Characterization and implementation of the MarathonRT template-switching reaction to expand the capabilities of RNA-Seq.

End-to-end RNA sequencing methods that capture 5'-sequence content without cumbersome library manipulations are of great interest, particularly for analysis of long RNAs. While template-switching methods have been developed for RNA sequencing by distributive short-read RTs, such as the MMLV RT enzymes used in SMART-Seq methods, they have not been adapted to leverage the power of ultraprocessive RTs, such as those that derive from group II self-splicing introns. To facilitate this transition, we dissected the individual processes that guide the enzymatic specificity and efficiency of the multi-step template switching reaction carried out by RT enzymes, in this case, by a well-characterized enzyme known as MarathonRT. Remarkably, this is the first study of its kind, for any RT. First, we characterized and optimized the enzymatic nontemplated addition (NTA) reaction that occurs when the RT enzyme extends past the RNA 5'-terminus, and we determined the nucleotide specificity of the NTA reaction. We then evaluated the binding specificity of specialized template-switching oligonucleotides, optimizing their sequences and chemical properties to guide efficient template switching reaction. Having dissected and optimized these individual steps, we then unified them into a procedure for performing RNA sequencing with MarathonRT enzymes, using a well-characterized RNA reference set. The resulting reads span a six-log range in transcript concentration and accurately represent the input RNA identities in both length and composition. We also performed RNA-seq starting from total human RNA and poly(A)-enriched RNA, with short and long-read sequencing demonstrating that MarathonRT enhances the discovery of unseen RNA molecules by conventional RT. Altogether, by employing mechanistic enzymology on RT enzymes and using them to modify RNA-seq technologies, we have generated a new pipeline for rapid, accurate sequencing of complex RNA libraries containing mixtures of long RNA transcripts.

Direct tracking of reverse-transcriptase speed and template sensitivity: implications for sequencing and analysis of long RNA molecules.

Although reverse-transcriptase (RT) enzymes are critical reagents for research and biotechnology, their mechanical properties are not well understood. In particular, we know little about their relative speed and response to structural obstacles in the template. Commercial retroviral RTs stop at many positions along mixed sequence templates, resulting in truncated cDNA products that complicate downstream analysis. By contrast, group II intron-encoded RTs appear to copy long RNAs with high processivity and minimal stops. However, their speed, consistency and pausing behavior have not been explored. Here, we analyze RT velocity as the enzyme moves through heterogeneous sequences and structures that are embedded within a long noncoding RNA transcript. We observe that heterogeneities in the template are highly disruptive to primer extension by retroviral RTs. However, sequence composition and template structure have negligible effects on behavior of group II intron RTs, such as MarathonRT (MRT). Indeed, MRT copies long RNAs in a single pass, and displays synchronized primer extension at a constant speed of 25 nt/sec. In addition, it passes through stable RNA structural motifs without perturbation of velocity. Taken together, the results demonstrate that consistent, robust translocative behavior is a hallmark of group II intron-encoded RTs, some of which operate at high velocity.

Ancestral archaea expanded the genetic code with pyrrolysine.

The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and in vivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Candidatus Methanomethylophilus alvus. We show that, compared with the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 pyrrolysine tRNA (tRNAPyl) sequences from diverse archaea and bacteria. This analysis suggests that the PylRS•tRNAPyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the coevolutionary history of PylRS and tRNAPyl and reveal the emergence of tRNAPyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNAPyl species with the more common G73-containing tRNAPyl will enable future efforts to engineer PylRS systems for further genetic code expansion.

Understanding Acute Respiratory Distress Syndrome in High-Altitude Environments: A Comprehensive Review of Diagnosis and Treatment.

