Identification of inhibitors of A to l editing

RNA-editing ist ein konservierter Mechanismus zur Diversifizierung der RNA von der genetisch codierten Information. Adenosin zu Inosin (A zu I) Editing bewirkt eine Informationsänderung an der RNA von A zu G und wird durch Doppelsträngige RNA spezifische Adenosin Deaminasen (ADARs)positionsabhängig katalysiert. Die biologische Bedeutung von Editing ist weitreichend und umfasst antivirale Mechanismen, alternatives Splicing, selektive Codonumwandlung, als auchModulierung der microRNA Prozessierung. Defekte in der Regulation des RNA Editings werden mit verschiedenen neuronalen Erkrankungen des Menschen in Verbindung gebracht wie Depression und Epilepsie. Auch wurde vermindertes RNA Editing als Eigenschaft von Tumorgewebe identifiziert. Bislang wird angenommen, dass die Aktivität von ADAR2 über RNA Splicing und post-translationelle Modifikationen reguliert wird. Um potentielle Modulatoren der ADAR2 Proteinaktivität zu identifizieren, wurde ein Editing System in Hefe konstruiert. Dieses System enthält ein mutiertes URA3 Gen, in dessen 5‘ Region ein UAG Stop Codon liegt, dieses jedoch durch ADAR2 in ein codierendes Tryptophan Codon editiert wird. Die Konvertierung dieses internen Stop Codons gestattet in Folge die Expression von URA3. Durch Anwesenheit des Selektionsmittels 5-FOA (5-Fluororotsäure) wird nun das Wachstum der URA3 exprimierenden Zellen jedoch verhindert. Um Editing inhibierende Proteine zu finden, wurde eine humane cDNA Bibliothek in das Systemtransformiert. Durch 5-FOA können Kolonien nur aus jenen Transformanten hervorgehen, in denen ADAR2 Editing inhibiert wurde. DiecDNA möglicher Editinginhibitoren wurde aus den gewonnenen Transformanten isoliert. Um die Relevanz potentieller ADAR2 Inhibitoren in Säugerzellen zu bestimmen, wurde für die Sekundäranalyse ein Zellkulturreportersystem in Säugerzellen etabliert und eingesetzt. Letztlich konnten drei RNA bindende Proteine, RPS-14 (kleine ribosomale Untereinheit, Protein 14), SFRS-9 (Splicing Faktor, Arginin/Serin-reich 9), und DDX-15 (DEAH-box protein 15), als wahrscheinliche Editing-Inhibitoren identifiziert werden. Mittels Kolokalisations- und Koimmunopräzipitationsexperimenten konnten RPS-14 und SFRS-9 als ADAR2-Interaktoren identifiziert werden. Weiters konnte für RPS-14, SFRS-9 und DDX-15 eine Beteiligung in der Editing-Inhibierung an endogenen ADAR2 Substraten nachgewiesen werden. Zudem konnte die Editing inhibierende Aktivität von DDX-15im Fadenwurm Caenorhabditis elegans wiedergefunden werden, Indiz für einen konservierten Mechanismus. Unsere Resulate lassen daher auf die erfolgreiche Identifikation von drei bisher unbekannten Inhibitoren des ADAR2 vermittelten RNA Editings, RPS-14, SFRS-9 und DDX-15, schliessen.RNA editing is a conserved posttranscriptional mechanism diversifying RNA and proteins from limited genetic information. A to I editing is carried out by ADARs (Bass). Editing has profound effect in various biological processes as anti-viral defense, alternation of splicing, codon changes and modulation of microRNA processing. Editing deregulation has been linked to CNS diseases as depression and epilepsy. Recently, global hypoediting has been found in tumor tissues indicating a correlation between editing and cancer. Until now, splicing and post translational modifications are proposed to modulate ADAR2 activity. To identify potential cellular modulators of ADAR 2 activity a yeast-editing assay was established. In this assay, an amber stop codon located in a stem loop in the 5’ region of the URA3 gene can be edited into a tryptophan codon by ADAR2. Conversion of the amber codon to a tryptophan codon thereby allows URA3 expression. However, addition of the drug 5-FOA selectively kills the cells expressing URA3. To identify cellular proteins that inhibit editing, a human cDNA library was transformed into the reporter strain. In the presence of 5-FOA only colonies that fail to express URA3, i.e colonies in which editing was inhibited could grow. A tissue culture reporter assay was established for secondary screening of the potential inhibitors in the mammalian system. Three RNA binding candidate proteins RPS-14 (small subunit ribosomal protein 14), SFRS-9 (splicing factor, arginine/serine rich 9), and DDX-15 (DEAH-box protein 15, pre-mRNA processing factor) were isolated from this screen as inhibitors of editing in mammalian cells. Further co-localization and co-immunoprecipitation studies are done in order to dissect the mode of interaction. RPS-14 and SFRS-9 were found to interact with ADAR2. RPS-14, SFRS-9, and DDX-15 show editing inhibition on endogenous substrates. DDX-15 entailed ADAR2 activity inhibition is conserved in Caenorhabditis elegans when compared to other RNA helicases. Our results indicate that RPS-14, SFRS-9 and DDX-15 are potential cellular inhibitors of ADAR2 mediated editing, proving the functionality of the candidates in mammalian cells

