Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Incidence of Kidney Stones After Bariatric Surgeries: Comparing Roux-en-Y Gastric Bypass and Sleeve Gastrectomy.

INTRODUCTION/PURPOSE: The two most common procedures performed to treat obesity are Roux-En-Y gastric bypass (RNYGB) and laparoscopic sleeve gastrectomy (LSG). Due to changes in enteric absorption, bariatric surgery increases rates of nephrolithiasis. As population-based data are limited, we aimed to compare the incidence of kidney stones after RNYGB and LSG. MATERIALS AND METHODS: We queried Explorys (Cleveland, OH), a database that aggregated data from 26 healthcare systems. We identified patients who were newly diagnosed with nephrolithiasis 3, 6, and 12 months after their RNYGB or LSG. Additionally, a multivariate analysis was conducted to investigate the association of nephrolithiasis with RNYGB as compared to LSG. This analysis adjusted for other risk factors, including age above 65, male gender, Caucasian race, diabetes mellitus, hypertension, primary hyperparathyroidism, gout, and obesity. RESULTS: From 1999 to 2019, there were 11,480 patients who underwent RNYGB and 22,770 patients who underwent LSG. The incidence of nephrolithiasis in the RNYGB cohort at all three time points was higher than in the LSG cohort (3 months, 7.1% vs. 2.4%; 6 months, 6.6% vs. 2.0%; 1 year, 5.8% vs. 1.4%; P < 0.001). After the multivariate analysis, it was found that, though both RNYGB and LSG were independently associated with the development of nephrolithiasis, the risk of nephrolithiasis was higher in those who underwent RNYGB compared to those who underwent LSG (OR 1.594, 95% CI 1.494 to 1.701, P < 0.001). CONCLUSION: RNYGB is associated with a higher risk of nephrolithiasis when compared to LSG.

Recoding UAG to selenocysteine in Saccharomyces cerevisiae.

Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system; however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel Sec biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNASer was mutated to resemble A. salmonicida tRNASec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these Sec pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded Sec. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of Sec.

Mistranslation of the genetic code by a new family of bacterial transfer RNAs.

The correct coupling of amino acids with transfer RNAs (tRNAs) is vital for translating genetic information into functional proteins. Errors during this process lead to mistranslation, where a codon is translated using the wrong amino acid. While unregulated and prolonged mistranslation is often toxic, growing evidence suggests that organisms, from bacteria to humans, can induce and use mistranslation as a mechanism to overcome unfavorable environmental conditions. Most known cases of mistranslation are caused by translation factors with poor substrate specificity or when substrate discrimination is sensitive to molecular changes such as mutations or posttranslational modifications. Here we report two novel families of tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that adopted dual identities by integrating the anticodons AUU (for Asn) or AGU (for Thr) into the structure of a distinct proline tRNA. These tRNAs are typically encoded next to a full-length or truncated version of a distinct isoform of bacterial-type prolyl-tRNA synthetase. Using two protein reporters, we showed that these tRNAs translate asparagine and threonine codons with proline. Moreover, when expressed in Escherichia coli, the tRNAs cause varying growth defects due to global Asn-to-Pro and Thr-to-Pro mutations. Yet, proteome-wide substitutions of Asn with Pro induced by tRNA expression increased cell tolerance to the antibiotic carbenicillin, indicating that Pro mistranslation can be beneficial under certain conditions. Collectively, our results significantly expand the catalog of organisms known to possess dedicated mistranslation machinery and support the concept that mistranslation is a mechanism for cellular resiliency against environmental stress.

Gastrointestinal Discontinuity After Emergency Laparotomy.

During laparotomy, patients requiring intestinal resection may be temporarily left in gastrointestinal discontinuity (GID). We performed this study to determine predictors of futility for patients initially left in GID after emergency bowel resection. We divided the patients into 3 groups: never restored continuity and died (group 1), restored continuity and died (group 2), and restored continuity and survived (group 3). We compared the 3 groups for differences in demographics, acuity at presentation, hospital course, laboratory data, comorbidities, and outcomes. From a total of 120 patients, 58 patients died and 62 survived. We identified 31 patients in group 1, 27 patients in group 2, and 62 patients in group 3. On multivariate logistic regression, only lactate (P = .002) and use of vasopressors (P = .014) remained significant to predict survival. The results of this study can be used to identify futile situations which can direct end-of-life decisions.