Approximately 2% of the global population lives above 1500 m, where low atmospheric pressure, decreased oxygen levels, harsh cold and dry conditions, strong radiation, and the effects of climate change present significant health challenges. Residents of these high-altitude areas display physiological adaptions, including smaller body size, enlarged ribs, improved oxygen delivery in hypoxic conditions, and adjustments in oxygen utilization and metabolism. Both acute and chronic hypoxia prevalent in such regions can trigger various diseases by stimulating hypoxia-inducible factors, boosting inflammatory responses, and impairing mitochondrial function.Acute Respiratory Distress Syndrome (ARDS) - a critical respiratory condition associated with high morbidity and mortality - occurs more frequently among the health risks in these environments. Hypoxia is a critical predisposing and aggravating factor for high-altitude ARDS. Despite similarities with its low-altitude counterpart, ARDS in high-altitude areas displays unique pathophysiology and clinical manifestations due to the specific environmental conditions.This review aims to shed light on how high-altitude environments influence the diagnosis and treatment of ARDS, providing a comprehensive understanding of the distinct challenges inherent to these regions.

Effects of Cadmium Stress on Bacterial and Fungal Communities in the Whitefly Bemisia tabaci.

Heavy metal contamination is among the most prominent environmental problems in China, posing serious threats to both ecosystem and human health. Among the diverse heavy metal contaminants, cadmium is the most serious. The whitefly Bemisia tabaci is a cosmopolitan pest capable of causing severe damage to a broad range of agricultural crops, especially vegetables. At present, little is known about the effects of cadmium stress on B. tabaci, including on its bacterial and fungal communities. In the current study, we investigated the effects of cadmium on bacterial and fungal communities in whiteflies. Meta-barcode sequencing of the 16S rRNA gene revealed that the whitefly bacterial community contained 264 operational taxonomic units (OTUs) belonging to 201 known genera and 245 known species. The top five most frequent bacterial genera were Rickettsia, Rhodococcus, Candidatus Portiera, Candidatus Hamiltonella, and Achromobacter. Meta-barcode sequencing of the fungal ITS locus revealed that the whitefly fungal community contained 357 OTUs belonging to 187 known genera and 248 known species. The top five most frequent fungal genera were Wallemia, unclassified_f_Dipodascaceae, Apiotrichum, Penicillium, and unclassified_o_Saccharomycetales. Cadmium exposure reduced the fungal OTU richness but increased the bacterial Shannon and Simpson diversity indices in whiteflies. In addition, upon exposure to cadmium, the microbial community composition in whiteflies changed significantly, with increased prevalence of the bacterial genera Rhodococcus and Exiguobacterium and fungal genus Wallemia. Our results indicate that the whitefly microbiota likely contributed to their adaptation and resistance to cadmium and suggested that whiteflies may contain microbes that could help remediate cadmium contamination in natural environments and agricultural fields.

First report of Fusarium fujikuroi causing bulb rot on Lilium lancifolium in China.

