In vitro evolution driven by epistasis reveals alternative cholesterol-specific binding motifs of perfringolysin O.

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to the complex makeup of biological membranes. Cholesterol, the major modulator of bilayer structure in mammalian cell membranes, is recognized by various proteins, including the well-studied cholesterol-dependent cytolysins. Here, we use in vitro evolution to investigate the molecular adaptations that preserve the cholesterol specificity of perfringolysin O, the prototypical cholesterol-dependent cytolysin from Clostridium perfringens.

Uncovering translation roadblocks during the development of a synthetic tRNA.

Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other.

Indirect Routes to Aminoacyl-tRNA: The Diversity of Prokaryotic Cysteine Encoding Systems.

Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product.

Bacterial translation machinery for deliberate mistranslation of the genetic code.

Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism.

Intein-based Design Expands Diversity of Selenocysteine Reporters.

The presence of selenocysteine in a protein confers many unique properties that make the production of recombinant selenoproteins desirable. Targeted incorporation of Sec into a protein of choice is possible by exploiting elongation factor Tu-dependent reassignment of UAG codons, a strategy that has been continuously improved by a variety of means. Improving selenoprotein yield by directed evolution requires selection and screening markers that are titratable, have a high dynamic range, enable high-throughput screening, and can discriminate against nonspecific UAG decoding.

Biological Nanopores: Engineering on Demand.

Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques.

Vegetation of Ventriculoatrial Shunt Managed via Multidisciplinary Surgical Approach.

Introduction: The popularity of the ventriculoatrial shunt as a means for cerebrospinal fluid diversion was temporally limited, overcome by the success of the peritoneum as a site for distal drainage. Nevertheless, it remains an important tool for patients for whom ventriculoperitoneal shunting is not an option.

Clinical Presentation: We present the case of a 9-year-old girl with a ventriculoatrial shunt, who had undergone multiple revisions.

Engineering aminoacyl-tRNA synthetases for use in synthetic biology.

Within the broad field of synthetic biology, genetic code expansion (GCE) techniques enable creation of proteins with an expanded set of amino acids. This may be invaluable for applications in therapeutics, bioremediation, and biocatalysis. Central to GCE are aminoacyl-tRNA synthetases (aaRSs) as they link a non-canonical amino acid (ncAA) to their cognate tRNA, allowing ncAA incorporation into proteins on the ribosome.

Plasticity and Constraints of tRNA Aminoacylation Define Directed Evolution of Aminoacyl-tRNA Synthetases.

Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l-α-AA.

Versatility of Synthetic tRNAs in Genetic Code Expansion.

Transfer RNA (tRNA) is a dynamic molecule used by all forms of life as a key component of the translation apparatus. Each tRNA is highly processed, structured, and modified, to accurately deliver amino acids to the ribosome for protein synthesis. The tRNA molecule is a critical component in synthetic biology methods for the synthesis of proteins designed to contain non-canonical amino acids (ncAAs).

Designing seryl-tRNA synthetase for improved serylation of selenocysteine tRNAs.

Selenocysteine (Sec) lacks a cognate aminoacyl-tRNA synthetase. Instead, seryl-tRNA synthetase (SerRS) produces Ser-tRNA , which is subsequently converted by selenocysteine synthase to Sec-tRNA . Escherichia coli SerRS serylates tRNA poorly; this may hinder efficient production of designer selenoproteins in vivo.

Upgrading aminoacyl-tRNA synthetases for genetic code expansion.

Synthesis of proteins with non-canonical amino acids via genetic code expansion is at the forefront of synthetic biology. Progress in this field has enabled site-specific incorporation of over 200 chemically and structurally diverse amino acids into proteins in an increasing number of organisms. This has been facilitated by our ability to repurpose aminoacyl-tRNA synthetases to attach non-canonical amino acids to engineered tRNAs.

Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids.

