Search

Rationale: Transesophageal and transbronchial, ultrasound-guided, fine-needle aspiration of enlarged mediastinal lymph nodes have become popular, but have never been compared directly. Objectives: To compare the relative diagnostic yield and ability of the transesophageal and transbronchial approaches to reach abnormal mediastinal lymph nodes. Methods: A total of 160 patients with enlarged lymph nodes in one of eight mediastinal lymph node stations underwent transbronchial and transesophageal biopsies in a crossover design. Each of the eight stations was allocated 20 patients. Two needle punctures were done with each approach. Measurements: Percentage of successful biopsies, percentage of patients diagnosed, and biopsy time were measured from when the lymph node was identified with ultrasound. Main Results: Among the 106 men and 54 women (mean age 53.2 years), transbronchial aspiration was successful in 85%, and transesophageal aspiration was successful in 78% (p = 0.2). For each station, the number of positive samples for the transbronchial/transesophageal approaches was: 2R: 19/13; 2L: 16/19; 3: 17/15; 4R: 19/12; 4L: 17/20; 7: 19/20; 10R: 18/9; and 10L: 17/18. Combining both approaches produced successful biopsies in 97% and diagnoses in 94% of patients. Mean biopsy times were 3.2 minutes for the transbronchial approach and 4.1 minutes for the transesophageal approach. The transbronchial approach was superior in nodes 2R, 4R, and 10R. No complications were encountered. Conclusions: In experienced hands, enlarged mediastinal lymph nodes may be aspirated with either the transbronchial or transesophageal approach. These nonsurgical approaches have similar diagnostic yields, although the transbronchial approach is superior for right-sided lymph nodes. Combining both approaches provides results similar those of mediastinoscopy. [ABSTRACT FROM AUTHOR]

BACKGROUND: Lung abscesses commonly respond well to antibiotic therapy. In patients in whom conventional therapy fails, either percutaneous catheter drainage or surgical resection are usually considered, but are frequently problematic. This study describes our experience with endoscopic lung abscess drainage in patients in whom antibiotic therapy fails. METHODS: Patients in whom antibiotic therapy for lung abscess (enlarging cavity or lack of improvement of clinical status) was unsuccessful were considered candidates if an airway connection to the cavity was present. Treatment decisions were made in a multidisciplinary chest conference. Pigtail catheters were placed via a guidewire approach into the cavities. The abscesses were flushed twice daily with gentamycin solution. If fungal infection was suspected, once-daily amphotericin B was added to the regimen. RESULTS: Forty-two patients, from January 2000 to May 2002 (17 woman and 25 men) were included in this study (mean age, 48.9 years). Catheter placement was successful in 38 patients and led to successful therapy after a mean of 6.2 days of treatment (range, 3 to 21 days). Two patients required transient ventilation after catheter placement; there were no other complications. CONCLUSIONS: Endoscopic lung abscess drainage in selected patients in whom antibiotic therapy fails is feasible and successful in experienced hands. This treatment represents an additional option for the chest physician other than percutaneous catheter drainage or surgical resection. [ABSTRACT FROM AUTHOR]

Central airway obstruction is a problem facing all medical and surgical subspecialists caring for patients with chest diseases. The incidence of this disorder appears to be rising because of the epidemic of lung cancer; however, benign causes of central airway obstruction are being seen more frequently as well. The morbidity is significant and if left untreated, death from suffocation is a frequent outcome. Management of these patients is difficult, but therapeutic and diagnostic tools are now available that are beneficial to most patients and almost all airway obstruction can be relieved expeditiously. This review examines current approaches in the workup and treatment of patients suffering from airway impairment. Although large, randomized, comparative studies are not available, data show significant improvement in patient outcomes and quality of life with treatment of central airway obstruction. Clearly, more studies assessing the relative utility of specific airway interventions and their impact on morbidity and mortality are needed. Currently, the most comprehensive approach can be offered at centers with expertise in the management of complex airway disorders and availability of all endoscopic and surgical options. [ABSTRACT FROM AUTHOR]

Study Objective: Our group performed a randomized trial to assess whether the addition of endobronchial ultrasound (EBUS) guidance will lead to better results than standard transbronchial needle aspiration (TBNS). EBUS guidance seems to be beneficial in increasing the yield of TBNA but has not been proven to be superior to conventional procedures in a randomized trial.Methods: Consecutive patients who were referred for TBNA were randomized to an EBUS-guided and a conventional TBNA arm. Patients with subcarinal lymph nodes were randomized and analyzed separately (group A) from all other stations (group B). A positive result was defined as either lymphocytes or a specific abnormality on cytology.Results: Two hundred patients were examined (100 patients each in groups A and B). Half of the patients underwent EBUS-guided TBNA rather than conventional TBNA. In group A, the yield of conventional TBNA was 74% compared to 86% in the EBUS group (difference not significant). In group B, the overall yields were 58% and 84%, respectively. This difference was statistically highly significant (p < 0.001). The average number of passes was four.Conclusion: EBUS guidance significantly increases the yield of TBNA in all stations except in the subcarinal region. It should be considered to be a routine adjunct to TBNA. On-site cytology may be unnecessary, and the number of necessary needle passes required is low. [ABSTRACT FROM AUTHOR]

