Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs\' ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on plasmid replication proteins (Rep) in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with \"two Reps in one plasmid.\" Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.

Intoxications with organophosphorus compounds (OPCs) effect a severe impairment of cholinergic neurotransmission that, as a result of overstimulation may lead to desensitization of nicotinic acetylcholine receptors (nAChRs) and finally to death due to respiratory paralysis. So far, therapeutics, that are capable to address and revert desensitized neuromuscular nAChRs into their resting, i.e. functional state are still missing. Still, among a class of compounds termed bispyridinium salts, which are characterized by the presence of two pyridinium subunits, constituents have been identified, that can counteract organophosphate poisoning by resensitizing desensitized nAChRs. According to comprehensive modeling studies this effect is mediated by an allosteric binding site at the nAChR termed MB327-PAM-1 site. For MB327, the most prominent representative of the bispyridinium salts and all other analogues studied so far, the affinity for the aforementioned binding site and the intrinsic activity measured in ex vivo and in in vivo experiments are distinctly too low, to meet the criteria to be fulfilled for therapeutic use. Hence, in order to identify new compounds with higher affinities for the MB327-PAM-1 binding site, as a basic requirement for an enhanced potency, two compound libraries, the ChemDiv library with 60 constituents and the Tocriscreen Plus library with 1280 members have been screened for hit compounds addressing the MB327-PAM-1 binding site, utilizing the [2H6]MB327 MS Binding Assay recently developed by us. This led to the identification of a set of 10 chemically diverse compounds, all of which exhibit an IC50 value of ≤ 10 µM (in the [2H6]MB327 MS Binding Assay), which had been defined as selection criteria. The three most affine ligands, which besides a quinazoline scaffold share similarities with regard to the substitution pattern and the nature of the substituents, are UNC0638, UNC0642 and UNC0646. With binding affinities expressed as pKi values of 6.01 ± 0.10, 5.97 ± 0.05 and 6.23 ± 0.02, respectively, these compounds exceed the binding affinity of MB327 by more than one log unit. This renders them promising starting points for the development of drugs for the treatment of organophosphorus poisoning by addressing the MB327-PAM-1 binding site of the nAChR.

The specificity of biological systems makes it possible to develop biosensors targeting specific metabolites, toxins, and pollutants in complex medical or environmental samples without interference from structurally similar compounds. For the last two decades, great efforts have been devoted to creating proteins or nucleic acids with novel properties through synthetic biology strategies. Beyond augmenting biocatalytic activity, expanding target substrate scopes, and enhancing enzymes\' enantioselectivity and stability, an increasing research area is the enhancement of molecular specificity for genetically encoded biosensors. Here, we summarize recent advances in the development of highly specific biosensor systems and their essential applications. First, we describe the rational design principles required to create libraries containing potential mutants with less promiscuity or better specificity. Next, we review the emerging high-throughput screening techniques to engineer biosensing specificity for the desired target. Finally, we examine the computer-aided evaluation and prediction methods to facilitate the construction of ligand-specific biosensors.

Yeasts are single-celled eukaryotic organisms classified as fungi, mostly in the phylum Ascomycota. Of about 1500 named species, Saccharomyces cerevisiae, also known as baker\'s yeast, domesticated by humans in the context of cooking and brewing, is a profound genetic tool for exploring functions of novel effector proteins from Wolbachia and prokaryotes in general. Wolbachia is a Gram-negative alpha-proteobacterium that infects up to ~75% of all insects as an obligate intracellular microbe (Jeyaprakash A, Hoy MA, Insect Mol Biol 9:393-405, 2000). Wolbachia\'s lifestyle presents unique challenges for researchers. Wolbachia cannot be axenically cultured and has never been genetically manipulated. Furthermore, many Wolbachia genes have no known function or well-annotated orthologs in other genomes. Yet given the effects of Wolbachia on host phenotypes, which have considerable practical applications for pest control, they undoubtedly involve secreted effector proteins that interact with host gene products. Studying these effectors is challenging with Wolbachia\'s current genetic limitations. However, some of the constraints to working with Wolbachia can be overcome by expressing candidate proteins in S. cerevisiae. This approach capitalizes on yeast\'s small genome (~6500 genes), typical eukaryotic cellular organization, and the sophisticated suite of genetic tools available for its manipulation in culture. Thus, yeast can serve as a powerful mock eukaryotic host background to study Wolbachia effector function. Specifically, yeast is used for recombinant protein expression, drug discovery, protein localization studies, protein interaction mapping (yeast two-hybrid system), modeling chromosomal evolution, and examining interactions between proteins responsible for complex phenotypes in less tractable prokaryotic systems. As an example, the paired genes responsible for Wolbachia-mediated cytoplasmic incompatibility (CI) encode novel proteins with limited homology to other known proteins, and no obvious function. This article details how S. cerevisiae was used as an initial staging ground to explore the molecular basis of one of Wolbachia\'s trademark phenotypes (CI).