Synthesis of proteins with noncanonical amino acids (ncAAs) enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in , involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs) and tRNAs (o-tRNAs). Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the metabolism remains largely unexplored.

An archaeal aminoacyl-tRNA synthetase complex for improved substrate quality control.

Aminoacyl-tRNA synthetases (aaRSs) decode genetic information by coupling tRNAs with cognate amino acids. In the archaeon Methanothermobacter thermautotrophicus arginyl- and seryl-tRNA synthetase (ArgRS and SerRS, respectively) form a complex which enhances serylation and facilitates tRNA recycling through its association with the ribosome. Yet, the way by which complex formation participates in Arg-tRNA synthesis is still unresolved.

RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea.

The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNA is first acylated with phosphoserine (Sep) by -phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNA by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, , TACK, and Asgard) and two groups of bacteria (" Parcubacteria" and ).

The central role of tRNA in genetic code expansion.

Background: The development of orthogonal translation systems (OTSs) for genetic code expansion (GCE) has allowed for the incorporation of a diverse array of non-canonical amino acids (ncAA) into proteins. Transfer RNA, the central molecule in the translation of the genetic message into proteins, plays a significant role in the efficiency of ncAA incorporation.

Scope Of Review: Here we review the biochemical basis of OTSs for genetic code expansion.

Pyrrolysyl-tRNA synthetase, an aminoacyl-tRNA synthetase for genetic code expansion.

Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties.

Bioinformatic Analysis Reveals Archaeal tRNA and tRNA Identities in Bacteria.

The tRNA identity elements for some amino acids are distinct between the bacterial and archaeal domains. Searching in recent genomic and metagenomic sequence data, we found some candidate phyla radiation (CPR) bacteria with archaeal tRNA identity for Tyr-tRNA and Trp-tRNA synthesis. These bacteria possess genes for tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase (TrpRS) predicted to be derived from DPANN superphylum archaea, while the cognate tRNA and tRNA genes reveal bacterial or archaeal origins.

Whether to Proceed With Deep Brain Stimulator Battery Change in a Patient With Signs of Potential Sepsis and Parkinson Hyperpyrexia Syndrome: A Case Report.

Parkinsonism-hyperpyrexia syndrome (PHS) is a neurologic emergency associated with anti- Parkinson medication withdrawal; however, its clinical presentation mimics sepsis. We describe the case of a 69-year-old man with advanced Parkinson disease who presented for exchange of the depleted battery in his subthalamic deep brain stimulator. The patient's preoperative symptoms of fever, rigidity, altered consciousness, and autonomic instability presented a dilemma whether to proceed with battery exchange to treat PHS or postpone surgery due to potential sepsis.

Use of jet ventilation in thoracoscopic tracheo-esophageal fistula repair-can both surgeons and anesthesiologists be happy?

Laparoscopic and open thoracic surgery in the neonate typically results in hypercapnea and low cardiac output with often poor surgical visualization as the anesthesiologist attempts to correct the respiratory derangements usually seen. We describe three cases in which jet ventilation provided not only superior ventilation with a return to normocapnea but also ideal operating conditions. In addition, jet ventilation utilizes lower mean airway pressures which typically results in improved cardiac output.

Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution.

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria.

Identification of amino acids in the N-terminal domain of atypical methanogenic-type Seryl-tRNA synthetase critical for tRNA recognition.

Seryl-tRNA synthetase (SerRS) from methanogenic archaeon Methanosarcina barkeri, contains an idiosyncratic N-terminal domain, composed of an antiparallel beta-sheet capped by a helical bundle, connected to the catalytic core by a short linker peptide. It is very different from the coiled-coil tRNA binding domain in bacterial-type SerRS. Because the crystal structure of the methanogenic-type SerRSxtRNA complex has not been obtained, a docking model was produced, which indicated that highly conserved helices H2 and H3 of the N-terminal domain may be important for recognition of the extra arm of tRNA(Ser).