Inflammatory cells are generally assumed to be the primary source of most growth factors/cytokines that participate in wound healing. Correspondingly, most attempts to enhance wound healing have been directed at the wound itself. However, certain key factors such as insulin-like growth factor-I (IGF-I) and related proteins are present in blood in sufficient quantities to suggest significant contributions from outside the wound. Because little is known of the dynamics of IGF family members in wounds, particularly in human wounds, we serially measured the mRNAs and proteins of the IGF family in fluid and tissues taken from acute as well as chronically inflamed human wounds and compared them to their corresponding concentrations in blood. We also measured transforming growth factor-beta1 and -beta3, vascular endothelial growth factor, interleukin-1beta, matrix metalloproteinases and selected isomers/receptors, all of which are associated with inflammation. All IGF proteins reached their highest concentrations immediately after injury. No difference between IGF-I mRNA expression between acute and inflamed wounds was found. As a group, IGF-related proteins, in contrast to transforming growth factor-beta, vascular endothelial growth factor, and interleukin-1beta, are highly correlated to and are generally below their concentrations in blood and are not elevated by inflammation. The IGF family therefore appears to enter wounds, even inflamed wounds, mainly from blood. If blood IGF-I is low, wound levels are lower. This data suggests that healing impairment due to IGF-I deficiency can be readily detected and is, at least in part, easily and safely correctable. [ABSTRACT FROM AUTHOR]

Study Objectives: Conventional transbronchial needle aspiration (TBNA) is a valuable procedure but remains underutilized. Recently, imaging guidance such as CT fluoroscopy has created considerable interest. As CT fluoroscopy is cumbersome and exposes patients and staff to radiation, we have examined the feasibility of endobronchial ultrasound (EBUS) in providing imaging support for TBNA.Design: Prospective consecutive patient enrollment.Setting: University-related tertiary referral centers for pulmonary diseases.Results: From January 1999 to January 2000, 242 consecutive patients were entered into this prospective study (82 women and 160 men; mean age, 60.0 years). Indications for TBNA were diagnosis of enlarged lymph nodes and cancer staging. The average lymph node size was 1.7 cm (SD, 0.47; range, 0.8 to 4.3 cm). Target lymph nodes were visualized with EBUS, followed by TBNA in standard fashion. All targets could be visualized with EBUS. In 207 patients, the lymph nodes were successfully sampled (86%). This was independent of lymph node size and location. A firm diagnosis or cancer stage could be obtained in 172 patients (72%). There were no complications associated with the use of EBUS.Conclusion: EBUS is simply performed and if used for TBNA guidance affords an excellent yield independent of lymph node location. Randomized trials comparing standard TBNA and imaging-guided TBNA by CT fluoroscopy and EBUS are indicated. [ABSTRACT FROM AUTHOR]

Study Objective: To provide current data on the long-term outcome and incidence of lung cancer in a large cohort of patients with hemoptysis of unknown origin.Design: A retrospective chart review followed by a telephone interview for follow-up.Setting: A university-affiliated tertiary referral center for pulmonary diseases.Patients: Seven hundred twenty-two patients who presented with hemoptysis from January 1990 to December 1993. One hundred thirty-five patients were identified as having hemoptysis of unknown origin.Results: One hundred thirty-five patients (19%) had hemoptysis of unknown origin; follow-up data were obtained in 115 patients, of whom 100 were still alive. The mean time of observation was 6.6 years after initial presentation. Lung cancer developed in 7 of 115 patients (6%) and was unresectable once detected; all of these patients were smokers > 40 years old, and malignancy developed within 3 years after first presentation.Conclusions: Hemoptysis of unknown origin is present in a minority of patients presenting with hemoptysis if evaluated at a referral center for pulmonary diseases. Lung cancer seems to be increasing in these patients compared to previous studies, and closer follow-up or additional testing may be indicated in the defined population at risk. [ABSTRACT FROM AUTHOR]

Even if a myriad of approaches has been developed to identify the subcellular localization of a protein, the easiest and fastest way remains to fuse the protein to Green Fluorescent Protein (GFP) and visualize its location using fluorescence microscopy. However, this strategy is not well suited to visualize the organellar pools of proteins that are simultaneously localized both in the cytosol and in organelles because the GFP signal of a cytosolic pool of the protein (cytosolic echoform) will inevitably mask or overlay the GFP signal of the organellar pool of the protein (organellar echoform). To solve this issue, we engineered a dedicated yeast strain expressing a Bi-Genomic Mitochondrial-Split-GFP. This split-GFP is bi-genomic because the first ten ß-strands of GFP (GFP ß1-10 ) are encoded by the mitochondrial genome and translated by mitoribosomes whereas the remaining ß-strand of GFP (GFP ß11 ) is fused to the protein of interest encoded by the nucleus and expressed by cytosolic ribosomes. Consequently, if the GFP ß11 -tagged protein localizes into mitochondria, GFP will be reconstituted by self-assembly GFP ß1-10 and GFP ß11 thereby generating a GFP signal restricted to mitochondria and detectable by regular fluorescence microscopy. In addition, because mitochondrial translocases and import mechanisms are evolutionary well conserved, the BiG Mito-Split-GFP yeast strain can be used to probe mitochondrial importability of proteins regardless of their organismal origins and can thus serve to identify unsuspected mitochondrial echoforms readily from any organism., (Copyright © 2024. Published by Elsevier Inc.)

The objective of the present review is to provide an insight into modifications of microbial cell walls and membrane constituents by using the aminoacyl-tRNA as amino acid donor. In bacteria, phospholipids are modified by Multiple peptide resistance Factor enzymes and peptidoglycan precursors by so called fem ligases. Although these modifications were thought to be restricted to procaryotes, we discovered enzymes that modify ergosterol (the main component of fungal membrane) with glycine and aspartate. The focus of this review is to present the molecular mechanisms underlying all these processes together with the structure of the enzymes and their substrates. This article also reviews how substrates are recognized and modified and how the products are subsequently exported in various organisms. Finally, the physiological outcome and the discoveries of each family of enzymes is also discussed., (Copyright © 2022. Published by Elsevier B.V.)