Is atomic rearrangement of type IV PHA synthases responsible for increased PHA production?

<div><p><b>Background</b>: Type IV PHA synthase is a key enzyme responsible for catalyzing the formation of non-toxic, biocompatible, and biodegradable short-chain-length polyhydroxyalkanoates (scl-PHA) under the growth-limiting conditions in the members of the genus <i>Bacillus</i>. <b>Results</b>: The comparative <i>in vitro</i> and <i>in silico</i> analysis of the <i>phaC</i> subunit of type IV PHA synthases among <i>Bacillus cereus</i> FA11, <i>B. cereus</i> FC11, and <i>B. cereus</i> FS1 was done in our study to determine its structural and functional properties. Conserved domain analysis demonstrated that <i>phaC</i> subunit belongs to the alpha/beta (α/β) hydrolase fold. The catalytic triad comprising of cysteine (Cys), histidine (His), and aspartate (Asp) was found to be present at the active site. A shorter inter-atomic distance was found between the carboxyl (–COO) group of Asp and amino (NH₂) group of His. Furthermore, slightly long inter-atomic distances between sulfhydryl (SH) group of Cys and NH₂ group of His may be pointing toward the broader substrate specificity of type IV PHA synthases. However, a shorter distance between the SH group of Cys and NH₂ group of His in case of <i>B. cereus</i> FC11 leads to a higher enzymatic activity and maximum PHA yield (49.26%). <b>Conclusion</b>: The <i>in silico</i> study verifies that the close proximity between SH group of Cys and NH₂ group of His in <i>phaC</i> subunit of type IV PHA synthases can be crucial for synthesis of scl-PHA. However, the catalytic activity of type IV PHA synthases declines as the distance between the sulfur (S) atom of the SH group of Cys and the nitrogen (N) atom of NH₂ group of His increases.</p></div

Modulation of ADAR mRNA expression in patients with congenital heart defects.

