Biomedical subjects

M Mergeay

Publications and source records attributed to M Mergeay.

At least 19 recordsLinked to original sources

PubMed2004

Effect of curcuma on radiation-induced apoptosis in human cancer cells.

There have been considerable efforts to search for naturally occurring substances for the intervention of carcinogenesis. Many components from dietary or medicinal plants have been identified that possess substantial chemopreventive properties. Curcuma, a yellow pigment from Curcuma longa, exhibits anti-inflammatory, antitumor, and antioxidative properties. Although its precise mode of action has not been elucidated so far, studies have shown that chemopreventive action of curcuma might be due to its ability to induce apoptosis (programmed cell death) in cancer cells. This original study was conducted in order to estimate whether curcuma enhances the radiation sensitivity of cancer cells. For this purpose, curcuma (concentrations ranging from 0 to 200 microM) was applied to human cancer cell cultures (HeLa, K-562 and IM-9) with or without X-irradiation (doses comprised between 0 and 8 Gy). Cell proliferation was monitored by trypan blue exclusion. For the estimation of apoptosis, changes in cell morphology and flow cytometry analysis (DNA content and presence of the sub-G1 peak) were performed. Microscopic examination of the curcuma-treated cells (with concentrations above 100 microM) showed a characteristic morphology of apoptosis. Furthermore, cells treated with curcuma exhibited a sub-G1 peak from which the magnitude was proportional to the concentration of curcuma. X-irradiation alone induced polyploidisation and apoptosis of the three cell lines, proportional to the doses of irradiation with a marked difference in radiation sensitivity between the cell lines (IM-9 < K-562 < HELA). However, when radiation and curcuma were applied together, our results showed that in HELA, K-562 and IM-9, curcuma showed a radiation sensitising effect only at the dose of 200 micro M. This result may open a perspective of synergical therapy at the condition to also address the intrinsic toxicity of curcuma on normal cells.

Apoptosis↗

PubMed2002

Isolation, characterization, and identification of bacteria associated with the zinc hyperaccumulator Thlaspi caerulescens subsp. calaminaria.

We investigated bacterial populations associated with the Zn hyperaccumulator Thlaspi caerulescens subsp. calaminaria grown in a soil collected from an abandoned Zn-Pb mine and smelter in Plombières, Belgium. The bacterial population of the nonrhizospheric soil consisted of typical soil bacteria, some exhibiting multiple heavy-metal resistance characteristics that often are associated with polluted substrates: 7.8% and 4% of the population survived in the presence of elevated levels of Zn (1 mM) and Cd (0.8 mM), respectively. For the bacterial population isolated from the rhizosphere, the comparable survival rates were 88 and 78%. This observation indicates a selective enrichment of the metal-resistant strains due to an increased availability of the metals in soils near the roots compared with nonrhizospheric soil. The endophytic inhabitants of the roots and shoots were isolated, identified, and characterized. Although similar endophytic species were isolated from both compartments, those from the rhizoplane and roots showed lower resistance to Zn and Cd than the endophytic bacteria isolated from the shoots. In addition, root endophytic bacteria had additional requirements. Contrary to the rootresiding inhabitants, the shoot represented a niche rich in metal-resistant bacteria and even seemed to contain species that were exclusively abundant there. These differences in the characteristics of the bacterial microflora associated with T. caerulescens might possibly reflect altered metal speciation in the different soils and plant compartments studied.

Journal Article↗

PubMed2001

A microbial biosensor to predict bioavailable nickel in soil and its transfer to plants.