Background: Aminoacyl-tRNA synthetases (ARS) are key enzymes catalysing the first reactions in protein synthesis, with increasingly recognised pleiotropic roles in tumourgenesis, angiogenesis, immune response and lifespan. Germline mutations in several ARS genes have been associated with both recessive and dominant neurological diseases. Recently, patients affected with microcephaly, intellectual disability and ataxia harbouring biallelic variants in the seryl-tRNA synthetase encoded by seryl-tRNA synthetase 1 ( SARS1 ) were reported., Methods: We used exome sequencing to identify the causal variant in a patient affected by complex spastic paraplegia with ataxia, intellectual disability, developmental delay and seizures, but without microcephaly. Complementation and serylation assays using patient's fibroblasts and an Saccharomyces cerevisiae model were performed to examine this variant's pathogenicity., Results: A de novo splice site deletion in SARS1 was identified in our patient, resulting in a 5-amino acid in-frame insertion near its active site. Complementation assays in S. cerevisiae and serylation assays in both yeast strains and patient fibroblasts proved a loss-of-function, dominant negative effect. Fibroblasts showed an abnormal cell shape, arrested division and increased beta-galactosidase staining along with a senescence-associated secretory phenotype (raised interleukin-6, p21, p16 and p53 levels)., Conclusion: We refine the phenotypic spectrum and modes of inheritance of a newly described, ultrarare neurodevelopmental disorder, while unveiling the role of SARS1 as a regulator of cell growth, division and senescence., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

The yeast mitochondrial ATP synthase is an assembly of 28 subunits of 17 types of which 3 (subunits 6, 8, and 9) are encoded by mitochondrial genes, while the 14 others have a nuclear genetic origin. Within the membrane domain (FO) of this enzyme, the subunit 6 and a ring of 10 identical subunits 9 transport protons across the mitochondrial inner membrane coupled to ATP synthesis in the extra-membrane structure (F1) of ATP synthase. As a result of their dual genetic origin, the ATP synthase subunits are synthesized in the cytosol and inside the mitochondrion. How they are produced in the proper stoichiometry from two different cellular compartments is still poorly understood. The experiments herein reported show that the rate of translation of the subunits 9 and 6 is enhanced in strains with mutations leading to specific defects in the assembly of these proteins. These translation modifications involve assembly intermediates interacting with subunits 6 and 9 within the final enzyme and cis-regulatory sequences that control gene expression in the organelle. In addition to enabling a balanced output of the ATP synthase subunits, these assembly-dependent feedback loops are presumably important to limit the accumulation of harmful assembly intermediates that have the potential to dissipate the mitochondrial membrane electrical potential and the main source of chemical energy of the cell., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

A wide range of bacteria possess virulence factors such as aminoacyl-tRNA transferases (ATTs) that are capable of rerouting aminoacyl-transfer RNAs away from protein synthesis to conjugate amino acids onto glycerolipids. We recently showed that, although these pathways were thought to be restricted to bacteria, higher fungi also possess ergosteryl-3β-O-L-aspartate synthases (ErdSs), which transfer the L-Asp moiety of aspartyl-tRNA Asp onto the 3β-OH group of ergosterol (Erg), yielding ergosteryl-3β-O-L-aspartate (Erg-Asp). Here, we report the discovery, in fungi, of a second type of fungal sterol-specific ATTs, namely, ergosteryl-3β-O-glycine (Erg-Gly) synthase (ErgS). ErgS consists of a freestanding DUF2156 domain encoded by a gene distinct from and paralogous to that of ErdS. We show that the enzyme only uses Gly-tRNA Gly produced by an independent glycyl-tRNA synthetase (GlyRS) to transfer glycine onto the 3β-OH of Erg, producing Erg-Gly. Phylogenomics analysis also show that the Erg-Gly synthesis pathway exists only in Ascomycota, including species of biotechnological interest, and more importantly, in human pathogens, such as Aspergillus fumigatus. The discovery of a second type of Erg-aa not only expands the repertoire of this particular class of fungal lipids but suggests that Erg-aa synthases might constitute a genuine subfamily of lipid-modifying ATTs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Proving with certainty that a GFP-tagged protein is imported inside mitochondria by visualizing its fluorescence emission with an epifluorescence microscope is currently impossible using regular GFP-tagging. This is particularly true for proteins dual localized in the cytosol and mitochondria, which have been estimated to represent up to one third of the established mitoproteomes. These proteins are usually composed of a surpassingly abundant pool of the cytosolic isoform compared to the mitochondrial isoform. As a consequence, when tagged with a regular GFP, the fluorescence emission of the cytosolic isoform will inevitably eclipse that of the mitochondrial one and prevent the detection of the mitochondrial echoform. To overcome this technical limit, we engineered a yeast strain expressing a new type of GFP called Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). In this strain, one moiety of the GFP is encoded by the mitochondrial DNA while the second moiety of the GFP can be tagged to any nuclear-encoded protein (suspected to be dual localized or bona fide mitochondrial). By doing so, only mitochondrial proteins or echoforms of dual localized proteins, regardless of their organismal origin, trigger GFP reconstitution that can be visualized by regular fluorescence microscopy. The strength of the BiG Mito-Split-GFP system is that proof of the mitochondrial localization of a given protein rests on a simple and effortless microscopy observation., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aminoacylated ergosterol such as 1-ergosteryl aspartate (Erg-Asp) is a new lipid component recently discovered in fungi. In order to study physiological functions of this novel sterol derivative and to develop potential antifungal agents, we established the method to synthesize aminoacylated ergosterol derivatives. Herein, we report the synthesis of Erg-Asp as well as some other aminoacylated ergosterols (Erg-Gly, Erg-Ala, Erg-Leu, Erg-Ile, and Erg-Val) using Boc protected amino acids., (Copyright © 2021 Elsevier Inc. All rights reserved.)