Lilium lancifolium Thunb., commonly known as Juandan lily and tiger lily, is widely cultivated in China for its edible bulbs and medicinal properties, with a commercial value worth of ~RMB 6 billion Yuan per year. Bulb rot is an increasingly common disease on L. lancifolium, significantly impacting both the quantity and quality of the main product, the scaled bulbs. Typically, the causal pathogens invade the plant through wounds in the root or the ends of the bulb, causing the roots and bulb to brown and rot, which can eventually lead to stem wilt and death of the whole plants. During pathogenesis, the infected bulbs typically turn from white to brown, with sunken lesions and later the scales flaking off from the base of the bulb (Figure 1A and 1B). Plants growing from infected bulbs are generally short, with discolored leaves, wilting, and death at an early stage. Bulb rot is commonly observed in fields with excess water and a history of continuous Juandan lily cultivation. For this study, wilted L. lancifolium plants with rotted bulbs were collected from Longshan in Hunan, Enshi in Hubei, Yixing in Jiangsu, and Lu'an in Anhui in 2018 and 2019. Infected bulbs were surface sterilized with 75% ethanol for 30 seconds, followed by disinfection with 2% sodium hypochlorite for 5 minutes, and then rinsing with sterile water three times. The surface-sterilized tissue was divided into small pieces of 0.5 × 0.5 cm in size, placed on potato dextrose agar (PDA) medium containing 50 mg/l streptomycin sulfate, and incubated at 25℃. Mycelia growing from diseased tissues were sub-cultured onto fresh PDA medium to obtain pure culture, which formed dense white hyphae after a few days (Figure 1C and 1D). Colonies on PDA produced abundant condia about 15 days after subculturing. Microconidia were abundant, solitary, thin walled, hyaline, ovoid, 0 to 1 septate, with an average size of 6.1 × 2.6 µm (n=50) (Figure 1E). Macroconidia had a curved apical cell and foot-like basal cell with 3 to 5 septa, with an average size of 35.4 × 4.3 µm (n=30) (Figure 1E). No chlamydospore was observed. These morphological characteristics of the causal pathogen were similar to those of Fusarium spp. (Leslie et al., 2006). To identify the Fusarium isolates to species level, DNA fragments of the internal transcribed spacer (ITS) regions of the ribosomal RNA gene cluster, translation elongation factor subunit 1-alpha (TEF1-α), and RNA polymerase II subunit 2 (RPB2) genes were amplified using primers ITS1/ITS4, EF1/EF2, and 7cF/11aR respectively and sequenced (Choi et al. 2018; Jiang et al. 2018; Choi et al. 2017). BLAST analyses showed that the ITS (GenBank Accession No. MT549849), TEF1-α (GenBank Accession No. MT553348), and RPB2 (Accession No. MW201686) sequences of our isolates shared the highest sequence identities (98-100%) with those of F. fujikuroi reference strains in GenBank. A phylogenetic tree showing the relationship between one of our strains, S106, and those of the closely related species within the F. fujikuroi species complex was constructed by the maximum likelihood method using MEGA X (Kumar et al. 2018) (Figure 2). Based on the morphological characteristics and DNA sequences, the strains were identified as F. fujikuroi sensu stricto. We used two methods, an ex vivo assay using Juandan lily bulb scales and an in vivo assay using potted Juandan lily plants, to confirm pathogenicity for one representative F. fujikuroi strain from each of the four geographic regions to fulfill Koch's postulates (Bian et al. 2016; Zeng et al. 2019). In the ex vivo assay, actively growing mycelia on PDA plates were cut into 5mm diameter fungal blocks as inocula. To prepare healthy Juandan lily bulb scales as test tissues, healthy fresh scales were first surface sterilized using 75% alcohol for 30 seconds, followed by treatment of 2% sodium hypochlorite for 5 minutes, and then rinsed with sterile water 3 times. The scales were punctured with sterilized dissecting needles, the 5mm mycelial blocks containing the PDA medium were then inoculated on the punctured wound of the scales. Sterile PDA culture medium without mycelia was inoculated on the punctured wound as a negative control. After inoculation, Juandan lily scales were placed in sterile culture dishes with two layers of sterilized filter paper and 5ml of sterile water in each dish. Six Juandan lily scales were placed in each dish, with different treatments placed in different dishes, and the dishes were placed in an incubator in the dark at 25℃. After 10 days of incubation, we found that the F. fujikuroi-inoculated Juandan lily bulb scales showed disease symptoms (brownish lesion) similar to those in the original field collected infected bulb samples (Figure 1F). However, such symptoms were not observed in the negative control group. The pathogenicity test was performed 3 times for each isolate, each with six repeats. In the in vivo pathogenicity test using potted lily plants, we prepared actively growing cultures of our F. fujikuroi strains by incubating them in a liquid medium, the potato dextrose broth, for 3 days in a shaker-incubator at 25℃ and 180rpm. The asexual spores conidia from the fungal cultures were harvested by filtration through eight layers of sterile cheese clothes and with spore concentrations adjusted to 1×107 conidia per ml. Healthy Juandan lily bulbs were selected and one bulb was planted in each pot containing sterilized soil. Each pot was inoculated with 1ml conidia suspension, at the base soil where the bulbs were planted. The pots were placed in a growth chamber at 25℃ with a 12 h light and 12 h dark cycle. Symptoms similar to those observed in diseased bulbs in the field were observed, with symptoms at 30 days after inoculations shown in Figure 3. Specifically, most of the roots, bulb plate and scale tissues of Juandan lily plants inoculated with F. fujikuroi conidia were rotten and turned black, with few new roots. In addition, the infected plants showed stunted growth (Figure 3). In contrast, the uninoculated plants grew normally, with dense new roots and healthy-looking bulbs, and no rot symptom (Figure 3). The fungi were re-isolated from the infected Juandan lily tissues from both pathogenicity assays, following the procedures described above for isolating and identifying the fungal cultures from infected field samples. These re-isolated fungi were shown to have colony morphology and DNA sequences at the three loci identical to those of our inoculated F. fujikuroi strains. Several Fusarium species have been reported as pathogens of lily plants in China, including F. oxysporum, F. solani and F. tricinctum (Li, et al., 1995; Li, et al., 2013). In addition, F. redolens has been reported previously in ornamental lily in Ukraine (Zerova, 1940). Indeed, Fusarium moniliforme, one of the disused synonyms of F. fujikuroi (Seifert et al. 2003), has been reported as a causal agent for diseases in lily. However, it's now known that the originally defined F. fujikuroi sensu lato is in fact a large species complex consisting of over 60 recognized species, including F. fujikuroi sensu stricto (Moussa et al. 2017; Choi et al. 2018). In addition, there are over 100 species in the genus Lilium as well as many other species with their common names including the word "lily" but are not in the Lilium genus. To our knowledge, this is the first confirmed report of bulb rot of Juandan lily L. lancifolium caused by F. fujikuroi sensu stricto in China. Our result should help with future monitoring and control of Juandan lily diseases.