Ralstonia eutropha strain AE2515 was constructed and optimised to serve as a whole-cell biosensor for the detection of bioavailable concentrations of Ni2+ and Co2+ in soil samples. Strain AE2515 is a Ralstonia eutropha CH34 derivative containing pMOL1550, in which the cnrYXH regulatory genes are transcriptionally fused to the bioluminescent luxCDABE reporter system. Strain AE2515 was standardised for its specific responses to Co2+ and Ni2+. The detection limits for AE2515 were 0.1 microM Ni2+ and 9 microM Co2+, respectively. The signal to noise (S/N) bioluminescence response and the metal cation concentration could be linearly correlated: for Ni2+ this was applicable within the range 0.1-60 microM, and between 9 and 400 microM for Co2+. The AE2515 biosensor strain was found to be highly selective for nickel and cobalt: no induction was observed with Zn(II), Cd(II), Mn(II), Cu(III) and Cr(VI). In mixed metal solutions, the bioluminescent response always corresponded to the nickel concentrations. Only in the presence of high concentrations of Co2+ (2 mM), the sensitivity to nickel was reduced due to metal toxicity. AE2515 was used to quantify the metal bioavailability in various nickel-enriched soils, which had been treated with additives for in situ metal immobilisation. The data obtained with strain AE2515 confirmed that the bioavailability of nickel was greatly reduced following the treatment of the soils with the additives beringite and steel shots. Furthermore, the data were found to correlate linearly with those on the biological accumulation of Ni2+ in specific parts of important agricultural crops, such as maize and potato. Therefore, the test can be used to assess the potential transfer of nickel to organisms of higher trophic levels, in this case maize and potato plants grown on nickel-enriched soils, and the potential risk of transfer of these elements to the food chain.

Biosensing Techniques↗

PubMed2001

Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient.

A polyphasic taxonomic study, including 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base ratio determinations, amplified 165 rDNA restriction analysis, whole-cell protein analyses and extensive biochemical characterization, was conducted to clarify the relationships of eight isolates from root nodules of Mimosa species and one isolate from sputum of a cystic fibrosis patient. All nine isolates were classified as a novel Ralstonia species, for which the name Ralstonia taiwanensis sp. nov. is proposed. The type strain is LMG 19424T (= CCUG 44338T). R. taiwanensis effectively nodulated the Mimosa species and is the first beta-proteobacterium that is known to be capable of root nodule formation and nitrogen fixation.

Anti-Bacterial Agents↗

PubMed2001

Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend.

Thirty-one heavy-metal-resistant bacteria isolated from industrial biotopes were subjected to polyphasic characterization, including 16S rDNA sequence analysis, DNA-DNA hybridizations, biochemical tests, whole-cell protein and fatty-acid analyses. All strains were shown to belong to the Ralstonia branch of the beta-Proteobacteria. Whole-cell protein profiles and DNA-DNA hybridizations revealed two clearly distinct groups, showing low similarity to known Ralstonia species. These two groups, of 8 and 17 isolates, were assigned to two new species, for which the names Ralstonia campinensis sp. nov. and Ralstonia metallidurans sp. nov. are proposed. The type strains are WS2T (= LMG 19282T = CCUG 44526T) and CH34T (= LMG 1195T = DSM 2839T), respectively. Six isolates were allocated to Ralstonia basilensis, which presently contains only the type strain; an emendation of the latter species description is therefore proposed.

Bacterial Proteins↗

PubMed2001

Occurrence of Tn4371-related mobile elements and sequences in (chloro)biphenyl-degrading bacteria.