COPI (coatomer complex I) coated vesicles are involved in Golgi-to-ER and intra-Golgi trafficking pathways, and mediate retrieval of ER resident proteins. Functions and components of the COPI-mediated trafficking pathways, beyond the canonical set of Sec/Arf proteins, are constantly increasing in number and complexity. In mammalian cells, GORAB, SCYL1 and SCYL3 proteins regulate Golgi morphology and protein glycosylation in concert with the COPI machinery. Here, we show that Cex1, homologous to the mammalian SCYL proteins, is a component of the yeast COPI machinery, by interacting with Sec27, Sec28 and Sec33 (Ret1/Cop1) proteins of the COPI coat. Cex1 was initially reported to mediate channeling of aminoacylated tRNA outside of the nucleus. Our data show that Cex1 localizes at membrane compartments, on structures positive for the Sec33 α-COP subunit. Moreover, the Wbp1 protein required for N-glycosylation and interacting via its di-lysine motif with the Sec27 β'-COP subunit is mis-targeted in cex1 Δ deletion mutant cells. Our data point to the possibility of developing Cex1 yeast-based models to study neurodegenerative disorders linked to pathogenic mutations of its human homologue SCYL1., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

A single nuclear gene can be translated into a dual localized protein that distributes between the cytosol and mitochondria. Accumulating evidences show that mitoproteomes contain lots of these dual localized proteins termed echoforms. Unraveling the existence of mitochondrial echoforms using current GFP (Green Fluorescent Protein) fusion microscopy approaches is extremely difficult because the GFP signal of the cytosolic echoform will almost inevitably mask that of the mitochondrial echoform. We therefore engineered a yeast strain expressing a new type of Split-GFP that we termed Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). Because one moiety of the GFP is translated from the mitochondrial machinery while the other is fused to the nuclear-encoded protein of interest translated in the cytosol, the self-reassembly of this Bi-Genomic-encoded Split-GFP is confined to mitochondria. We could authenticate the mitochondrial importability of any protein or echoform from yeast, but also from other organisms such as the human Argonaute 2 mitochondrial echoform., Competing Interests: GB, LE, YA, MH, KB, EB, JD, JR, JP, DT, BS, Jd, SF, RK, HB No competing interests declared, (© 2020, Bader et al.)

Diverting aminoacyl-transfer RNAs (tRNAs) from protein synthesis is a well-known process used by a wide range of bacteria to aminoacylate membrane constituents. By tRNA-dependently adding amino acids to glycerolipids, bacteria change their cell surface properties, which intensifies antimicrobial drug resistance, pathogenicity, and virulence. No equivalent aminoacylated lipids have been uncovered in any eukaryotic species thus far, suggesting that tRNA-dependent lipid remodeling is a process restricted to prokaryotes. We report here the discovery of ergosteryl-3β- O -l-aspartate (Erg-Asp), a conjugated sterol that is produced by the tRNA-dependent addition of aspartate to the 3β-OH group of ergosterol, the major sterol found in fungal membranes. In fact, Erg-Asp exists in the majority of "higher" fungi, including species of biotechnological interest, and, more importantly, in human pathogens like Aspergillus fumigatus We show that a bifunctional enzyme, ergosteryl-3β- O -l-aspartate synthase (ErdS), is responsible for Erg-Asp synthesis. ErdS corresponds to a unique fusion of an aspartyl-tRNA synthetase-that produces aspartyl-tRNA Asp (Asp-tRNA Asp )-and of a Domain of Unknown Function 2156 , which actually transfers aspartate from Asp-tRNA Asp onto ergosterol. We also uncovered that removal of the Asp modifier from Erg-Asp is catalyzed by a second enzyme, ErdH, that is a genuine Erg-Asp hydrolase participating in the turnover of the conjugated sterol in vivo. Phylogenomics highlights that the entire Erg-Asp synthesis/degradation pathway is conserved across "higher" fungi. Given the central roles of sterols and conjugated sterols in fungi, we propose that this tRNA-dependent ergosterol modification and homeostasis system might have broader implications in membrane remodeling, trafficking, antimicrobial resistance, or pathogenicity., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The aminoacylation reaction is one of most extensively studied cellular processes. The so-called "canonical" reaction is carried out by direct charging of an amino acid (aa) onto its corresponding transfer RNA (tRNA) by the cognate aminoacyl-tRNA synthetase (aaRS), and the canonical usage of the aminoacylated tRNA (aa-tRNA) is to translate a messenger RNA codon in a translating ribosome. However, four out of the 22 genetically-encoded aa are made "noncanonically" through a two-step or indirect route that usually compensate for a missing aaRS. Additionally, from the 22 proteinogenic aa, 13 are noncanonically used, by serving as substrates for the tRNA- or aa-tRNA-dependent synthesis of other cellular components. These nontranslational processes range from lipid aminoacylation, and heme, aa, antibiotic and peptidoglycan synthesis to protein degradation. This chapter focuses on these noncanonical usages of aa-tRNAs and the ways of generating them, and also highlights the strategies that cells have evolved to balance the use of aa-tRNAs between protein synthesis and synthesis of other cellular components., (© 2020 Elsevier Inc. All rights reserved.)