OBJECTIVE: New antibacterial agents are urgently needed to counter increasingly resistant bacteria. One approach to this problem is library screening for new antibacterial agents. Library screening efforts can be improved by increasing the information content of the screening effort. In this study, we screened the National Cancer Institute diversity set V against methicillin-resistant Staphylococcus aureus (MRSA) with several enhancements. One of these is to screen the library before and after microsomal metabolism as means to identify potential active metabolites. A second enhancement is to screen the library in the absence and presence of sub-minimum inhibitory concentration levels of another antibiotic, such as cefoxitin in this study. This identified four agents with synergistic activity with cefoxitin out of 16 agents with good MRSA activity alone. Finally, active agents from this effort were counter-screened in the presence of thymidine, which quickly identified three folate/thymidine biosynthesis inhibitors, and also screened for bactericidal vs bacteriostatic activity.

OBJECTIVE: During their life cycle, bacteria are exposed to a range of different stresses that need to be managed appropriately in order to ensure their growth and viability. This applies not only to bacteria in their natural habitats but also to bacteria employed in biotechnological production processes. Oxidative stress is one of these stresses that may originate either from bacterial metabolism or external factors. In biotechnological settings, it is of critical importance that production strains are resistant to oxidative stresses. Accordingly, this also applies to the major industrial cell factory Bacillus subtilis. In the present study, we, therefore, developed a screen for B. subtilis strains with enhanced oxidative stress tolerance. The results show that our approach is feasible and time-, space-, and resource-efficient. We, therefore, anticipate that it will enhance the development of more robust industrial production strains with improved robustness under conditions of oxidative stress.

Identifying the interactors of a protein is a key step in understanding its possible cellular function(s). Among the various methods that can be used to study protein-protein interactions (PPIs), the yeast two-hybrid (Y2H) assay is one of the most standardized, sensitive, and cost-effective in vivo methods available. The most commonly used GAL4-based Y2H system utilizes the yeast transcription factor GAL4 to detect interactions between soluble proteins. By virtue of involving a transcription factor, the protein-protein interactions occur in the nucleus. The split-ubiquitin Y2H system offers an alternative to the traditional GAL4-based Y2H system and takes advantage of the reconstitution of split-ubiquitin in the cytosol to identify interactions between two proteins. Moreover, new membranous and soluble interacting partner(s) can be identified by screening a target protein against proteins produced from a cDNA library using this system.

A new design concept for pH-sensing supramolecular fluorescent probes is reported. Supramolecular fluorescent pH probes based on pro-guest are designed and prepared. Pro-guests are designed to degrade under acidic condition and convert to competitive guests to displace encapsulated dyes, which leads to a significant enhancement in fluorescence intensity. A library of potential fluorescent pH probes is generated and screened to discover workable probes. These probes are capable of detecting the acidic pH in solution phase. We confirm that these supramolecular probes could detect the acidic environment in endosomal compartments in live cells.

Tumors of the urinary system include those in the urinary and reproductive systems, of which tumors of the prostate, bladder, and kidney have the highest incidence. In recent years, due to changes in dietary structure, prostate cancer has become the most common type of male genitourinary system cancer. Furthermore, due to tobacco consumption, increases in industrialization, and the age of the population, the incidence of bladder cancer in both males and females in both urban and rural areas, has shown an increasing trend. The incidence and mortality of kidney cancer have also increased and negatively affected the lives and health of all residents. While surgery, radiotherapy, and chemotherapy have greatly improved the cure and survival rates of patients with urinary tumors, we lack methods for early detection and effective long-term treatment. New tools and methods for diagnosis and treatment are thus urgently needed. Recently, CRISPR/Cas9 has become an efficient method to alter the genome in many organisms. It can be used to activate or inhibit gene expression, which greatly facilitates the editing of targeted genes, both in vivo and in vitro. It provides a powerful scientific research tool to analyze the mechanisms of disease occurrence and development and to develop advanced targeted drug delivery. The diagnosis and treatment of human tumors will consequently be improved as this technology will surely accelerate cancer research. In this article, we discuss how CRISPR/Cas9 technology can be used to research and treat genitourinary system tumors will consequently be improved as this technology will surely accelerate cancer research. Here, we review the current applications of CRISPR/Cas9 technology for genitourinary system tumor research and therapy.