Separation of mycolic acid isomers by cyclic ion mobility-mass spectrometry.

RATIONALE: Mycobacterial species contain high concentrations of mycolic acids in their cell wall. Mycobacteria can pose a threat to both human health and the environment. Mass spectrometry lipidomic characterization can identify bacterial species and suggest targets for microbiological interventions. Due to the complex structures of mycolic acids and the possibility of isobaric isomers, multiple levels of separation are required for complete characterization. In this study, cyclic ion mobility (cIM) mass spectrometry (MS) was used for the analysis, separation and fragmentation of mycolic acids isomers from the bacterial species Gordonia amarae and Mycobacterium bovis. METHODS: Mycolic acid isomers were interrogated from cultured G. amarae biomass and commercially available M. bovis mycolic acid extracts. These were infused into a cIM-enabled quadrupole time-of-flight MS. Ions of interest were non-simultaneously selected with the quadrupole and passed around the cyclic ion mobility device multiple times. Fragment ion analysis was then performed for the resolved isomers of the quadrupole-selected ions. RESULTS: Repeated passes of the cIM device successfully resolved otherwise overlapping MA isomers, allowing isomer isolation and producing an ion-specific post-mobility fragmentation spectrum without isomeric interference. CONCLUSIONS: Mycolic acids (MA) isomers from G. amarae and M. bovis were resolved, resulting in a high mobility resolution and low interference fragmentation analysis. These revealed varying patterns of MA isomers in the two species: G. amarae's most abundant ion of each set of MA has 1-2 conformations, while the MA + 2 m/z the most abundant ion of each set has 3-6 conformations. These were resolved after 70 passes of the cyclic device. M. bovis' most abundant ion of each keto-MA set has 2 conformations, while the keto-MA + 2 m/z has 1-2 conformations. These were resolved after 75 passes.

Biophysical Interaction Landscape of Mycobacterial Mycolic Acids and Phenolic Glycolipids with Host Macrophage Membranes.

Lipidic adjuvant formulations consisting of immunomodulatory mycobacterial cell wall lipids interact with host cells following administration. The impact of this cross-talk on the host membrane's structure and function is rarely given enough consideration but is imperative to rule out nonspecific perturbation underlying the adjuvant. In this work, we investigated changes in the plasma membranes of live mammalian cells after exposure to mycobacterial mycolic acid (MA) and phenolic glycolipids, two strong candidates for lipidic adjuvant therapy. We found that phenolic glycolipid 1 softened the plasma membrane, lowering membrane tension and stiffness, but MA did not significantly change the membrane characteristics. Further, phenolic glycolipid 1 had a fluidizing impact on the host plasma membrane, increasing the fluidity and the abundance of fluid-ordered-disordered coexisting lipid domains. Notably, lipid diffusion was not impacted. Overall, MA and, to a lesser extent, phenolic glycolipid 1, due to minor disruption of host cell membranes, may serve as appropriate lipids in adjuvant formulations.

[An improved extraction and nonradioactive thin-layer chromatography detection method of mycolic acid].

Mycolic acids (MAs), i.e. 2-alkyl, 3-hydroxy long-chain fatty acids, are the hallmark of the cell envelope of Mycobacterium tuberculosis and are related with antibiotic resistance and host immune escape. Nowadays, they've become hot target of new anti-tuberculosis drugs. There are two main methods to detect MAs, 14C metabolic labeling thin-layer chromatography (TLC) and liquid chromatograph mass spectrometer (LC-MS). However, the user qualification of 14C or the lack of standards for LC-MS hampered the easy use of this method. TLC is a common way to analyze chemical substance and can be used to analyze MAs. In this study, we used tetrabutylammonium hydroxide and methyl iodide to hydrolyze and formylate MAs from mycobacterium cell wall. Subsequently, we used diethyl ether to extract methyl mycolate. By this method, we can easily extract and analyze MA in regular biological labs. The results demonstrated that this method could be used to compare MAs of different mycobacterium in different growth phases, MAs of mycobacteria treated by anti-tuberculosis drugs or MAs of mycobacterium mutants. Therefore, we can use this method as an initial validation for the changes of MAs in researches such as new drug screening without using radioisotope or when the standards are not available.