Mutations in genes encoding aminoacyl-tRNA synthetases have been reported in several neurological disorders. KARS is a dual localized lysyl-tRNA synthetase and its cytosolic isoform belongs to the multiple aminoacyl-tRNA synthetase complex (MSC). Biallelic mutations in the KARS gene were described in a wide phenotypic spectrum ranging from nonsyndromic deafness to complex impairments. Here, we report on a patient with severe neurological and neurosensory disease investigated by whole-exome sequencing and found to carry biallelic mutations c.683C>T (p.Pro228Leu) and c.871T>G (p.Phe291Val), the second one being novel, in the KARS gene. The patient presented with an atypical clinical presentation with an optic neuropathy not previously reported. At the cellular level, we show that cytoplasmic KARS was expressed at a lower level in patient cells and displayed decreased interaction with MSC. In vitro, these two KARS variants have a decreased aminoacylation activity compared with wild-type KARS, the p.Pro228Leu being the most affected. Our data suggest that dysfunction of cytoplasmic KARS resulted in a decreased level of translation of the nuclear-encoded lysine-rich proteins belonging to the respiratory chain complex, thus impairing mitochondria functions., (© 2019 Wiley Periodicals, Inc.)

Prokaryotic and eukaryotic cytosolic aminoacyl-tRNA synthetases (aaRSs) are essentially known for their conventional function of generating the full set of aminoacyl-tRNA species that are needed to incorporate each organism's repertoire of genetically-encoded amino acids during ribosomal translation of messenger RNAs. However, bacterial and eukaryotic cytosolic aaRSs have been shown to exhibit other essential nonconventional functions. Here we review all the subcellular compartments that prokaryotic and eukaryotic cytosolic aaRSs can reach to exert either a conventional or nontranslational role. We describe the physiological and stress conditions, the mechanisms and the signaling pathways that trigger their relocation and the new functions associated with these relocating cytosolic aaRS. Finally, given that these relocating pools of cytosolic aaRSs participate to a wide range of cellular pathways beyond translation, but equally important for cellular homeostasis, we mention some of the pathologies and diseases associated with the dis-regulation or malfunctioning of these nontranslational functions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of >2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived. Although AlaRS, GlyRS, LeuRS, IleRS, ValRS are the most stable members of the family, GluRS, LysRS and CysRS often have paralogs, whereas AsnRS, GlnRS, PylRS and SepRS are often absent from many genomes. In the course of this analysis, highly conserved protein motifs and domains within each of the AARS loci were identified and used to build a web-based computational tool for the genome-wide detection of AARS coding sequences. This is based on hidden Markov models (HMMs) and is available together with a cognate database that may be used for specific analyses. The bioinformatics tools that we have developed may also help to identify new antibiotic agents and targets using these essential enzymes. These tools also may help to identify organisms with alternative pathways that are involved in maintaining the fidelity of the genetic code.

By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukaryotic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species. Hence, these supposedly cytosolic-restricted enzymes have instead the potential to be multi-localized. As expected, in all examples that were studied so far, when the cytosolic aaRS is imported inside an organelle that already contains its bona fide corresponding organellar-restricted aaRSs, the cytosolic form was proven to exert a nonconventional and essential function. Some of these essential functions include regulating homeostasis and protecting against various stresses. It thus becomes critical to assess meticulously the subcellular localization of each of these cytosolic aaRSs to unravel their additional roles. With this objective in mind, we provide here a review on what is currently known about cytosolic aaRSs multi-compartmentalization and we describe all commonly used protocols and procedures for identifying the compartments in which cytosolic aaRSs relocalize in yeast and human cells., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In this work, we describe the characterization of a quantity-limited sample (100 ng) of yeast mitochondria by shotgun bottom-up proteomics. Sample characterization was carried out by sheathless capillary electrophoresis, equipped with a high sensitivity porous tip and coupled to tandem mass spectrometry (CESI-MS-MS) and concomitantly with a state-of-art nano flow liquid chromatography coupled to a similar mass spectrometry (MS) system (nanoLC-MS-MS). With single injections, both nanoLC-MS-MS and CESI-MS-MS 60 min-long separation experiments allowed us to identify 271 proteins (976 unique peptides) and 300 proteins (1,765 unique peptides) respectively, demonstrating a significant specificity and complementarity in identification depending on the physicochemical separation employed. Such complementary, maximizing the number of analytes detected, presents a powerful tool to deepen a biological sample's proteomic characterization. A comprehensive study of the specificity provided by each separating technique was also performed using the different properties of the identified peptides: molecular weight, mass-to-charge ratio (m/z), isoelectric point (pI), sequence coverage or MS-MS spectral quality enabled to determine the contribution of each separation. For example, CESI-MS-MS enables to identify larger peptides and eases the detection of those having extreme pI without impairing spectral quality. The addition of peptides, and therefore proteins identified by both techniques allowed us to increase significantly the sequence coverages and then the confidence of characterization. In this study, we also demonstrated that the two yeast enolase isoenzymes were both characterized in the CESI-MS-MS data set. The observation of discriminant proteotypic peptides is facilitated when a high number of precursors with high-quality MS-MS spectra are generated., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In Staphylococcus aureus, a T-box riboswitch exists upstream of the glyS gene to regulate transcription of the sole glycyl-tRNA synthetase, which aminoacylates five tRNA(Gly) isoacceptors bearing GCC or UCC anticodons. Subsequently, the glycylated tRNAs serve as substrates for decoding glycine codons during translation, and also as glycine donors for exoribosomal synthesis of pentaglycine peptides during cell wall formation. Probing of the predicted T-box structure revealed a long stem I, lacking features previously described for similar T-boxes. Moreover, the antiterminator stem includes a 42-nt long intervening sequence, which is staphylococci-specific. Finally, the terminator conformation adopts a rigid two-stem structure, where the intervening sequence forms the first stem followed by the second stem, which includes the more conserved residues. Interestingly, all five tRNA(Gly) isoacceptors interact with S. aureus glyS T-box with different binding affinities and they all induce transcription readthrough at different levels. The ability of both GCC and UCC anticodons to interact with the specifier loop indicates ambiguity during the specifier triplet reading, similar to the unconventional reading of glycine codons during protein synthesis. The S. aureus glyS T-box structure is consistent with the recent crystallographic and NMR studies, despite apparent differences, and highlights the phylogenetic variability of T-boxes when studied in a genome-dependent context. Our data suggest that the S. aureus glyS T-box exhibits differential tRNA selectivity, which possibly contributes toward the regulation and synchronization of ribosomal and exoribosomal peptide synthesis, two essential but metabolically unrelated pathways., (© 2015 Apostolidi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