Adenosine (A) to inosine (I) RNA editing is a hydrolytic deamination reaction catalyzed by the adenosine deaminase (ADAR) enzyme acting on double-stranded RNA. This posttranscriptional process diversifies a plethora of transcripts, including coding and noncoding RNAs. Interestingly, few studies have been carried out to determine the role of RNA editing in vascular disease. The aim of this study was to determine the potential role of ADARs in congenital heart disease. Strong downregulation of ADAR2 and increase in ADAR1 expression was observed in blood samples from congenital heart disease (CHD) patients. The decrease in expression of ADAR2 was in line with its downregulation in ventricular tissues of dilated cardiomyopathy patients. To further decipher the plausible regulatory pathway of ADAR2 with respect to heart physiology, miRNA profiling of ADAR2 was performed on tissues from ADAR2-/- mouse hearts. Downregulation of miRNAs (miR-29b, miR-405, and miR-19) associated with cardiomyopathy and cardiac fibrosis was observed. Moreover, the upregulation of miR-29b targets COL1A2 and IGF1, indicated that ADAR2 might be involved in cardiac myopathy. The ADAR2 target vascular development associated protein-coding gene filamin B (FLNB) was selected. The editing levels of FLNB were dramatically reduced in ADAR2-/- mice; however, no observable changes in FLNB expression were noted in ADAR2-/- mice compared to wild-type mice. This study proposes that sufficient ADAR2 enzyme activity might play a vital role in preventing cardiovascular defects

Capillary-driven flow microfluidics combined with smartphone detection: an emerging tool for point-of-care diagnostics

Point-of-care (POC) or near-patient testing allows clinicians to accurately achieve real-time diagnostic results performed at or near to the patient site. The outlook of POC devices is to provide quicker analyses that can lead to well-informed clinical decisions and hence improve the health of patients at the point-of-need. Microfluidics plays an important role in the development of POC devices. However, requirements of handling expertise, pumping systems and complex fluidic controls make the technology unaffordable to the current healthcare systems in the world. In recent years, capillary-driven flow microfluidics has emerged as an attractive microfluidic-based technology to overcome these limitations by offering robust, cost-effective and simple-to-operate devices. The internal wall of the microchannels can be pre-coated with reagents, and by merely dipping the device into the patient sample, the sample can be loaded into the microchannel driven by capillary forces and can be detected via handheld or smartphone-based detectors. The capabilities of capillary-driven flow devices have not been fully exploited in developing POC diagnostics, especially for antimicrobial resistance studies in clinical settings. The purpose of this review is to open up this field of microfluidics to the ever-expanding microfluidic-based scientific community

Expression and applications of HriCFP in E. coli: a novel biosensing fluorescent protein

Metalloids and heavy metal contamination in the environment has become a global problem. Therefore, there is a dire need to develop effective and inexpensive approaches that can facilitate efficient monitoring of the hazardous level of these environmental pollutants. Microbial cell-based and fluorescent protein-based biosensors offer relatively convenient and inexpensive tools for the analysis of environmental pollutants as opposed to traditional instrumental approaches. Small size fluorescent proteins can withstand exposure to denaturants, high temperature and a wide pH range. These characteristics, along with their potential of sensing different toxic analytes, makes them a suitable candidate for developing on-site detection biosensors. The current study exploits the biosensing potential of a novel fluorescent protein called HriCFP. HriCFP was expressed in the prokaryotic system (gram-negative E. coli), which showed stable and discreet expression in bacterial cells. Whole-cell biosensors (WCB) were developed by immobilization of HriCFP expressing non-pathogenic E. coli via nitrocellulose membrane, low melting agarose and sodium silicate gel. These immobilized biosensors were tested for their sensitivity of detection for environmental pollutants, i.e., heavy metals (Cu(II), Hg(II), As(III)). These WCBs exhibited profound fluorescent quenching when exposed to a range of heavy metals. These biosensors remained active for 12 days at 4 °C, demonstrating their potential for long-term stability and storage. This study implies that HriCFP may have a significant advantage over other larger and multimeric proteins as it has a minimal impact on host strain metabolism and hence, increasing its sustainability for a longer period

Evolutionary Dynamics of Foot and Mouth Disease Virus Serotype A and Its Endemic Sub-Lineage A/ASIA/Iran-05/SIS-13 in Pakistan