Increasing liver stiffness is associated with higher incidence of hepatocellular carcinoma in hepatitis C infection and non-alcoholic fatty liver disease-A population-based study.

BACKGROUND & AIMS: Both non-alcoholic fatty liver disease (NAFLD) and hepatitis C virus (HCV) infection commonly result in hepatic fibrosis and may lead to cirrhosis. This study aims to determine the incidence of HCC in patients with HCV or NAFLD complicated by advanced fibrosis, inferred from measurements of liver stiffness. METHODS: Using Veterans Affairs (VA) Informatics and Computing Infrastructure (VINCI), we identified a nationwide cohort of patients with an existing diagnosis of HCV or NAFLD with liver transient elastography (TE) testing from 2015 to 2019. HCC cases, along with a random sample of non-HCC patients, were identified and validated, leading to calculation of incidence rates for HCC after adjustment for confounders. RESULTS: 26,161 patients carried a diagnosis of HCV and 13,629 were diagnosed with NAFLD at the time of testing. In those with HCV, rates of HCC increased with liver stiffness with incidences of 0.28 (95% CI 0.24, 0.34), 0.93 (95% CI 0.72, 1.17), 1.28 (95% CI 0.89, 1.79), and 2.79 (95% CI 2.47, 3.14)/100,000 person years for TE score ranges <9.5 kPa, 9.5-12.5 kPa, 12.5-14.5 kPa and >14.5 kPa, respectively, after a median follow-up of 2.3 years. HCC incidence also increased with higher TE liver stiffness measures in NAFLD after a median follow-up of 1.1 years. CONCLUSION: In this retrospective cohort, the incidence of HCC in HCV and NAFLD increases with higher TE liver stiffness measures, confirming that advanced fibrosis portends risk in viral and non-viral fibrotic liver diseases. Additional comparative studies are needed to determine the optimal cut point of TE liver stiffness to inform HCC screening guidelines and approaches.

Experimental Senecavirus A Infection of Bovine Cell Lines and Colostrum-Deprived Calves.

Senecavirus A (SVA) is a causative agent for vesicular disease in swine, which is clinically indistinguishable from other vesicular diseases of swine including foot-and-mouth disease (FMD). Recently, it was reported that buffalo in Guangdong, China were experiencing clinical symptoms similar to FMD including mouth ulcers and lameness tested positive for SVA. The objective of this study was to determine the susceptibility of cattle (Bos taurus) to SVA infection. Initial in vitro work using the PrimeFlow assay demonstrated that bovine cell lines and peripheral blood mononuclear cells from cattle were susceptible to SVA infection. Subsequently, six colostrum-deprived Holstein calves were challenged with SVA intranasally. No vesicular lesions were observed after challenge. Serum, oral, nasal, and rectal swabs tested for SVA nucleic acid did not support significant viral replication and there was no evidence of seroconversion. Therefore, demonstrating cattle from this study were not susceptible to experimental SVA infection.

Characterization of Senecavirus A Isolates Collected From the Environment of U.S. Sow Slaughter Plants.

Vesicular disease caused by Senecavirus A (SVA) is clinically indistinguishable from foot-and-mouth disease (FMD) and other vesicular diseases of swine. When a vesicle is observed in FMD-free countries, a costly and time-consuming foreign animal disease investigation (FADI) is performed to rule out FMD. Recently, there has been an increase in the number of FADIs and SVA positive samples at slaughter plants in the U.S. The objectives of this investigation were to: (1) describe the environmental burden of SVA in sow slaughter plants; (2) determine whether there was a correlation between PCR diagnostics, virus isolation (VI), and swine bioassay results; and (3) phylogenetically characterize the genetic diversity of contemporary SVA isolates. Environmental swabs were collected from three sow slaughter plants (Plants 1-3) and one market-weight slaughter plant (Plant 4) between June to December 2020. Of the 426 samples taken from Plants 1-3, 304 samples were PCR positive and 107 were VI positive. There was no detection of SVA by PCR or VI at Plant 4. SVA positive samples were most frequently found in the summer (78.3% June-September, vs. 59.4% October-December), with a peak at 85% in August. Eighteen PCR positive environmental samples with a range of Ct values were selected for a swine bioassay: a single sample infected piglets (n = 2). A random subset of the PCR positive samples was sequenced; and phylogenetic analysis demonstrated co-circulation and divergence of two genetically distinct groups of SVA. These data demonstrate that SVA was frequently found in the environment of sow slaughter plants, but environmental persistence and diagnostic detection was not indicative of whether a sampled was infectious to swine. Consequently, a more detailed understanding of the epidemiology of SVA and its environmental persistence in the marketing chain is necessary to reduce the number of FADIs and aide in the development of control measures to reduce the spread of SVA.

