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.

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.

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.

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.

The differential expression patterns and co-expression networks of paralogs as an indicator of the TNM stages of lung adenocarcinoma and squamous cell carcinoma.

Cancers constitute a severe threat to human health. Elucidating the association between the expression patterns of the paralogous genes and transcription factors (TF) and the progression of cancers by comprehensively investigating the expression patterns and co-expression networks will contribute to the in-depth understanding of the pathogenesis of cancers. Here, we identified the paralogous gene pairs and systematically analyzed the expression patterns of these paralogs and the known TFs to elucidate the associations with Tumor, Node, Metastasis (TNM) staging information across ten cancers. We found that the expression of ~60% paralogs was cancer-dependent, and more than 50% of the differentially expressed TFs pairs showed positive expression correlations. The down-regulation patterns of paralogs and TFs were closely associated with the M and N developmental stages of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Our results will help to understand the roles of paralogs and TFs in cancer progression and to screen prognostic biomarkers for early cancer diagnosis.

Direct biosynthesis of adipic acid from lignin-derived aromatics using engineered Pseudomonas putida KT2440.

Lignin is one largely untapped natural resource that can be exploited as a raw material for the bioproduction of value-added chemicals. Meanwhile, the current petroleum-based process for the production of adipic acid faces sustainability challenges. Here we report the successful engineering of Pseudomonas putida KT2440 strain for the direct biosynthesis of adipic acid from lignin-derived aromatics. The devised bio-adipic acid route features an artificial biosynthetic pathway that is connected to the endogenous aromatics degradation pathway of the host at the branching point, 3-ketoadipoyl-CoA, by taking advantage of the unique carbon skeleton of this key intermediate. Studies of the metabolism of 3-ketoadipoyl-CoA led to the discovery of crosstalk between two aromatics degradation pathways in KT2440. This knowledge facilitated the formulation and implementation of metabolic engineering strategies to optimize the carbon flux into the biosynthesis of adipic acid. By optimizing pathway expression and cultivation conditions, an engineered strain AA-1 produced adipic acid at 0.76 g/L and 18.4% molar yield under shake-flask conditions and 2.5 g/L and 17.4% molar yield under fermenter-controlled conditions from common aromatics that can be derived from lignin. This represents the first example of the direct adipic acid production from model compounds of lignin depolymerization.

MicroRNA-328-3p Protects Vascular Endothelial Cells Against Oxidized Low-Density Lipoprotein Induced Injury via Targeting Forkhead Box Protein O4 (FOXO4) in Atherosclerosis.

BACKGROUND Endothelial cell (EC) injury is underlies for the pathogenesis of atherosclerosis (AS). MicroRNAs (miRNAs) have been indicated play important role in modulating AS occurrence and development. However, how miR-328-3p modulates EC injury molecular level for AS remains unclear. MATERIAL AND METHODS MiR-328-3p and forkhead box protein O4 (FOXO4) expression were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell viability was analyzed by Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was utilized to analyze the apoptotic rate. The migration and invasion abilities were measured by Transwell assay. Western blot was applied to examine the expression of C-caspase 3, Beclin, LC3-I, and LC3-II. The levels of interleukin (IL)-1ß, IL-10, and tumor necrosis factor-alpha (TNF-alpha) were tested by enzyme-linked immunosorbent assay (ELISA) assay and western blot. RESULTS MiR-328-3p expression was downregulated in oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs). Overexpressed miR-328-3p obviously alleviated ox-LDL induced inhibition on cell viability, migration and invasion, stimulation on apoptosis, autophagy as well as inflammation in HUVECs. FOXO4 was elevated in ox-LDL HUVECs, and functional assay indicated that FOXO4 aggravated ox-LDL induced HUVECs impairment. In addition, FOXO4 was a target of miR-328-3p in HUVECs; rescue experiments suggested miR-328-3p could protect HUVECs against ox-LDL induced injury via regulating FOXO4. CONCLUSIONS MiR-328-3p protected vascular endothelial cells against ox-LDL induced injury via targeting FOXO4, suggesting a novel insight for atherosclerosis treatment.

Aortic sinus aneurysm rupture into the right atrium in a 29-year-old patient.