Genome Sequence Resource for the Ramie Oomycete Pathogen Phytopythium vexans HF1.

The oomycete Phytopythium vexans is a causative agent of patch canker, damping-off, and crown, stem, and root rot in many economically important plants. P. vexans HF1 was isolated in China, where it caused brown root rot of ramie, a fiber crop broadly cultivated in Asia. The genome of HF1 was sequenced by a combination of technologies producing short (Illumina HiSeq X) and long (PacBio RS) reads. The genome is 41.73 Mbp long, assembled into 44 contigs. It has a GC content of 58.17% and contains 13,051 predicted coding genes, including 1,461 putative virulence genes and 220 putative antimicrobial resistance genes. This genome sequence provides a resource for determining the molecular mechanisms of disease development in this pathosystem.

Errata: Continuous directed evolution of aminoacyl-tRNA synthetases.

This corrects the article DOI: 10.1038/nchembio.2474.

Protein aggregation caused by aminoglycoside action is prevented by a hydrogen peroxide scavenger.

Protein mistranslation causes growth arrest in bacteria, mitochondrial dysfunction in yeast, and neurodegeneration in mammals. It remains poorly understood how mistranslated proteins cause such cellular defects. Here we demonstrate that streptomycin, a bactericidal aminoglycoside that increases ribosomal mistranslation, induces transient protein aggregation in wild-type Escherichia coli. We further determined the aggregated proteome using label-free quantitative mass spectrometry. To identify genes that reduce cellular mistranslation toxicity, we selected from an overexpression library protein products that increased resistance against streptomycin and kanamycin. The selected proteins were significantly enriched in members of the oxidation-reduction pathway. Overexpressing one of these proteins, alkyl hydroperoxide reductase subunit F (a protein defending bacteria against hydrogen peroxide), but not its inactive mutant suppressed aggregated protein formation upon streptomycin treatment and increased aminoglycoside resistance. This work provides in-depth analyses of an aggregated proteome caused by streptomycin and suggests that cellular defense against hydrogen peroxide lowers the toxicity of mistranslation.

Genome-Wide Identification and Expression Analysis of Udp-Glucuronosyltransferases in the Whitefly Bemisia Tabaci (Gennadius) (HemipterA: Aleyrodidae).