Characterization of Mycobacterium tuberculosis Mycolic Acids by Multiple-Stage Linear Ion-Trap Mass Spectrometry.

Mycobacterium tuberculosis (Mtb) cells are known to synthesize very long chain (C60-90) structurally complex mycolic acids with various functional groups. In this study, we applied linear ion-trap (LIT) multiple-stage mass spectrometry (MSn), combined with high-resolution mass spectrometry to study the mechanisms underlying the fragmentation processes of mycolic acid standards desorbed as lithiated adduct ions by ESI. This is followed by structural characterization of a Mtb mycolic acid family (Bovine strain). Using the insight fragmentation processes gained from the study, we are able to achieve a near complete characterization of the whole mycolic acid family, revealing the identity of the α-alkyl chain, the location of the functional groups including methyl, methoxy, and keto groups along the meroaldehyde chain in each lipid species. This study showcased the power of LIT MSn toward structural determination of complex lipids in a mixture, which would be otherwise very difficult to define using other analytical techniques.

Biomarcadores en tuberculosis / Biomarkers in tuberculosis

Resumen Los métodos diagnósticos clásicos para la tuberculosis son de baja sensibilidad o son muy lentos en la obtención de resultados (baciloscopía, cultivo de Koch). De ahí nace la necesidad de nuevos métodos diagnósticos para esta enfermedad. Los biomarcadores surgen como una opción a esta problemática, con un buen rendimiento diagnóstico, costo y accesibilidad. Ellos permiten identificar la respuesta inflamatoria y/o metabólica del huésped, extrapolando la presencia de Mycobacterium tuberculosis; o identifican moléculas propias del patógeno. En la presente revisión se describen biomarcadores que presentan un buen rendimiento diagnóstico basados en metodologías de investigación de alto nivel (estudio de cohortes, prospectivos, muestreo consecutivo o aleatorizado, comparación de rendimiento diagnóstico frente a cultivo). Es necesario el desarrollo de estas nuevas técnicas con el fin de realizar el diagnóstico precoz de la enfermedad y lograr así su tan ansiada eliminación.

The classical laboratory diagnostic methods for tuberculosis have a low sensitivity or take a long time to know their results. New methods are underway. Biomarkers are a good option to improve our diagnostic approach to this disease. They have good performance, low cost and accessibility. They identify a patient's inflammatory or metabolic response to Mycobacterium Tuberculosis or identifies molecules that are typical of the pathogen. In this paper we sum up the biomarkers with a good diag-nostic performance described in well design investigations. Early diagnosis with these new techniques should contribute to the elimination of the disease.

Metabolic Labeling of Live Mycobacteria with Trehalose-Based Probes.

The mycobacterial cell envelope includes a unique outer membrane, also known as the mycomembrane, which is the major defense barrier that confers intrinsic drug tolerance to Mycobacterium tuberculosis (Mtb) and related bacteria. The mycomembrane is typified by long-chain mycolic acids that are esterified to various acceptors, including: (1) trehalose, forming trehalose mono- and di-mycolate; (2) arabinogalactan, forming arabinogalactan-linked mycolates; and (3) in some species, protein serine residues, forming O-mycoloylated proteins. Synthetic trehalose and trehalose monomycolate analogs have been shown to specifically and metabolically incorporate into mycomembrane components, facilitating their analysis in native contexts and opening new avenues for the specific detection and therapeutic targeting of mycobacterial pathogens in complex settings. This chapter highlights trehalose-based probes that have been developed to date, briefly discusses their applications, and describes protocols for their use in mycobacteria research.

Mycobacterium vicinigordonae sp. nov., a slow-growing scotochromogenic species isolated from sputum.