A 29-year-old man presented with chest tightness and shortness of breath. Physical examination showed jugular venous distention. Echocardiography showed a large saccular anechoic structure in the right atrium, swinging with the cardiac cycle. The operator initially mistook the lesion for a right atrial cystic tumor; however, color Doppler imaging showed abundant, high-velocity blood flow signals, leading to the diagnosis of giant aortic sinus rupture into the right atrium. Aortic sinus aneurysm is a rare and usually asymptomatic lesion. However, rupture leads to chest tightness and dyspnea, suggesting left or right heart failure. Surgery is the primary treatment.

Characterization of Carboxylic Acid Reductases for Biocatalytic Synthesis of Industrial Chemicals.

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids into aldehydes, which can serve as common biosynthetic precursors to many industrial chemicals. This work presents the systematic biochemical characterization of five carboxylic acid reductases from different microorganisms, including two known and three new ones, by using a panel of short-chain dicarboxylic acids and hydroxy acids, which are common cellular metabolites. All enzymes displayed broad substrate specificities. Higher catalytic efficiencies were observed when the carbon chain length, either of the dicarboxylates or of the terminal hydroxy acids, was increased from C2 to C6 . In addition, when substrates of the same carbon chain length are compared, carboxylic acid reductases favor hydroxy acids over dicarboxylates as their substrates. Whole-cell bioconversions of eleven carboxylic acid substrates into the corresponding alcohols were investigated by coupling the CAR activity with that of an aldehyde reductase in Escherichia coli hosts. Alcohol products were obtained in yields ranging from 0.5 % to 71 %. The de novo stereospecific biosynthesis of propane-1,2-diol enantiomer was successfully demonstrated with use of CARs as the key pathway enzymes. E. coli strains accumulated 7.0 mm (R)-1,2-PDO (1.0 % yield) or 9.6 mm (S)-1,2-PDO (1.4 % yield) from glucose. This study consolidates carboxylic acid reductases as promising enzymes for sustainable synthesis of industrial chemicals.

Improved Photoinduced Fluorogenic Alkene-Tetrazole Reaction for Protein Labeling.

The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents one elegant and rare example of fluorophore-forming bioorthogonal chemistry. This is an attractive reaction for imaging applications in live cells that requires less intensive washing steps and/or needs spatiotemporal resolutions. In the present work, as an effort to improve the fluorogenic property of the alkene-tetrazole reaction, an aromatic alkene (styrene) was investigated as the dipolarophile. Over 30-fold improvement in quantum yield of the reaction product was achieved in aqueous solution. According to our mechanistic studies, the observed improvement is likely due to an insufficient protonation of the styrene-tetrazole reaction product. This finding provides useful guidance to the future design of alkene-tetrazole reactions for biological studies. Fluorogenic protein labeling using the styrene-tetrazole reaction was demonstrated both in vitro and in vivo. This was realized by the genetic incorporation of an unnatural amino acid containing the styrene moiety. It is anticipated that the combination of styrene with different tetrazole derivatives can generally improve and broaden the application of alkene-tetrazole chemistry in real-time imaging in live cells.

Synthetic biology approach for the development of conditionally replicating HIV-1 vaccine.

While the combined antiretroviral therapy has resulted in a significant decrease in HIV-1 related morbidity and mortality, the HIV-1 pandemic has not been substantially averted. To curtail the 2.4 million new infections each year, a prophylactic HIV-1 vaccine is urgently needed. This review first summarizes four major completed clinical efficacy trials of prophylactic HIV-1 vaccine and their outcomes. Next, it discusses several other approaches that have not yet advanced to clinical efficacy trials, but provided valuable insights into vaccine design. Among them, live-attenuated vaccines (LAVs) provided excellent protection in a non-human primate model. However, safety concerns have precluded the current version of LAVs from clinical application. As the major component of this review, two synthetic biology approaches for improving the safety of HIV-1 LAVs through controlling HIV-1 replication are discussed. Particular focus is on a novel approach that uses unnatural amino acid-mediated suppression of amber nonsense codon to generate conditionally replicating HIV-1 variants. The objective is to attract more attention towards this promising research field and to provoke creative designs and innovative utilization of the two control strategies.

Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking.

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD(+)-dependent oxidation steps. Human UGDH apoprotein is purified as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate cross-linking of monomeric subunits into predominantly obligate oligomeric species. Optimal cross-linking was achieved by encoding p-benzoyl-l-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing the enzyme to long wavelength ultraviolet (UV) radiation in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% higher-molecular mass species. The activity of the cross-linked enzyme was reduced by almost 60% relative to that of the un-cross-linked UGDH mutant, and UV exposure had no effect on the activity of the wild-type enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer.