Foot and mouth disease (FMD) causes severe economic losses to the livestock industry of endemic countries, including Pakistan. Pakistan is part of the endemic pool 3 for foot and mouth disease viruses (FMDV), characterized by co-circulating O, A, and Asia 1 serotypes, as designated by the world reference laboratory for FMD (WRL-FMD). FMDV serotype A lineage ASIA/Iran-05 is widespread in buffalos and cattle populations and was first reported in Pakistan in 2006. This lineage has a high turnover, with as many as 10 sub-lineages reported from Pakistan over the years. In this study, we reconstructed the evolutionary, demographic, and spatial history of serotype A and one of its sub-lineages, A/ASIA/Iran-05/SIS-13, prevalent in Pakistan. We sequenced nearly complete genomes of three isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13. We estimated recombination patterns and natural selection acting on the serotype A genomes. Source and transmission routes in Pakistan were inferred, and the clustering pattern of isolates of the SIS-13 sub-lineage were mapped on a tree. We hereby report nearly complete genome sequences of isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13, along with purported recombinant genomes, and highlight that complete coding sequences can better elucidate the endemic history and evolutionary pressures acting on long-term co-circulating FMDV strains

Increased expression levels of Syntaxin 1A and Synaptobrevin 2/Vesicle-Associated Membrane Protein-2 are associated with the progression of bladder cancer

Abstract Gene expression is tightly regulated in time and space through a multitude of factors consisting of signaling molecules. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNARE) are membrane proteins responsible for the intercellular trafficking of signals through endocytosis and exocytosis of vesicles. Altered expression of SNARE proteins in cellular communication is the major hallmark of cancer phenotypes as indicated in recent studies. SNAREs play an important role in maintaining cell growth and epithelial membrane permeability of the bladder and are not only involved in cancer progression but also metastatic cell invasion through SNARE-mediated trafficking. Synaptobrevin2/Vesicle associated membrane protein-2 (v-SNARE) and Syntaxin (t-SNARE) form a vesicular docking complex during endocytosis. Some earlier studies have shown a critical role of SNARE in colon, lungs, and breast cancer progression and metastasis. In this study, we analyzed the relative expression of the STX1A and VAMP2 (SYB2) for their possible association in the progression and metastasis of bladder cancer. The profiling of the genes showed a significant increase in STX1A and VAMP2 expression (p < 0.001) in high-grade tumor cells compared to normal and low-grade tumors. These findings suggest that elevated expression of STX1A and VAMP2 might have caused the abnormal progression and invasion of cancer cells leading to the transformation of cells into high-grade tumor in bladder cancer

A natural antisense lncRNA controls breast cancer progression by promoting tumor suppressor gene mRNA stability.

The human genome encodes thousands of long noncoding RNA (lncRNA) genes; the function of majority of them is poorly understood. Aberrant expression of a significant number of lncRNAs is observed in various diseases, including cancer. To gain insights into the role of lncRNAs in breast cancer progression, we performed genome-wide transcriptome analyses in an isogenic, triple negative breast cancer (TNBC/basal-like) progression cell lines using a 3D cell culture model. We identified significantly altered expression of 1853 lncRNAs, including ~500 natural antisense transcript (NATs) lncRNAs. A significant number of breast cancer-deregulated NATs displayed co-regulated expression with oncogenic and tumor suppressor protein-coding genes in cis. Further studies on one such NAT, PDCD4-AS1 lncRNA reveal that it positively regulates the expression and activity of the tumor suppressor PDCD4 in mammary epithelial cells. Both PDCD4-AS1 and PDCD4 show reduced expression in TNBC cell lines and in patients, and depletion of PDCD4-AS1 compromised the cellular levels and activity of PDCD4. Further, tumorigenic properties of PDCD4-AS1-depleted TNBC cells were rescued by exogenous expression of PDCD4, implying that PDCD4-AS1 acts upstream of PDCD4. Mechanistically, PDCD4-AS1 stabilizes PDCD4 RNA by forming RNA duplex and controls the interaction between PDCD4 RNA and RNA decay promoting factors such as HuR. Our studies demonstrate crucial roles played by NAT lncRNAs in regulating post-transcriptional gene expression of key oncogenic or tumor suppressor genes, thereby contributing to TNBC progression