Covalent Immune Proximity-Induction Strategy Using SuFEx-Engineered Bifunctional Viral Peptides.

Covalent antibody recruiting molecules (cARMs) constitute a proximity-inducing chemical strategy to modulate the recognition and elimination of cancer cells by the immune system. Recognition is achieved through synthetic bifunctional molecules that use covalency to stably bridge endogenous hapten-specific antibodies like anti-dinitrophenyl (anti-DNP), with tumor antigens on cancer cell surfaces. To recruit these antibodies, cARMs are equipped with the native hapten-binding molecule. The majority of cancer-killing immune machinery, however, recognizes epitopes on protein ligands and not small molecule haptens (e.g., Fc receptors, pathogen-specific antibodies). To access this broader class of immune machinery for recruitment, we developed a covalent immune proximity-inducing strategy. This strategy uses synthetic bifunctional electrophilic peptides derived from the native protein ligand. These bifunctional peptides are engineered to contain both a tumor-targeting molecule and a sulfonyl (VI) fluoride exchange (SuFEx) electrophile. As a proof of concept, we synthesized bifunctional electrophilic peptides derived from glycoprotein D (gD) on herpes simplex virus (HSV), to recruit gD-specific serum anti-HSV antibodies to cancer cells expressing the prostate-specific membrane antigen (PSMA). We demonstrate that serum anti-HSV antibodies can be selectively and irreversibly targeted by these electrophilic peptides and that the reaction rate can be uniquely enhanced by tuning SuFEx chemistry without a loss in selectivity. In cellular assays, electrophilic peptides demonstrated enhanced anti-tumor immunotherapeutic efficacy compared to analogous peptides lacking electrophilic functionality. This enhanced efficacy was especially prominent in the context of (a) natural anti-HSV antibodies isolated from human serum and (b) harder to treat tumor cells associated with lower PSMA expression levels. Overall, we demonstrate a new covalent peptide-based approach to immune proximity induction and reveal the potential utility of anti-viral antibodies in synthetic tumor immunotherapy.

Prevalence of Obstructive Sleep Apnea Is Increased in Patients With Inflammatory Bowel Disease: A Large, Multi-Network Study.

Background: Crohn's disease (CD) and ulcerative colitis (UC) involve an inflammatory state where sleep dysregulation is common. Little is known about implications, if any, of inflammatory bowel disease (IBD) on the development of obstructive sleep apnea (OSA). This study aims to investigate if IBD patients are at higher risk for OSA. Methods: This retrospective multivariate analysis utilized a commercial database named Explorys (IBM Watson). We identified patients from 1/2015 to 1/2020 with UC and CD. Cohorts of these patients with and without OSA were then created and prevalence values were obtained. A multivariate analysis was used to correct for several potential confounding variables. Results: The overall prevalence of OSA was 7.8% in UC and 7.2% in CD, as compared with a prevalence of 4.3% in non-IBD patients (odds ratio [OR] for UC: 1.9 [95% CI 1.86-1.94, P < .0001], OR for CD: 1.72 [95% CI 1.69-1.76, P < .0001]). In multivariate analysis, age above 65, Caucasian race, male sex, obesity, smoking, hypertension, and diabetes were all independent risk factors for the development of OSA, with obesity being the most significant. After controlling for the listed variables in the multivariate analysis, IBD was an independent risk factor associated with OSA (OR 1.46, 95% CI 1.43-1.48). Conclusions: In this large population-based study, IBD was independently associated with increased prevalence of OSA. This has implications for screening for OSA in IBD, as well as management of other risk factors for OSA in IBD.

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. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNAProA, that are predominately found in plant pathogens from Streptomyces species. We then show that tRNAProA has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNAProA can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.

Differential Chloride Secretory Capacity in Transepithelial Ion Transport Properties in Chronic Rhinosinusitis.

