Genetic Code Expansion in Pseudomonas putida KT2440.

Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs. Using superfolder green fluorescent protein (sfGFP) as an example, this method lays down a solid foundation for future work to study and enhance the biological functions of KT2440.

Arthroscopic Lateral Meniscus Root Repair With Reverse Suture Anchor Technique.

The stability of the knee joint is crucially dependent on the integrity of the lateral meniscus posterior root, which is often accompanied by anterior cruciate ligament injury. Anchor suture repair for lateral meniscus posterior root injury not only achieves better biomechanical effects but also ensures favorable prognosis. However, the placement of anchors often requires the establishment of a posterior approach, and the insertion of an anchor is a technical challenge. In light of this, we have applied the technique of reverse anchor fixation for repairing the lateral meniscus posterior root, which not only simplifies the procedure but also effectively reduces the "bungee effect."

Engineering carboxylic acid reductases and unspecific peroxygenases for flavor and fragrance biosynthesis.

Emerging consumer demand for safer, more sustainable flavors and fragrances has created new challenges for the industry. Enzymatic syntheses represent a promising green production route, but the broad application requires engineering advancements for expanded diversity, improved selectivity, and enhanced stability to be cost-competitive with current methods. This review discusses recent advances and future outlooks for enzyme engineering in this field. We focus on carboxylic acid reductases (CARs) and unspecific peroxygenases (UPOs) that enable selective productions of complex flavor and fragrance molecules. Both enzyme types consist of natural variants with attractive characteristics for biocatalytic applications. Applying protein engineering methods, including rational design and directed evolution in concert with computational modeling, present excellent examples for property improvements to unleash the full potential of enzymes in the biosynthesis of value-added chemicals.

Production of Optically Pure (S)-3-Hydroxy-γ-butyrolactone from d-Xylose Using Engineered Escherichia coli.

Biocatalysis has advantages in asymmetric synthesis due to the excellent stereoselectivity of enzymes. The present study established an efficient biosynthesis pathway for optically pure (S)-3-hydroxy-γ-butyrolactone [(S)-3HγBL] production using engineered Escherichia coli. We mimicked the 1,2,4-butanetriol biosynthesis route and constructed a five-step pathway consisting of d-xylose dehydrogenase, d-xylonolactonase, d-xylonate dehydratase, 2-keto acid decarboxylase, and aldehyde dehydrogenase. The engineered strain harboring the five enzymes could convert d-xylose to 3HγBL with glycerol as the carbon source. Stereochemical analysis by chiral GC proved that the microbially synthesized product was a single isomer, and the enantiomeric excess (ee) value reached 99.3%. (S)-3HγBL production was further enhanced by disrupting the branched pathways responsible for d-xylose uptake and intermediate reduction. Fed-batch fermentation of the best engineered strain showed the highest (S)-3HγBL titer of 3.5 g/L. The volumetric productivity and molar yield of (S)-3HγBL on d-xylose reached 50.6 mg/(L·h) and 52.1%, respectively. The final fermentation product was extracted, purified, and confirmed by NMR. This process utilized renewable d-xylose as the feedstock and offered an alternative approach for the production of the valuable chemical.

Engineering of SH2 Domains for the Recognition of Protein Tyrosine O-Sulfation Sites.

Protein engineering has brought advances to industrial processes, biomaterials, nanotechnology, biosensors, and biomedical applications. This chapter will focus on the engineering of Src Homology 2 domains (SH2) to act as an antibody mimetic for the recognition of sulfotyrosine-containing peptides or proteins. In comparison to anti-sulfotyrosine antibodies, SH2 mutants have much smaller size and can be heterologously expressed and purified in large quantity at low cost. This chapter will describe the use of phage display to identify a sulfotyrosine-binding SH2 mutant and the subsequent enrichment of sulfotyrosine-containing peptides in complex biological samples.

CHI3L1 promotes myocardial fibrosis via regulating lncRNA TUG1/miR-495-3p/ETS1 axis.