A slow-growing, scotochromogenic mycobacterial strain (24T) was isolated from the sputum of a Chinese male human. Phylogenetic analysis using the 16S rRNA gene assigned strain 24T to the Mycobacterium gordonae complex, which includes Mycobacterium gordonae and Mycobacterium paragordonae. The phenotypic characteristics, unique mycolic acid profile and the results of phylogenetic analysis based on hsp65 and rpoB sequences strongly supported the taxonomic status of strain 24T as a representative of a species distinct from the other members of the M. gordonae complex. The genomic G+C content of strain 24T was 65.40mol%. Genomic comparisons showed that strain 24T and M. gordonae ATCC 14470T had an average nucleotide identity (ANI) value of 81.00 % and a DNA-DNA hybridization (DDH) value of 22.80 %, while the ANI and DDH values between strain 24Tand M. paragordonae 49 061T were 80.98 and 22.80 %, respectively. In terms of phylogenetic, phenotypic and chemotaxonomic features, strain 24T is distinguishable from its closest phylogenetic relatives and represents a novel species of the genus Mycobacterium, therefore the name Mycobacterium vicinigordonae sp. nov. is proposed. The type strain is 24T (=CMCC 93559T=DSM 105979T).

Mechanisms of Resistance Associated with the Inhibition of the Dehydration Step of Type II Fatty Acid Synthase in Mycobacterium tuberculosis.

Isoxyl (ISO) and thiacetazone (TAC) are two antitubercular prodrugs that abolish mycolic acid biosynthesis and kill Mycobacterium tuberculosis (Mtb) through the inhibition of the essential type II fatty acid synthase (FAS-II) dehydratase HadAB. While mutations preventing ISO and TAC either from being converted to their active form or from covalently modifying their target are the most frequent spontaneous mutations associated with high-level resistance to both drugs, the molecular mechanisms underlying the high-level ISO and TAC resistance of Mtb strains harboring missense mutations in the second, nonessential, FAS-II dehydratase HadBC have remained unexplained. Using a combination of genetic, biochemical, and biophysical approaches and molecular dynamics simulation, we here show that all four reported resistance mutations in the HadC subunit of HadBC alter the stability and/or specific activity of the enzyme, allowing it in two cases (HadBCV85I and HadBCK157R) to compensate for a deficiency in HadAB in whole Mtb bacilli. The analysis of the mycolic acid profiles of Mtb strains expressing the mutated forms of HadC further points to alterations in the activity of the mycolic acid biosynthetic complex and suggests an additional contributing resistance mechanism whereby HadC mutations may reduce the accessibility of HadAB to ISO and TAC. Collectively, our results highlight the importance of developing optimized inhibitors of the dehydration step of FAS-II capable of inhibiting both dehydratases simultaneously, a goal that may be achievable given the structural resemblance of the two enzymes and their reliance on the same catalytic subunit HadB.

Localization of Cyclopropane Modifications in Bacterial Lipids via 213 nm Ultraviolet Photodissociation Mass Spectrometry.

Subtle structural features in bacterial lipids such as unsaturation elements can have vast biological implications. Cyclopropane rings have been correlated with tolerance to a number of adverse conditions in bacterial phospholipids. They have also been shown to play a major role in Mycobacterium tuberculosis ( M. tuberculosis or Mtb) pathogenesis as they occur in mycolic acids (MAs) in the mycobacterial cell. Traditional collisional activation methods allow elucidation of basic structural features of lipids but fail to reveal the presence and position of cyclopropane rings. Here, we employ 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for structural characterization of cyclopropane rings in bacterial phospholipids and MAs. Upon UVPD, dual cross-ring C-C cleavages on both sides of the cyclopropane ring are observed for cyclopropyl lipids, resulting in diagnostic pairs of fragment ions spaced 14 Da apart, thus enabling cyclopropane localization. These diagnostic pairs of ions corresponding to dual cross-ring cleavage are observed in both negative and positive ion modes and afford localization of multiple cyclopropane rings within a single lipid. This method was integrated with liquid chromatography (LC) for LC/UVPD-MS analysis of cyclopropyl glycerophospholipids in Escherichia coli ( E. coli) and for analysis of MAs in Mycobacterium bovis ( M. bovis) and M. tuberculosis lipid extracts.