BACKGROUND: Epithelial ion transport regulates hydration of airway mucosal surfaces, and thus promotes effective mucociliary clearance (MCC). Decreased transepithelial Cl- transport may contribute to epithelial dysfunction by abrogating MCC and increasing mucus viscosity in chronic rhinosinusitis (CRS). The objective of the current study is to evaluate Cl- channel transport properties from cultures of human sinonasal epithelia. METHODS: Human sinonasal epithelia (HSNE) from patients undergoing sinus surgery were cultured at an air-liquid interface to confluence and full differentiation. The epithelial monolayers were mounted in Ussing Chambers to investigate pharmacological manipulation of ion transport. Epithelial Na+ channel (via Amiloride), CFTR (via forskolin), and Ca2+-activated Cl- channel (CaCC, via UTP) transport were investigated among three different patient groups: Control, CRS and CRS with polyposis. CFTR mRNA levels were evaluated with quantitative RT-PCR. RESULTS: HSNE cultures from 18 patients (Control = 9, CRS = 6, CRS with polyposis = 3) were evaluated in 142 experiments. Summary data from the 18 patients demonstrated that stimulated CFTR-mediated anion transport (Δ ISC) was significantly lower with CRS (7.58+/-2.24 µA/cm2) compared to control (25.86+/-3.44 µA/cm2) and CRS with polyposis (20.16+/-4.0 µA/cm2) (p = 0.004). No statistically significant difference was found for CaCC anion transport between groups (p = 0.39). Significantly decreased mRNA (relative expression) was noted in CRS cultures (CRS = 40.83+/-1.76 vs. control = 116.2+/-24.27, p = 0.03). CONCLUSIONS: A substantial decrease in the Cl- secretory capacity of HSNE monolayers was demonstrated in CRS subjects. Data suggest that CFTR may contribute more to abnormal ion transport in CRS than CaCC.

Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro.

Here, we report that wild type Escherichia coli ribosomes accept and elongate precharged initiator tRNAs acylated with multiple benzoic acids, including aramid precursors, as well as malonyl (1,3-dicarbonyl) substrates to generate a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work expands the scope of ribozyme- and ribosome-catalyzed chemical transformations, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for the biosynthesis of polyketide-peptide natural products.

A cysteinyl-tRNA synthetase variant confers resistance against selenite toxicity and decreases selenocysteine misincorporation.

Selenocysteine (Sec) is the 21st genetically encoded amino acid in organisms across all domains of life. Although structurally similar to cysteine (Cys), the Sec selenol group has unique properties that are attractive for protein engineering and biotechnology applications. Production of designer proteins with Sec (selenoproteins) at desired positions is now possible with engineered translation systems in Escherichia coli However, obtaining pure selenoproteins at high yields is limited by the accumulation of free Sec in cells, causing undesired incorporation of Sec at Cys codons due to the inability of cysteinyl-tRNA synthetase (CysRS) to discriminate against Sec. Sec misincorporation is toxic to cells and causes protein aggregation in yeast. To overcome this limitation, here we investigated a CysRS from the selenium accumulator plant Astragalus bisulcatus that is reported to reject Sec in vitro Sequence analysis revealed a rare His → Asn variation adjacent to the CysRS catalytic pocket. Introducing this variation into E. coli and Saccharomyces cerevisiae CysRS increased resistance to the toxic effects of selenite and selenomethionine (SeMet), respectively. Although the CysRS variant could still use Sec as a substrate in vitro, we observed a reduction in the frequency of Sec misincorporation at Cys codons in vivo We surmise that the His → Asn variation can be introduced into any CysRS to provide a fitness advantage for strains burdened by Sec misincorporation and selenium toxicity. Our results also support the notion that the CysRS variant provides higher specificity for Cys as a mechanism for plants to grow in selenium-rich soils.

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). The multiple interactions and maturation requirements of a tRNA pose engineering challenges, but also offer tunable features. Major advances in the field of genetic code expansion have repeatedly demonstrated the central importance of suppressor tRNAs for efficient incorporation of ncAAs. Here we review the current status of two fundamentally different translation systems (TSs), selenocysteine (Sec)- and pyrrolysine (Pyl)-TSs. Idiosyncratic requirements of each of these TSs mandate how their tRNAs are adapted and dictate the techniques used to select or identify the best synthetic variants.