Abnormal levels of CHI3L1 and lnc TUG1 are often associated with myocardial fibrosis, and their specific expressions may be closely related to the process of myocardial fibrosis. In addition, CHI3L1 was found to significantly up-regulate the expression of lncTUG1. Therefore, this study further explored the major role of CHI3L1 in regulating the progression of myocardial fibrosis. Myocardial fibrosis in mice was established using an angiotensin (Ang II) model, and the degree of myocardial fibrosis was assessed by qPCR, western blot and pathological techniques. HL-1 cells with overexpression and silencing of CHI3L1 were constructed, and the cell migration ability was detected using the Transwell method. Biological information was used to predict the potential target miRNA of lnc TUG1, and the interaction between them was verified by dual luciferase reporter assay. Using functional rescue assay and the rAAV9 technique, CHI3L1 was verified to affect the fibrotic process of myocardial cells by regulating the lnc TUG1/miR-495-3p/ETS1 axis in vitro and in vivo. The myocardial fibrosis index in the model group was significantly upregulated, and expression of both CHI3L1 and lnc TUG1 was upregulated. Pathological results revealed fibrosis and collagen deposition in the myocardium. Overexpression of lnc TUG1 reversed the inhibitory effect of CHI3L1 silencing on myocardial fibrosis. Mechanistically, CH3L1 upregulates the expression of lnc TUG1, and lnc TUG1 weakens the inhibition of ETS1 through sponge absorption of miR-495-3p, promoting the process of myocardial fibrosis.

Genetic Code Expansion in Mammalian Cells Through Quadruplet Codon Decoding.

Genetic code expansion enables the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins both in vitro and in vivo. In addition to a widely applied nonsense suppression strategy, the use of quadruplet codons could further expand the genetic code. A general approach to genetically incorporate ncAAs in response to quadruplet codons is achieved by utilizing an engineered aminoacyl-tRNA synthetase (aaRS) together with a tRNA variant containing an expanded anticodon loop. Here we provide a protocol to decode quadruplet UAGA codon with a ncAA in mammalian cells. We also describe microscopy imaging and flow cytometry analysis of ncAA mutagenesis in response to quadruplet codons.

Site-Specific Incorporation of Sulfotyrosine into Proteins in Mammalian Cells.

Protein tyrosine O-sulfation (PTS) plays a crucial role in numerous extracellular protein-protein interactions. It is involved in diverse physiological processes and the development of human diseases, including AIDS and cancer. To facilitate the study of PTS in live mammalian cells, an approach for the site-specific synthesis of tyrosine-sulfated proteins (sulfoproteins) was developed. This approach takes advantage of an evolved Escherichia coli tyrosyl-tRNA synthetase to genetically encode sulfotyrosine (sTyr) into any proteins of interest (POI) in response to a UAG stop codon. Here, we give a step-by-step account of the incorporation of sTyr in HEK293T cells using the enhanced green fluorescent protein as an example. This method can be widely applied to incorporating sTyr into any POI to investigate the biological functions of PTS in mammalian cells.

Co-translational Installation of Posttranslational Modifications by Non-canonical Amino Acid Mutagenesis.

Protein posttranslational modifications (PTMs) play critical roles in regulating cellular activities. Here we provide a survey of genetic code expansion (GCE) methods that were applied in the co-translational installation and studies of PTMs through noncanonical amino acid (ncAA) mutagenesis. We begin by reviewing types of PTM that have been installed by GCE with a focus on modifications of tyrosine, serine, threonine, lysine, and arginine residues. We also discuss examples of applying these methods in biological studies. Finally, we end the piece with a short discussion on the challenges and the opportunities of the field.

Proximity-enhanced protein crosslinking through an alkene-tetrazine reaction.

The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for selective labeling of biomolecules. In this work, we exploit the slow reaction between a non-conjugated terminal alkene and a tetrazine, and apply this reaction to achieving a proximity-enhanced protein crosslinking. In one protein subunit, a terminal alkene-containing amino acid was site-specifically incorporated in response to an amber nonsense codon. In another protein subunit, a tetrazine moiety was introduced through the attachment to a cysteine residue. Fast protein crosslinking was achieved due to a large increase in effective molarity of the two reactants that were brought to close proximity by the two interacting protein subunits. Such a proximity-enhanced protein crosslinking is useful for the study of protein-protein interactions.

Genetic Code Expansion in Pseudomonas putida KT2440.