Metabolic engineering of Escherichia coli for the production of hydroxy fatty acids from glucose.

BACKGROUND: Hydroxy fatty acids (HFAs) are valuable chemicals for a broad variety of applications. However, commercial production of HFAs has not been established so far due to the lack of low cost routes for their synthesis. Although the microbial transformation pathway of HFAs was extensively studied decades ago, these attempts mainly focused on converting fatty acids or vegetable oils to their hydroxyl counterparts. The use of a wider range of feedstocks to produce HFAs would reduce the dependence on oil crops and be expected to cut down the manufacturing cost. RESULTS: In this study, the industrially important microorganism Escherichia coli was engineered to produce HFAs directly from glucose. Through the coexpression of the acetyl-CoA carboxylase (ACCase) and the leadless acyl-CoA thioesterase ('TesA), and knockout of the endogenous acyl-CoA synthetase (FadD), an engineered E. coli strain was constructed to efficiently synthesize free fatty acids (FFAs). Under shake-flask conditions, 244.8 mg/L of FFAs were obtained by a 12 h induced culture. Then the fatty acid hydroxylase (CYP102A1) from Bacillus megaterium was introduced into this strain and high-level production of HFAs was achieved. The finally engineered strain BL21ΔfadD/pE-A1'tesA&pA-acc accumulated up to 58.7 mg/L of HFAs in the culture broth. About 24 % of the FFAs generated by the thioesterase were converted to HFAs. Fatty acid composition analysis showed that the HFAs mainly consisted of 9-hydroxydecanoic acid (9-OH-C10), 11-hydroxydodecanoic acid (11-OH-C12), 10-hydroxyhexadecanoic acid (10-OH-C16) and 12-hydroxyoctadecanoic acid (12-OH-C18). Fed-batch fermentation of this strain further increased the final titer of HFAs to 548 mg/L. CONCLUSIONS: A robust HFA-producing strain was successfully constructed using glucose as the feedstock, which demonstrated a novel strategy for bioproduction of HFAs. The results of this work suggest that metabolically engineered E. coli has the potential to be a microbial cell factory for large-scale production of HFAs.

Sulfotyrosine dipeptide: Synthesis and evaluation as HIV-entry inhibitor.

Human immunodeficiency virus type 1 (HIV-1) is responsible for the worldwide AIDS pandemic. Due to the lack of prophylactic HIV-1 vaccine, drug treatment of the infected patients becomes essential to reduce the viral load and to slow down progression of the disease. Because of drug resistance, finding new antiviral agents is necessary for AIDS drug therapies. The interaction of gp120 and co-receptor (CCR5/CXCR4) mediates the entry of HIV-1 into host cells, which has been increasingly exploited in recent years as the target for new antiviral agents. A conserved co-receptor binding site on gp120 that recognizes sulfotyrosine (sTyr) residues represents a structural target to design novel HIV entry inhibitors. In this work, we developed an efficient synthesis of sulfotyrosine dipeptide and evaluated it as an HIV-1 entry inhibitor.

Biosynthetic pathway for acrylic acid from glycerol in recombinant Escherichia coli.

Acrylic acid is an important industrial feedstock. In this study, a de novo acrylate biosynthetic pathway from inexpensive carbon source glycerol was constructed in Escherichia coli. The acrylic acid was produced from glycerol via 3-hydroxypropionaldehyde, 3-hydroxypropionyl-CoA, and acrylyl-CoA. The acrylate production was improved by screening and site-directed mutagenesis of key enzyme enoyl-CoA hydratase and chromosomal integration of some exogenous genes. Finally, our recombinant strain produced 37.7 mg/L acrylic acid under shaking flask conditions. Although the acrylate production is low, our study shows feasibility of engineering an acrylate biosynthetic pathway from inexpensive carbon source. Furthermore, the reasons for limited acrylate production and further strain optimization that should be performed in the future were also discussed.

Construction of a live-attenuated HIV-1 vaccine through genetic code expansion.

A safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) is urgently needed to combat the worldwide AIDS pandemic, but still remains elusive. The fact that uncontrolled replication of an attenuated vaccine can lead to regaining of its virulence creates safety concerns precluding many vaccines from clinical application. We introduce a novel approach to control HIV-1 replication, which entails the manipulation of essential HIV-1 protein biosynthesis through unnatural amino acid (UAA*)-mediated suppression of genome-encoded blank codon. We successfully demonstrate that HIV-1 replication can be precisely turned on and off in vitro.

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.

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.

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."