therapeutic targets because they are unique to the disease. We describe the use of a novel approach based on the catalytic RNA subunit of RNase P to destroy specifically the tumor-specific fusion genes created as a result of chromosome abnormalities. Using as a target model the abnormal BCR-ABL p190 and p210 products, we constructed M1-RNA with guide sequences that recognized the oncogenic messengers at the fusion point (M1-p190-GS and M1-p210-GS). To test the effectiveness and the specificity of M1-p190-GS and M1-p210-GS, we studied in vitro and in vivo effects of these RNA enzymes againstBCR-ABLp190 andBCR-ABLp210, bearing in mind that both fusion genes share the ABL sequence but differ in the sequence coming from the BCR gene. We showed that M1-p190-GS and M1-p210-GS can act as sequence-specific endonucleases and can exclusively cleave target RNA that forms a base pair with the guide sequence (GS). We also demonstrated that when M1-p190-GS and M1-p210-GS were expressed in proper mammalian cell models, they abolished the effect of BCR-ABL by specifically decreasing the amount of the target BCR-ABL mRNA and preventing the function of theBCR-ABL oncogenes. These data clearly demonstrate the usefulness of the catalytic activity of M1-GS RNA to cleave specifically the chimeric molecules created by chromosomal abnormalities in human cancer and to represent a novel approach to cancer treatment.

Download Full-text

Degradation and erosion mechanisms of bioresorbable porous acellular vascular grafts: an in vitro investigation

Journal of The Royal Society Interface ◽

10.1098/rsif.2017.0102 ◽

2017 ◽

Vol 14 (132) ◽

pp. 20170102 ◽

Cited By ~ 8

Author(s):

Piyusha S. Gade ◽

Keewon Lee ◽

Blaise N. Pfaff ◽

Yadong Wang ◽

Anne M. Robertson

Keyword(s):

Mechanical Properties ◽

Mass Loss ◽

Mechanistic Model ◽

Vascular Grafts ◽

Damage Function ◽

Surface Erosion ◽

Outer Diameter ◽

In Vitro Investigation

A fundamental mechanism of in situ tissue regeneration from biodegradable synthetic acellular vascular grafts is the effective interplay between graft degradation, erosion and the production of extracellular matrix. In order to understand this crucial process of graft erosion and degradation, we conducted an in vitro investigation of grafts ( n = 4 at days 1, 4, 7, 10 each) exposed to enzymatic degradation. Herein, we provide constitutive relationships for mass loss and mechanical properties based on much-needed experimental data. Furthermore, we formulate a mathematical model to provide a physics-based framework for understanding graft erosion. A novel finding is that despite their porous nature, grafts lost mass exponentially via surface erosion demonstrating a 20% reduction in outer diameter and no significant change in apparent density. A diffusion based, concentration gradient-driven mechanistic model of mass loss through surface erosion was introduced which can be extended to an in vivo setting through the use of two degradation parameters. Furthermore, notably, mechanical properties of degrading grafts did not scale with mass loss. Thus, we introduced a damage function scaling a neo-Hookean model to describe mechanical properties of the degrading graft; a refinement to existing mass-dependent growth and remodelling (G&R) models. This framework can be used to improve accuracy of well-established G&R theories in biomechanics; tools that predict evolving structure–function relationships of neotissues and guide graft design.

Download Full-text