Pseudomonas putida KT2440 is an emerging microbial chassis for biobased chemical production from renewable feedstocks and environmental bioremediation. However, tools for studying, engineering, and modulating protein complexes and biosynthetic enzymes in this organism are largely underdeveloped. Genetic code expansion for the incorporation of unnatural amino acids (unAAs) into proteins can advance such efforts and, furthermore, enable additional controls of biological processes of the strain. In this work, we established the orthogonality of two widely used archaeal tRNA synthetase and tRNA pairs in KT2440. Following the optimization of decoding systems, four unAAs were incorporated into proteins in response to a UAG stop codon at 34.6-78% efficiency. In addition, we demonstrated the utility of genetic code expansion through the incorporation of a photocross-linking amino acid, p-benzoyl-l-phenylalanine (pBpa), into glutathione S-transferase (GstA) and a chemosensory response regulator (CheY) for protein-protein interaction studies in KT2440. This work reported the successful genetic code expansion in KT2440 for the first time. Given the diverse structure and functions of unAAs that have been added to protein syntheses using the archaeal systems, our research lays down a solid foundation for future work to study and enhance the biological functions of KT2440.

Parecoxib sodium alleviates ischemia reperfusion-induced pulmonary injury via inhibiting ERK/NF-κB and further activating the HIF-1α pathway.

INTRODUCTION: The lungs are extremely vulnerable to ischemia/reperfusion (I/R), which is characterized by intense inflammation, oxidative stress, alveolar damage, and vascular permeability. Parecoxib sodium (Pare) has been shown to exert protective effects against multiple I/R-induced tissue injuries. However, its role in I/R-induced lung injury remains unknown. This study aimed to reveal the roles and mechanisms of Pare in pulmonary I/R injury. METHODS: Sixty-six rats were randomly divided into three groups: The sham-operated group, the pulmonary I/R group, and the Pare-pretreated I/R group. Pare at 10 mg/kg or saline (vehicle control) were intraperitoneally administered to rats once per day for 5 consecutive days before ischemia. Serum and tissue samples were harvested following 2 h of reperfusion. The oxygenation index (OI) and alveolar-arterial oxygen partial pressure difference (PA-aO2 ) were analyzed. The levels or activities of malondialdehyde, superoxidase dismutase, catalase, glutathione peroxidase, intracellular reactive oxygen species, tumor necrosis factor-α, interleukin (IL)-6, and IL-8 were examined. The mitochondrial membrane potential was measured. The protein expression levels of the extracellular signal-regulated kinase (ERK), nuclear factor-κB (NF-κB) and their phosphorylated forms, and hypoxia-inducible factor-1α (HIF-1α) were detected. Histological changes were observed using hematoxylin and eosin staining. Moreover, the survival rate following pulmonary I/R injury was recorded daily. RESULTS: Pare significantly increased the OI, decreased the PA-aO2 , increased the levels of antioxidants, while decreasing the levels of oxidants, and alleviated mitochondrial dysfunction and the histopathological damage induced by I/R. Furthermore, Pare inhibited the expression of proinflammatory cytokines, suppressed the activation of ERK and NF-κB, further increased HIF-1α expression, and significantly improved the rat survival rate. CONCLUSIONS: Pare pretreatment attenuated lung I/R injury by inhibiting oxidative stress and the inflammatory response possibly via inhibiting the activation of the ERK/NF-κB pathway and further activating the HIF-1α pathway.

Expanding the chemical diversity of M13 bacteriophage.

Bacteriophage M13 virions are very stable nanoparticles that can be modified by chemical and genetic methods. The capsid proteins can be functionalized in a variety of chemical reactions without loss of particle integrity. In addition, Genetic Code Expansion (GCE) permits the introduction of non-canonical amino acids (ncAAs) into displayed peptides and proteins. The incorporation of ncAAs into phage libraries has led to the discovery of high-affinity binders with low nanomolar dissociation constant (K D) values that can potentially serve as inhibitors. This article reviews how bioconjugation and the incorporation of ncAAs during translation have expanded the chemistry of peptides and proteins displayed by M13 virions for a variety of purposes.

Noncanonical amino acid mutagenesis in response to recoding signal-enhanced quadruplet codons.

While amber suppression is the most common approach to introduce noncanonical amino acids into proteins in live cells, quadruplet codon decoding has potential to enable a greatly expanded genetic code with up to 256 new codons for protein biosynthesis. Since triplet codons are the predominant form of genetic code in nature, quadruplet codon decoding often displays limited efficiency. In this work, we exploited a new approach to significantly improve quadruplet UAGN and AGGN (N = A, U, G, C) codon decoding efficiency by using recoding signals imbedded in mRNA. With representative recoding signals, the expression level of mutant proteins containing UAGN and AGGN codons reached 48% and 98% of that of the wild-type protein, respectively. Furthermore, this strategy mitigates a common concern of reading-through endogenous stop codons with amber suppression-based system. Since synthetic recoding signals are rarely found near the endogenous UAGN and AGGN sequences, a low level of undesirable suppression is expected. Our strategy will greatly enhance the utility of noncanonical amino acid mutagenesis in live-cell studies.