In eukaryotic cells, oxidative phosphorylation involves multisubunit complexes of mixed genetic origin. Assembling these complexes requires an organelle-independent synchronizing system for the proper expression of nuclear and mitochondrial genes. Here we show that proper expression of the F1FO ATP synthase (complex V) depends on a cytosolic complex (AME) made of two aminoacyl-tRNA synthetases (cERS and cMRS) attached to an anchor protein, Arc1p. When yeast cells adapt to respiration the Snf1/4 glucose-sensing pathway inhibits ARC1 expression triggering simultaneous release of cERS and cMRS. Free cMRS and cERS relocate to the nucleus and mitochondria, respectively, to synchronize nuclear transcription and mitochondrial translation of ATP synthase genes. Strains releasing asynchronously the two aminoacyl-tRNA synthetases display aberrant expression of nuclear and mitochondrial genes encoding subunits of complex V resulting in severe defects of the oxidative phosphorylation mechanism. This work shows that the AME complex coordinates expression of enzymes that require intergenomic control., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and ancient enzymes, mostly known for their essential role in generating aminoacylated tRNAs. During the last two decades, many aaRSs have been found to perform additional and equally crucial tasks outside translation. In metazoans, aaRSs have been shown to assemble, together with non-enzymatic assembly proteins called aaRSs-interacting multifunctional proteins (AIMPs), into so-called multi-synthetase complexes (MSCs). Metazoan MSCs are dynamic particles able to specifically release some of their constituents in response to a given stimulus. Upon their release from MSCs, aaRSs can reach other subcellular compartments, where they often participate to cellular processes that do not exploit their primary function of synthesizing aminoacyl-tRNAs. The dynamics of MSCs and the expansion of the aaRSs functional repertoire are features that are so far thought to be restricted to higher and multicellular eukaryotes. However, much can be learnt about how MSCs are assembled and function from apparently 'simple' organisms. Here we provide an overview on the diversity of these MSCs, their composition, mode of assembly and the functions that their constituents, namely aaRSs and AIMPs, exert in unicellular organisms., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Yeast mitochondrial Gln-mtRNAGln is synthesized by the transamidation of mischarged Glu-mtRNAGln by a non-canonical heterotrimeric tRNA-dependent amidotransferase (AdT). The GatA and GatB subunits of the yeast AdT (GatFAB) are well conserved among bacteria and eukaryota, but the GatF subunit is a fungi-specific ortholog of the GatC subunit found in all other known heterotrimeric AdTs (GatCAB). Here we report the crystal structure of yeast mitochondrial GatFAB at 2.0 Å resolution. The C-terminal region of GatF encircles the GatA-GatB interface in the same manner as GatC, but the N-terminal extension domain (NTD) of GatF forms several additional hydrophobic and hydrophilic interactions with GatA. NTD-deletion mutants displayed growth defects, but retained the ability to respire. Truncation of the NTD in purified mutants reduced glutaminase and transamidase activities when glutamine was used as the ammonia donor, but increased transamidase activity relative to the full-length enzyme when the donor was ammonium chloride. Our structure-based functional analyses suggest the NTD is a trans-acting scaffolding peptide for the GatA glutaminase active site. The positive surface charge and novel fold of the GatF-GatA interface, shown in this first crystal structure of an organellar AdT, stand in contrast with the more conventional, negatively charged bacterial AdTs described previously., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondria originate from the α-proteobacterial domain of life. Since this unique event occurred, mitochondrial genomes of protozoans, fungi, plants and metazoans have highly derived and diverged away from the common ancestral DNA. These resulting genomes highly differ from one another, but all present-day mitochondrial DNAs have a very reduced coding capacity. Strikingly however, ATP production coupled to electron transport and translation of mitochondrial proteins are the two common functions retained in all mitochondrial DNAs. Paradoxically, most components essential for these two functions are now expressed from nuclear genes. Understanding how mitochondrial translation evolved in various eukaryotic models is essential to acquire new knowledge of mitochondrial genome expression. In this review, we provide a thorough analysis of the idiosyncrasies of mitochondrial translation as they occur between organisms. We address this by looking at mitochondrial codon usage and tRNA content. Then, we look at the aminoacyl-tRNA-forming enzymes in terms of peculiarities, dual origin, and alternate function(s). Finally we give examples of the atypical structural properties of mitochondrial tRNAs found in some organisms and the resulting adaptive tRNA-protein partnership., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Diagnosis of bronchogenic cysts is possible with computed tomography, where the cysts are seen usually as well-circumscribed lesions of water density. However, many of the cysts have a soft-tissue density thus rendering them indistinguishable from neoplasms. In this article, we describe a case of bronchogenic cyst presenting as soft-tissue mass that was evaluated and diagnosed by endobronchial ultrasound (EBUS). We discuss the ultrasound image characteristics of the cyst and its histopathology findings. EBUS seems to be a valuable tool in the diagnosis of bronchogenic cysts and also enables their complete aspiration.