Comparative label-free lipidomic analysis of Mycobacterium tuberculosis during dormancy and reactivation.

Mycobacterium tuberculosis employs several strategies to combat and adapt to adverse conditions encountered inside the host. The non-replicative dormant state of the bacterium is linked to drug resistance and slower response to anti-tubercular therapy. It is known that alterations in lipid content allow dormant bacteria to acclimatize to cellular stress. Employing comparative lipidomic analysis we profiled the changes in lipid metabolism in M. tuberculosis using a modified Wayne's model of hypoxia-induced dormancy. Further we subjected the dormant bacteria to resuscitation, and analyzed their lipidomes until the lipid profile was similar to that of normoxially grown bacteria. An enhanced degradation of cell wall-associated and cytoplasmic lipids during dormancy, and their gradual restoration during reactivation, were clearly evident. This study throws light on distinct lipid metabolic patterns that M. tuberculosis undergoes to maintain its cellular energetics during dormancy and reactivation.

Identification of mycolic acid forms using surface-enhanced Raman scattering as a fast detection method for tuberculosis.

BACKGROUND: Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, based on the WHO Global Tuberculosis Report in 2017. TB causes massive health care burdens in many parts of the world, specifically in the resource constrained developing world. Most deaths from TB could be prevented with cost effective early diagnosis and appropriate treatment. PURPOSE: Conventional TB detection methods are either too slow as it takes a few weeks for diagnosis or they lack the specificity and accuracy. Thus the objective of this study was to develop a fast and efficient detection for TB using surface enhanced Raman scattering (SERS) technique. METHODS: SERS spectra for different forms of mycolic acids (MAs) that are both synthetic origin and actual extracts from the mycobacteria species were obtained by label-free direct detection mode. Similarly, we collected SERS spectra for γ-irradiated whole bacteria (WB). Measurements were done using silver (Ag) coated silicon nanopillar (Ag SNP) as SERS substrate. RESULTS: We report the SERS based detection of MA, which is a biomarker for mycobacteria species including Mycobacterium tuberculosis. For the first time, we also establish the SERS spectral characterization of the three major forms of MA - αMA, methoxy-MA, and keto-MA, in bacterial extracts and also in γ-irradiated WB. We validated our findings by mass spectrometry. SERS detection of these three forms of MA could be useful in differentiating pathogenic and nonpathogenic Mycobacterium spp. CONCLUSIONS: We have demonstrated the direct detection of three major forms of MA - αMA, methoxy-MA, and keto-MA, in two different types of MA extracts from MTB bacteria, namely delipidated MA and undelipidated MA and finally in γ-irradiated WB. In the near future, this study could pave the way for a fast and efficient detection method for TB, which is of high clinical significance.

Catenulobactins A and B, Heterocyclic Peptides from Culturing Catenuloplanes sp. with a Mycolic Acid-Containing Bacterium.

The production of two new heterocyclic peptide isomers, catenulobactins A (1) and B (2), in cultures of Catenuloplanes sp. RD067331 was significantly increased when it was cocultured with a mycolic acid-containing bacterium. The planar structures and absolute configurations of the catenulobactins were determined based on NMR/MS and chiral-phase GC-MS analyses. Catenulobactin B (2) displayed Fe(III)-chelating activity and moderate cytotoxicity against P388 murine leukemia cells.

Identification of a Desaturase Involved in Mycolic Acid Biosynthesis in Mycobacterium smegmatis.