Genetic encoding of a nonhydrolyzable phosphotyrosine analog in mammalian cells.

Protein tyrosine phosphorylation plays a critical role in signal transduction. We report the genetic incorporation of a phosphotyrosine (pTyr) analog, p-carboxymethyl-L-phenylalanine (CMF), into proteins in mammalian cells. This nonhydrolyzable pTyr analog can facilitate biological studies by removing complications caused by the dynamic interconversion between the phosphorylated and non-phosphorylated isoforms of a protein.

Machine learning for artemisinin resistance in malaria treatment across in vivo-in vitro platforms.

Drug resistance has been rapidly evolving with regard to the first-line malaria treatment, artemisinin-based combination therapies. It has been an open question whether predictive models for this drug resistance status can be generalized across in vivo-in vitro transcriptomic measurements. In this study, we present a model that predicts artemisinin treatment resistance developed with transcriptomic information of Plasmodium falciparum. We demonstrated the robustness of this model across in vivo clearance rate and in vitro IC50 measurement and based on different microarray and data processing modalities. The validity of the algorithm is further supported by its first placement in the DREAM Malaria challenge. We identified transcription biomarkers to artemisinin treatment resistance that can predict artemisinin resistance and are conserved in their expression modules. This is a critical step in the research of malaria treatment, as it demonstrated the potential of a platform-robust, personalized model for artemisinin resistance using molecular biomarkers.

Long-term survival of patients with unoperated single ventricle heart defect: Four case reports and literature review.

A single ventricle (SV) heart defect is a rare complex congenital cardiac malformation, accounting for approximately 1%-2% of congenital heart diseases. Surgical intervention is the mainstay treatment for SV patients, although patients who do not receive surgical intervention may also survive. We followed up four adult patients who had SV since birth without surgical intervention and they had a good prognosis. The common characteristics of four long-term SV survivors were investigated by reviewing their medical records and the literature, and the current treatment methods for SV patients were also reviewed. The clinical presentation and long-term prognosis of SV patients without surgical intervention depend on the presence or absence of pulmonary blood flow obstruction, pulmonary vascular resistance, ventricular shape and function, aortic blood flow obstruction, and the atrioventricular valve shape and function. While the Fontan operation has become a common and effective method for SV treatment, long-term outcomes are fraught with morbidity and mortality. In our opinion, such patients with balanced hemodynamic condition could be followed and treated conservatively. Major cardiac surgery which leads to gross hemodynamic adjustments should be avoided. However, additional prospective study will be necessary to verify this assertion. This report aims to improve the prognosis as well as quality of life of SV patients.

Genetic Code Expansion Through Quadruplet Codon Decoding.

Noncanonical amino acid mutagenesis has emerged as a powerful tool for the study of protein structure and function. While triplet nonsense codons, especially the amber codon, have been widely employed, quadruplet codons have attracted attention for the potential of creating additional blank codons for noncanonical amino acids mutagenesis. In this review, we discuss methodologies and applications of quadruplet codon decoding in genetic code expansion both in vitro and in vivo.

A computational prognostic model of lncRNA signature for clear cell renal cell carcinoma with genome instability.

Long non-coding RNAs (lncRNAs) play a critical role in genomic instability and prognosis of cancer patients, but the methods to identify genomic instability-related lncRNAs have yet to be established. In the present study, to assess the prognostic value of lncRNAs associated with genomic instability in clear cell renal cell carcinoma (ccRCC).A computational framework was established based on the mutation hypothesis and combined lncRNA expression and somatic mutation profiles of the ccRCC genome. Furthermore, a prognostic model was developed using the genome instability-derived lncRNA signature GILncSig based on three lncRNA genes (LINC02471, LINC01234, and LINC00460) and verified using multiple independent patient cohorts.This study established an effective computational method to study the role of lncRNAs in genomic instability, with potential applications in identifying new genomic instability-related cancer biomarkers.