Youth Baseball Coach Awareness of Pitch Count Guidelines and Overuse Throwing Injuries Remains Deficient.

BACKGROUND: Baseball is one of the most popular youth sporting activities. Youth pitchers remain at high risk for shoulder and elbow overuse injuries despite well-established recommendations on pitch count limits. The purpose of this investigation was to conduct surveys of youth baseball coaches evaluating the current state of understanding and compliance with youth pitch count guidelines and risk factors for overuse injuries. METHODS: A total of 82 youth baseball coaches in surrounding suburban areas outside 2 separate Midwestern metropolitan cities were asked to answer an anonymous 13-question survey. Survey questions solicited demographic information of youth athletes coached, while assessing for incidence, knowledge of, and compliance with established recommendations on pitch count limits and upper extremity overuse injuries in youth pitchers. RESULTS: In total, 61 of 82 (74%) coaches returned surveys. The majority of coaches reported coaching male athletes (89%, n=54) primarily between the ages of 11 to 12 years (51%, n=31). A total of 56% (n=34) of coaches reported "always" keeping track of pitch counts; however, 92% (n=56) reporting not keeping track of pitches based on established recommendations by the American Sports Medicine Institute, whereas 56% were noncompliant with age appropriate pitch count recommendations as established by the 2006 US Baseball Medical and Safety Advisory Committee. Only 13% (n=8) of coaches were able to correctly identify risk factors for overuse injuries while acknowledging the necessity of off-season strengthening. Thirty-eight percent (n=23) of coaches reported sitting out an athlete because of overuse injury, with the highest rates found in those coaching the youngest (≤8 and younger) and oldest (≥17 and older) age groups. Meanwhile, 15% (n=9) of coaches were noncompliant with age-based recommendations against throwing breaking pitches. CONCLUSIONS: Knowledge regarding pitch count guidelines established to prevent overuse injuries to the shoulder and elbow in youth pitchers remains deficient in the cohort of coaches surveyed. LEVEL OF EVIDENCE: Level IV-Case Series.

Visualizing tRNA-dependent mistranslation in human cells.

High-fidelity translation and a strictly accurate proteome were originally assumed as essential to life and cellular viability. Yet recent studies in bacteria and eukaryotic model organisms suggest that proteome-wide mistranslation can provide selective advantages and is tolerated in the cell at higher levels than previously thought (one error in 6.9 × 10-4 in yeast) with a limited impact on phenotype. Previously, we selected a tRNAPro containing a single mutation that induces mistranslation with alanine at proline codons in yeast. Yeast tolerate the mistranslation by inducing a heat-shock response and through the action of the proteasome. Here we found a homologous human tRNAPro (G3:U70) mutant that is not aminoacylated with proline, but is an efficient alanine acceptor. In live human cells, we visualized mistranslation using a green fluorescent protein reporter that fluoresces in response to mistranslation at proline codons. In agreement with measurements in yeast, quantitation based on the GFP reporter suggested a mistranslation rate of up to 2-5% in HEK 293 cells. Our findings suggest a stress-dependent phenomenon where mistranslation levels increased during nutrient starvation. Human cells did not mount a detectable heat-shock response and tolerated this level of mistranslation without apparent impact on cell viability. Because humans encode ∼600 tRNA genes and the natural population has greater tRNA sequence diversity than previously appreciated, our data also demonstrate a cell-based screen with the potential to elucidate mutations in tRNAs that may contribute to or alleviate disease.

Protective and antifungal properties of Nanodisk-Amphotericin B over commercially available Amphotericin B.