Mycolic acids are unique long chain fatty acids found in the cell walls of mycobacteria including the tubercle bacillus, Mycobacterium tuberculosis. The introduction of double bonds in mycolic acids remains poorly understood, however, genes encoding two potential aerobic desaturases have been proposed to be involved in this process. Here we show that one of these genes, desA1, is essential for growth of the saprophytic Mycobacterium smegmatis. Depletion of desA1 in a M. smegmatis conditional mutant led to reduction of mycolic acid biosynthesis and loss of viability. The DesA1-depleted cells exhibited two other phenotypes: using 14[C]-labelling, we detected the accumulation of minor mycolic acid-related species that migrated faster in a silver TLC plate. Spiral Time of Flight Mass Spectroscopic analysis suggested the presence of species with sizes corresponding to what were likely monoenoic derivatives of α-mycolic acids. Additionally, conditional depletion led to the presence of free fatty acyl species of lengths ~C26-C48 in the lysing cells. Cell viability could be rescued in the conditional mutant by Mycobacterium tuberculosis desA1, highlighting the potential of desA1 as a new drug target in pathogenic mycobacteria.

Williamsia herbipolensis sp. nov., isolated from the phyllosphere of Arabidopsis thaliana.

A Gram-stain-positive, non-endospore-forming actinobacterium (ARP1T) was isolated from the phyllosphere of Arabidopsis thaliana. On the basis of 16S rRNA gene sequence phylogeny strain ARP1T was placed into the genus Williamsia and the closest related species were Williamsia phyllosphaerae (98.5 % 16S rRNA gene sequence similarity), Williamsia deligens (98.5 %), Williamsia maris (98.3 %) and Williamsia serinedens (98.2 %). Genome-based comparison indicated a clear distinction to the type strains of those species with pairwise average nucleotide identities (ANI) between 76.4-78.4 %. The quinone system of strain ARP1T consisted predominantly of menaquinones MK-9(H2), MK-7(H2) and MK-8(H2), and the polar lipid profile contained the major compound diphosphatidylglycerol, and moderate amounts of phosphatidylethanolamine, phosphatidylglycerol and numerous unidentified lipids. Mycolic acids were present. These chemotaxonomic traits and the major fatty acids, which were C16 : 1ω7c, C16 : 0, C18 : 0, C18 : 1ω9c and tuberculostearic acid supported the affiliation of strain ARP1T to the genus Williamsia. Genotypic, physiological and biochemical testing revealed clear differences of strain ARP1T to the most closely related species of the genus Williamsia. Therefore strain ARP1T represents a novel species of this genus, for which the name Williamsia herbipolensis sp. nov. is proposed. The type strain is ARP1T (=DSM 46872T=LMG 28679T).

Bioorthogonal Chemical Reporters for Selective In†Situ Probing of Mycomembrane Components in Mycobacteria.

The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non-covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria-specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non-covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective in situ detection of the major MM components.

[Identification of Hydrocarbon-Oxidizing Dietzia Bacteria from Petroleum Reservoirs Based on Phenotypic Properties and Analysis of the 16S rRNA and gyrB Genes].

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol%, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002(T), respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classiffication of strain 32d as a D. maris strain and of strain 263, as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

Lipid biomarkers provide evolutionary signposts for the oldest known cases of tuberculosis.

Studies on the evolution of tuberculosis, and the influence of this disease on human and animal development and interaction, require the accumulation of indisputable biomarker evidence. Ideally, the determination of full genomes would provide all the necessary information, but for very old specimens DNA preservation may be compromised and only limited DNA amplification may be a possibility. Mycobacterium tuberculosis is characterised by the presence of unusual cell envelope lipids, with specific biomarker potential. Lipid biomarker recognition has been decisive in pinpointing the oldest known cases of human and animal tuberculosis; the former are a woman and child from a pre-pottery settlement at Atlit-Yam, Israel (∼9,000 ka) and the latter is an extinct Bison antiquus from Natural Trap Cave, Wyoming (∼17,000 ka). Including some new data, it is demonstrated how analysis of a combination of mycolic, mycocerosic and mycolipenic acid and phthiocerol biomarkers provide incontrovertible evidence for tuberculosis in these landmark specimens.