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is an important agricultural pest worldwide. Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) are one of the largest and most ubiquitous groups of proteins. Because of their role in detoxification, insect UGTs are attracting increasing attention. In this study, we identified and analyzed UGT genes in B. tabaci MEAM1 to investigate their potential roles in host adaptation and reproductive capacity. Based on phylogenetic and structural analyses, we identified 76 UGT genes in the B. tabaci MEAM1 genome. RNA-seq and real-time quantitative PCR (RT-qPCR) revealed differential expression patterns of these genes at different developmental stages and in association with four host plants (cabbage, cucumber, cotton and tomato). RNA interference results of selected UGTs showed that, when UGT352A1, UGT352B1, and UGT354A1 were respectively silenced by feeding on dsRNA, the fecundity of B. tabaci MEAM1 was reduced, suggesting that the expressions of these three UGT genes in this species may be associated with host-related fecundity. Together, our results provide detailed UGTs data in B.tabaci and help guide future studies on the mechanisms of host adaptation by B.tabaci.

Continuous directed evolution of aminoacyl-tRNA synthetases.

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing noncanonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.

Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase.

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion using noncanonical amino acids, yet its structure and function are not completely understood. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon independent: the anticodon does not contact the enzyme. Then, using standard microbiological culture equipment, we established a new method for laboratory evolution-a noncontinuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. Finally, we employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a noncanonical amino acid.

Genome-Wide Analysis of Carboxylesterases (COEs) in the Whitefly, Bemisia tabaci (Gennadius).

The whitefly (Bemisia tabaci), an important invasive pest that causes severe damage to crops worldwide, has developed resistance to a variety of insecticides. Carboxylesterases (COEs) are important multifunctional enzymes involved in the growth, development, and xenobiotic metabolism of insects. However, systematic studies on the COEs of B. tabaci are scarce. Here, 42 putative COEs in different functional categories were identified in the Mediterranean species of B. tabaci (B. tabaci MED) based on a genome database and neighbor-joining phylogeny. The expression patterns of the COEs were affected by the development of B. tabaci. The expression levels of six COEs were positively correlated with the concentration of imidacloprid to which B. tabaci adults were exposed. The mortality of B. tabaci MED adults fed dsBTbe5 (67.5%) and dsBTjhe2 (58.4%) was significantly higher than the adults fed dsEGFP (41.1%) when treated with imidacloprid. Our results provide a basis for functional research on COEs in B. tabaci and provide new insight into the imidacloprid resistance of B. tabaci.

The invasive MED/Q Bemisia tabaci genome: a tale of gene loss and gene gain.

BACKGROUND: Sweetpotato whitefly, Bemisia tabaci MED/Q and MEAM1/B, are two economically important invasive species that cause considerable damages to agriculture crops through direct feeding and indirect vectoring of plant pathogens. Recently, a draft genome of B. tabaci MED/Q has been assembled. In this study, we focus on the genomic comparison between MED/Q and MEAM1/B, with a special interest in MED/Q's genomic signatures that may contribute to the highly invasive nature of this emerging insect pest. RESULTS: The genomes of both species share similarity in syntenic blocks, but have significant divergence in the gene coding sequence. Expansion of cytochrome P450 monooxygenases and UDP glycosyltransferases in MED/Q and MEAM1/B genome is functionally validated for mediating insecticide resistance in MED/Q using in vivo RNAi. The amino acid biosynthesis pathways in MED/Q genome are partitioned among the host and endosymbiont genomes in a manner distinct from other hemipterans. Evidence of horizontal gene transfer to the host genome may explain their obligate relationship. Putative loss-of-function in the immune deficiency-signaling pathway due to the gene loss is a shared ancestral trait among hemipteran insects. CONCLUSIONS: The expansion of detoxification genes families, such as P450s, may contribute to the development of insecticide resistance traits and a broad host range in MED/Q and MEAM1/B, and facilitate species' invasions into intensively managed cropping systems. Numerical and compositional changes in multiple gene families (gene loss and gene gain) in the MED/Q genome sets a foundation for future hypothesis testing that will advance our understanding of adaptation, viral transmission, symbiosis, and plant-insect-pathogen tritrophic interactions.