T-box riboswitches control transcription of downstream genes through the tRNA-binding formation of terminator or antiterminator structures. Previously reported T-boxes were described as single-specificity riboswitches that can bind specific tRNA anticodons through codon-anticodon interactions with the nucleotide triplet of their specifier loop (SL). However, the possibility that T-boxes might exhibit specificity beyond a single tRNA had been overlooked. In Clostridium acetobutylicum, the T-box that regulates the operon for the essential tRNA-dependent transamidation pathway harbors a SL with two potential overlapping codon positions for tRNA(Asn) and tRNA(Glu). To test its specificity, we performed extensive mutagenic, biochemical, and chemical probing analyses. Surprisingly, both tRNAs can efficiently bind the SL in vitro and in vivo. The dual specificity of the T-box is allowed by a single base shift on the SL from one overlapping codon to the next. This feature allows the riboswitch to sense two tRNAs and balance the biosynthesis of two amino acids. Detailed genomic comparisons support our observations and suggest that "flexible" T-box riboswitches are widespread among bacteria, and, moreover, their specificity is dictated by the metabolic interconnection of the pathways under control. Taken together, our results support the notion of a genome-dependent codon ambiguity of the SLs. Furthermore, the existence of two overlapping codons imposes a unique example of tRNA-dependent regulation at the transcriptional level.

In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.

Background: To retrospectively evaluate the outcome of patients with inoperable non-small-cell lung cancer treated with primary external beam radiotherapy combined with high-dose-rate endobronchial brachytherapy boost., Patients and Methods: Between 1988 and 2005, 35 patients with non-small-cell lung cancer (stage I-III) ineligible for surgical resection and/or chemotherapy, were primarily treated with external beam radiotherapy with a median total dose of 50 Gy (range, 46-60). A median of 3 fractions high-dose-rate endobronchial brachytherapy was applied as a boost after external beam radiotherapy, the median total dose was 15 Gy (range, 8-20). High-dose-rate endobronchial brachytherapy was carried out with iridium-192 sources (370 GBq) and prescribed to 1 cm distance from the source axis., Results: With a median follow-up of 26 months from the first fraction of high-dose-rate endobronchial brachytherapy, the 1-, 2- and 5-year overall (local progression-free) survival rates were 76% (76%), 61% (57%) and 28% (42%), respectively. Complete or partial remission rates 6 to 8 weeks after treatment were 57% and 17%, respectively. Significant prognostic favorable factors were a complete remission 6-8 weeks after treatment and a negative nodal status. In patients without mediastinal node involvement, a long-term local control could be achieved with 56% 5-year local progression-free survival. Common Toxicity Criteria grade 3 toxicities were hemoptysis (n = 2) and necrosis (n = 1). One fatal hemoptysis occurred in combination with a local tumor recurrence., Conclusions: The combination of external beam radiotherapy with high-dose-rate endobronchial brachytherapy boost is an effective primary treatment with acceptable toxicity in patients with non-small-cell lung cancer ineligible for surgical resection and/or chemotherapy.

Both narrow band imaging (NBI) and autofluorescence imaging (AFI) are new techniques for the assessment of lung cancer. The major aim of this study was to investigate whether the combination of these two techniques improve sensitivity and specificity in the assessment of lung cancer extension. The study prospectively evaluated 118 patients with suspected lung cancer. All of the patients were examined using EVIS LUCERA SPECTRUM videobronchoscopy system. The narrow band imaging preceded autofluorescence imaging examination. In every patient, at least 1 but no more than 4 biopsies were taken from places visualized as pathologic, surrounding primary tumor, and at least 1 biopsy from places that appeared visually normal. Sensitivity, specificity, positive, and negative predictive value for autofluorescence imaging in the assessment of tumor extension were 89.2, 77.8, 87, and 81%, respectively. Sensitivity, specificity, positive, and negative predictive value for narrow band imaging were 90.4, 82.4, 91.8, and 79.7%, respectively. Corresponding values for combination of techniques were 93.7, 86.9, 94.5, and 85.1%. Combination of techniques significantly improves sensitivity (P = 0.034) with borderline effect on specificity (P = 0.056) of autofluorescence imaging. There was no significant improvement for sensitivity and specificity of NBI alone. The combination of techniques shows significantly better sensitivity and specificity in the assessment of lung cancer extension when compared to white light videobronchoscopy alone, but improvement is not so convincing when compared to the each technique alone.

Analysis of the Gram-positive Clostridium acetobutylicum genome reveals an inexplicable level of redundancy for the genes putatively involved in asparagine (Asn) and Asn-tRNA(Asn) synthesis. Besides a duplicated set of gatCAB tRNA-dependent amidotransferase genes, there is a triplication of aspartyl-tRNA synthetase genes and a duplication of asparagine synthetase B genes. This genomic landscape leads to the suspicion of the incoherent simultaneous use of the direct and indirect pathways of Asn and Asn-tRNA(Asn) formation. Through a combination of biochemical and genetic approaches, we show that C. acetobutylicum forms Asn and Asn-tRNA(Asn) by tRNA-dependent amidation. We demonstrate that an entire transamidation pathway composed of aspartyl-tRNA synthetase and one set of GatCAB genes is organized as an operon under the control of a tRNA(Asn)-dependent T-box riboswitch. Finally, our results suggest that this exceptional gene redundancy might be interconnected to control tRNA-dependent Asn synthesis, which in turn might be involved in controlling the metabolic switch from acidogenesis to solventogenesis in C. acetobutylicum.

Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.

Transbronchial lung biopsy is an indispensable method for the diagnosis of peripheral lung lesions; however, the diagnostic yield still remains unsatisfactory. Endobronchial ultrasound with guide sheath (EBUS-GS) is an excellent method for the decision of biopsy points and has contributed to improvements in diagnostic yield, but the decision of choosing the proper bronchus depends on the individual ability of each bronchoscopist. To clarify the usefulness of the virtual bronchoscopic navigation system (VBN), we evaluated the diagnostic yield and time required to determine the target lesion. Fifty-seven cases using EBUS-GS with VBN (VBN/EBUS-GS group) and 55 cases using EBUS-GS (EBUS-GS group) were compared. In the VBN/EBUS-GS group, computer software detects the air density in the bronchi from the computed tomography image and imports a detailed virtual Bronchoscopic Image. After inserting the starting position and the peripheral target lesion, the software depicts the most ideal route to the target lesion during the bronchoscopic procedure. EBUS is then used to confirm the accuracy of the route. Diagnostic yield was 84.2% for the VBN/EBUS-GS group and 80.0% for EBUS-GS group. The required time to determine the biopsy position was significantly less in the VBN/EBUS-GS group (5.54 ± 0.57 min in VBN/EBUS-GS group vs. 9.27 ± 0.86 min in EBUS-GS group, P<0.01). In conclusion, VBN proved useful in shortening the time needed to determine the biopsy position.

Recurrent respiratory papillomatosis (RRP), which is caused exclusively by human papilloma virus (HPV), is a rare condition characterized by recurrent growth of benign papillomata in the respiratory tract. The papillomata can occur anywhere in the aerodigestive tract but most frequently in the larynx, affecting both children and adults. The management of this entity remains still challenging since no specific definitive treatment exists. Nevertheless, novel surgical interventions as well as several adjuvant therapies have shown promising results in the long-term palliative management of this debilitating disease. Despite its mostly benign nature, RRP may cause significant morbidity and mortality because of its unpredictable clinical course and especially its tendency, albeit infrequent, for malignant transformation. In this article, we present two patients with RRP; one underwent bronchoscopic laser ablation in combination with inhaled interferon-alpha administration that led to a long-term regression of the disease while the other patient was diagnosed with transformation to squamous cell lung carcinoma with fatal outcome. We include a review of the current literature with special emphasis on RRP management and the potential role of HPV in the development of lung cancer.

Autofluorescence imaging videobronchoscopy (AFI) is one of the new systems of autofluorescence bronchoscopy designed for thorough examination of bronchial mucosa. The integration of autofluorescence and videobronchoscopy provides clear images of normal and pathologically altered bronchial mucosa. Major indications for AFI include evaluation of early-stage lung cancer and detection of precancerous lesions. However, in recent years, the indications for AFI are widening, and this tool might find its place in routine daily bronchoscopic practice. With new indications for AFI, such as evaluation of tumor extension or follow-up after surgical resection, this tool might be more often used by bronchoscopists. A sharp learning curve and clear distinction between healthy and pathologically altered mucosa make this technology acceptable for young and inexperienced bronchoscopists. One of the major disadvantages of AFI is low specificity in the detection of premalignant lesions and early-stage lung cancer. This disadvantage could be overcome with the appearance of new and improved technologies in autofluorescence, such as the addition of backscattered light analysis, ultraviolet spectra, fluorescence-reflectance or dual digital systems. Quantitative image analysis is also one of the ways to improve objectivity and minimize observer errors. However, one of the most appropriate solutions would be the addition of AFI to narrow band imaging, and merging the two technologies into one videobronchoscope., (© 2011 Expert Reviews Ltd)

Bronchial carcinoid tumors account for 1-2% of all primary lung tumors and are separated into 2 subgroups: typical and atypical carcinoids. Atypical carcinoids as intermediate-grade malignancies can metastasize more frequently, thus exhibiting poorer prognosis than the low-grade typical carcinoid tumors. Surgical resection remains the mainstay of treatment for pulmonary carcinoids. Bronchoscopic treatment using ablation techniques is an effective alternative to surgery in selected patients with typical carcinoid tumors. However, evidence is lacking regarding the effect of bronchoscopic resection of atypical carcinoid tumor and its recurrences. We report the case of a 73-year-old male with frequent endobronchial recurrences of a previously surgically resected atypical carcinoid tumor successfully treated using Nd:YAG laser photoresection. Furthermore, the therapeutic and local staging aspects of the disease are discussed emphasizing the efficacy of bronchoscopic resection strategies and the value of novel bronchoscopic imaging techniques in detailed inspection of the structures of the bronchial wall., (Copyright © 2011 S. Karger AG, Basel.)

Tracheal diverticulum is a rarely encountered entity, usually discovered incidentally as an outpouching at the right side on radiography or computed tomography (CT). We present the case of a male patient with a tracheal diverticulum detected during a work-up for hemoptysis. His thoracic CT scan showed a sac formation on the right dorsolateral wall of the trachea. Flexible bronchoscopy showed a collapsed entrance to the diverticulum, which could be opened by flexible forceps. The mucosal lining of the diverticulum seemed normal, without retained secretions or blood. Although with limited clinical consequences, tracheal diverticulum must be kept in mind as a reservoir for secretions that may cause infection, and also hemoptysis if infected.