Morphological and biomolecular evidence for tuberculosis in 8th century AD skeletons from Bélmegyer-Csömöki domb, Hungary.

Macromorphological analysis of skeletons, from 20 selected graves of the 8th century AD Bélmegyer-Csömöki domb, revealed 19 cases of possible skeletal tuberculosis. Biomolecular analyses provided general support for such diagnoses, including the individual without pathology, but the data did not show coherent consistency over the range of biomarkers examined. Amplification of ancient DNA fragments found evidence for the Mycobacterium tuberculosis complex DNA only in five graves. In contrast, varying degrees of lipid biomarker presence were recorded in all except two of the skeletons, though most lipid components appeared to be somewhat degraded. Mycobacterial mycolic acid biomarkers were absent in five cases, but the weak, possibly degraded profiles for the remainder were smaller and inconclusive for either tuberculosis or leprosy. The most positive lipid biomarker evidence for tuberculosis was provided by mycolipenic acid, with 13 clear cases, supported by five distinct possible cases. Combinations of mycocerosic acids were present in all but three graves, but in one case a tuberculosis-leprosy co-infection was indicated. In two specimens with pathology, no lipid biomarker evidence was recorded, but one of these specimens provided M. tuberculosis complex DNA fragments.

Ancient mycobacterial lipids: Key reference biomarkers in charting the evolution of tuberculosis.

Mycobacterium tuberculosis has a cell envelope incorporating a peptidoglycan-linked arabinogalactan esterified by long-chain mycolic acids. A range of "free" lipids are associated with the "bound" mycolic acids, producing an effective envelope outer membrane. The distribution of these lipids is discontinuous among mycobacteria and such lipids have proven potential for biomarker use in tracing the evolution of tuberculosis. A plausible evolutionary scenario involves progression from an environmental organism, such as Mycobacterium kansasii, through intermediate "smooth" tubercle bacilli, labelled "Mycobacterium canettii"; cell envelope lipid composition possibly correlates with such a progression. M. kansasii and "M. canettii" have characteristic lipooligosaccharides, associated with motility and biofilms, and glycosyl phenolphthiocerol dimycocerosates ("phenolic glycolipids"). Both these lipid classes are absent in modern M. tuberculosis sensu stricto, though simplified phenolic glycolipids remain in certain current biotypes. Dimycocerosates of the phthiocerol family are restricted to smaller phthiodiolone diesters in M. kansasii. Diacyl and pentaacyl trehaloses are present in "M. canettii" and M. tuberculosis, accompanied in the latter by related sulfated acyl trehaloses. In comparison with environmental mycobacteria, subtle modifications in mycolic acid structures in "M. canettii" and M. tuberculosis are notable. The probability of essential tuberculosis evolution taking place in Pleistocene megafauna, rather than Homo sapiens, is reemphasised.

Tuberculosis origin: The Neolithic scenario.

This paper follows the dramatic changes in scientific research during the last 20 years regarding the relationship between the Mycobacterium tuberculosis complex and its hosts - bovids and/or humans. Once the M. tuberculosis and Mycobacterium bovis genomes were sequenced, it became obvious that the old story of M. bovis evolving into the human pathogen should be reversed, as M. tuberculosis is more ancestral than M. bovis. Nevertheless, the timescale and geographical origin remained an enigma. In the current study human and cattle bone samples were examined for evidence of tuberculosis from the site of Atlit-Yam in the Eastern Mediterranean, dating from 9250 to 8160 (calibrated) years ago. Strict precautions were used to prevent contamination in the DNA analysis, and independent centers used to confirm authenticity of findings. DNA from five M. tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. These, together with pathological changes detected in some of the bones, confirm the presence of the disease in the Levantine populations during the Pre-pottery Neolithic C period, more than 8000 years ago.