Enzyme Inhibitor Antibiotics and Antibiotic-Associated Diarrhea in Critically Ill Patients.

BACKGROUND This study aimed to analyze the factors associated with the development of antibiotic-associated diarrhea (AAD) in critically ill patients receiving enzyme inhibitor antibiotics. MATERIAL AND METHODS A retrospective study of patients with and without AAD admitted to the intensive care unit (ICU) of the First Teaching Hospital of Xi'an Jiaotong University from February 1, 2014, to January 31, 2016, was undertaken. Relevant clinical data underwent univariate or multivariate regression analysis. RESULTS Of 184 patients who received enzyme inhibitor antibiotic therapy, 70 patients (38.04%) developed AAD, with a mean duration of onset of 6.97±3.64 days. AAD was associated with the use of enzyme inhibitor antibiotic therapy alone (OR, 1.142; 95% CI, 1.038-1.256; P=0.007), and in combination with antifungal agents (OR, 2.449; 95% CI, 1.116-5.372; P=0.025), quinolones (OR, 5.219; 95% CI, 1.746-15.601; P=0.003), and oxazolidinones (OR 2.895; 95% CI, 1.183-7.083; P=0.020). The mean duration of ICU stay was significantly increased in patients with AAD (19.00±11.49 days vs. 9.60±6.76 days) (P<0.001). Mean duration of antibiotic therapy (14.09±8.82 days vs. 8.10±4.91 days) (P<0.001) and duration of enzyme inhibitor antibiotic therapy (9.26±5.06 days vs. 6.61±3.24 days) (P<0.001) were significantly increased in patients with AAD. CONCLUSIONS Duration of use of enzyme inhibitor antibiotic therapy and the combined use of antifungals, quinolones, and oxazolidinones increased the incidence and duration of AAD and increased the length of stay in ICU.

Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog.

Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Anti-Inflammatory Effects of TRAF-Interacting Protein in Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by inflammatory cell infiltration, synovial inflammation, and cartilage destruction. Proliferative fibroblast-like synoviocytes (FLS) play crucial roles in both propagation of inflammation and joint damage because of their production of great amount of proinflammatory cytokines and proteolytic enzymes. In this study, we investigate the role of TRAF-interacting protein (TRIP) in regulating inflammatory process in RA-FLS. TRIP expression was attenuated in RA-FLS compared with osteoarthritis- (OA-) FLS. Overexpression of TRIP significantly inhibited the activation of NF-κB signaling and decreased the production of proinflammatory cytokines and matrix metalloproteinases (MMPs) in TNFα-stimulated RA-FLS. Furthermore, TRIP was found to interact with transforming growth factor ß-activated kinase 1 (TAK1) and promoting K48-linked polyubiquitination of TAK1 in RA-FLS. Our results demonstrate that TRIP has anti-inflammatory effects on RA-FLS and suggest TRIP as a potential therapeutic target for human RA.

Glutathione S-transferases are involved in thiamethoxam resistance in the field whitefly Bemisia tabaci Q (Hemiptera: Aleyrodidae).

The whitefly, Bemisia tabaci, has developed a high level of resistance to thiamethoxam, a second generation neonicotinoid insecticide that has been widely used to control this pest. In this study, we assessed the level of cross-resistance, the activities of detoxifying enzymes, and the expression profiles of 23 glutathione S-transferase (GST) genes in a thiamethoxam-resistant ant and -susceptible strain of Bemisia tabaci Q. The thiamethoxam-resistant strain showed a moderate level of cross-resistance to another nicotinoid insecticide imidacloprid, a low level of cross-resistance to acetamiprid and nitenpyram, and no significant cross-resistance to abamectin and bifenthrin. Among detoxifying enzymes, only GSTs had significantly higher activity in the resistant strain than in the susceptible strain. Seven of 23 GST genes were over-expressed in the resistant strain relative to the susceptible strain. Using the technology of RNA interference to knockdown a GST gene (GST14), the results showed that silencing GST14 increased the mortality of whiteflies to thiamethoxam in Bemisia tabaci.