OBJECTIVE: Amphotericin B (AMB), a potent antifungal agent, has been employed as topical and systemic therapy for sinonasal fungal infections. A novel formulation of nanodisc (ND) containing super aggregated AMB (ND-AMB) for the treatment of fungal infections has been recently developed to provide greater protection from AMB toxicity than current, clinically approved lipid-based formulations. The objective of the current study was to evaluate the safety and potency of ND-AMB for sinonasal delivery using an in vitro model. METHODS: Human sinonasal tissue was harvested during endoscopic sinus surgery and grown at air-liquid interface until well-differentiated. Cultures were exposed to ND-AMB vs AMB and changes in K+ permeability and resistance were measured and recorded via Ussing chamber assay. Ciliary beat frequency (CBF) was analyzed in parallel as well as cytotoxic assay. Potency was assessed using real-time PCR measurement of the Aspergillus fumigatus 18S rRNA. RESULTS: Ussing chamber studies revealed K+ currents that increased rapidly within 30 s of adding AMB (10 µg/mL) to the apical side, indicating apical membranes had become permeable to K+ ions. In contrast, negligible induction of K+ current was obtained following addition of ND-AMB [AMB = (107.7 ± 15.9) µA/cm2 AMB vs ND-AMB = (2.3 ± 0.7) µA/cm2 ND-AMB; P = 0.005]. ND-AMB also protected nasal epithelial cells from cytotoxicity of AMB (P < 0.05). There was no difference in ciliary beat frequency between the two groups (P = 0.96). The expression of A. fumigatus 18S rRNA with exposure of lower dose of ND-AMB was significantly lower compared to that with AMB (P < 0.05). CONCLUSIONS: Data from the present study suggests ND-AMB protects human nasal epithelia membranes from AMB toxicity by protecting against apical cell K+ permeability while maintaining uncompromised antifungal property compared to AMB. ND-AMB could provide a novel topical therapy for sinonasal fungal diseases.

Evolving Mistranslating tRNAs Through a Phenotypically Ambivalent Intermediate in Saccharomyces cerevisiae.

The genetic code converts information from nucleic acid into protein. The genetic code was thought to be immutable, yet many examples in nature indicate that variations to the code provide a selective advantage. We used a sensitive selection system involving suppression of a deleterious allele (tti2-L187P) in Saccharomyces cerevisiae to detect mistranslation and identify mechanisms that allow genetic code evolution. Though tRNASer containing a proline anticodon (UGG) is toxic, using our selection system we identified four tRNASerUGG variants, each with a single mutation, that mistranslate at a tolerable level. Mistranslating tRNALeuUGG variants were also obtained, demonstrating the generality of the approach. We characterized two of the tRNASerUGG variants. One contained a G26A mutation, which reduced cell growth to 70% of the wild-type rate, induced a heat shock response, and was lost in the absence of selection. The reduced toxicity of tRNASerUGG-G26A is likely through increased turnover of the tRNA, as lack of methylation at G26 leads to degradation via the rapid tRNA decay pathway. The second tRNASerUGG variant, with a G9A mutation, had minimal effect on cell growth, was relatively stable in cells, and gave rise to less of a heat shock response. In vitro, the G9A mutation decreases aminoacylation and affects folding of the tRNA. Notably, the G26A and G9A mutations were phenotypically neutral in the context of an otherwise wild-type tRNASer These experiments reveal a model for genetic code evolution in which tRNA anticodon mutations and mistranslation evolve through phenotypically ambivalent intermediates that reduce tRNA function.

Mistranslation: from adaptations to applications.

BACKGROUND: The conservation of the genetic code indicates that there was a single origin, but like all genetic material, the cell's interpretation of the code is subject to evolutionary pressure. Single nucleotide variations in tRNA sequences can modulate codon assignments by altering codon-anticodon pairing or tRNA charging. Either can increase translation errors and even change the code. The frozen accident hypothesis argued that changes to the code would destabilize the proteome and reduce fitness. In studies of model organisms, mistranslation often acts as an adaptive response. These studies reveal evolutionary conserved mechanisms to maintain proteostasis even during high rates of mistranslation. SCOPE OF REVIEW: This review discusses the evolutionary basis of altered genetic codes, how mistranslation is identified, and how deviations to the genetic code are exploited. We revisit early discoveries of genetic code deviations and provide examples of adaptive mistranslation events in nature. Lastly, we highlight innovations in synthetic biology to expand the genetic code. MAJOR CONCLUSIONS: The genetic code is still evolving. Mistranslation increases proteomic diversity that enables cells to survive stress conditions or suppress a deleterious allele. Genetic code variants have been identified by genome and metagenome sequence analyses, suppressor genetics, and biochemical characterization. GENERAL SIGNIFICANCE: Understanding the mechanisms of translation and genetic code deviations enables the design of new codes to produce novel proteins. Engineering the translation machinery and expanding the genetic code to incorporate non-canonical amino acids are valuable tools in synthetic biology that are impacting biomedical research. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.