Tn4371, a 55-kb transposable element involved in the degradation and biphenyl or 4-chlorobiphenyl identified in Ralstonia eutropha A5, displays a modular structure including a phage-like integrase gene (int), a Pseudomonas-like (chloro)biphenyl catabolic gene cluster (bph), and RP4- and Ti-plasmid-like transfer genes (trb) (C. Merlin, D. Springael, and A. Toussaint, Plasmid 41:40-54, 1999). Southern blot hybridization was used to examine the presence of different regions of Tn4371 in a collection of (chloro)biphenyl-degrading bacteria originating from different habitats and belonging to different bacterial genera. Tn4371-related sequences were never detected on endogenous plasmids. Although the gene probes containing only bph sequences hybridized to genomic DNA from most strains tested, a limited selection of strains, all beta-proteobacteria, displayed hybridization patterns similar to the Tn4371 bph cluster. Homology between Tn4371 and DNA of two of those strains, originating from the same area as strain A5, extended outside the catabolic genes and covered the putative transfer region of Tn4371. On the other hand, none of the (chloro)biphenyl degraders hybridized with the outer left part of Tn4371 containing the int gene. The bph catabolic determinant of the two strains displaying homology to the Tn4371 transfer genes and a third strain isolated from the A5 area could be mobilized to a R. eutropha recipient, after insertion into an endogenous or introduced IncP1 plasmid. The mobilized DNA of those strains included all Tn4371 homologous sequences previously identified in their genome. Our observations show that the bph genes present on Tn4371 are highly conserved between different (chloro)biphenyl-degrading hosts, isolated globally but belonging mainly to the beta-proteobacteria. On the other hand, Tn4371-related mobile elements carrying bph genes are apparently only found in isolates from the environment that provided the Tn4371-bearing isolate A5.

Bacteria↗

PubMed2001

Nickel-resistance-based minitransposons: new tools for genetic manipulation of environmental bacteria.

The ncc and nre nickel resistance determinants from Ralstonia eutropha-like strain 31A were used to construct mini-Tn5 transposons. Broad host expression of nickel resistance was observed for the nre minitransposons in members of the alpha, beta, and gamma subclasses of the Proteobacteria, while the ncc minitransposons expressed nickel resistance only in R. eutropha-like strains.

Conjugation, Genetic↗

PubMed2001

Dissemination of catabolic plasmids among desiccation-tolerant bacteria in soil microcosms.

The dissemination of catabolic plasmids was compared to bioaugmentation by strain inoculation in microcosm experiments. When Rhodococcus erythropolis strain T902, bearing a plasmid with trichloroethene and isopropylbenzene degradation pathways, was used as the inoculum, no transconjugant was isolated but the strain remained in the soil. This plasmid had a narrow host range. Pseudomonas putida strain C8S3 was used as the inoculum in a second approach. It bore a broad host range conjugative plasmid harboring a natural transposon, RP4::Tn4371, responsible for biphenyl and 4-chlorobiphenyl degradation pathways. The inoculating population slowly decreased from its original level (10(6) colony-forming units [CFU]/g of dry soil) to approx 3 x 10(2) CFU/g of dry soil after 3 wk. Transconjugant populations degrading biphenyl appeared in constant humidity soil (up to 2 x 10(3) CFU/g) and desiccating soil (up to 10(4) CFU/g). The feasibility of plasmid dissemination as a bioaugmentation technique was demonstrated in desiccating soils. The ecologic significance of desiccation in bioaugmentation was demonstrated: it upset the microbial ecology and the development of transconjugants.

Bacteria↗

PubMed2000

Factors influencing the biosorption of gadolinium by micro-organisms and its mobilisation from sand.

The present work was devoted to the study of the biosorption capacities of various microbial species (Bacillus subtilis, Pseudomonas aeruginosa, Ralstonia metallidurans CH34 previously Alcaligenes eutrophus CH34, Mycobacterium smegmatis, Saccharomyces cerevisiae) for ions of the lanthanide gadolinium (Gd3+). The uptake by sand of this element was also measured. Saturation curves and Scatchard models were established for all biosorbants used in this work. The results enabled us to determine the binding affinities and the maximum capacities for biosorption of Gd3+, which ranged from 350 micromol g(-1) for B. subtilis to 5.1 micromol g(-1) for S. cerevisiae. This study demonstrated the usefulness of optimisation of experimental conditions in biosorption investigations. Experimental results showed that biosorption could be influenced by the growth stage and by the composition of the growth medium of microbial cells. Finally, particular attention was given to the transfer of gadolinium ions from a loaded sand to a bacterial suspension.

Adsorption↗

PubMed2000

Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils.

Metal concentrations in soils are locally quite high, and are still increasing due to many human activities, leading to elevated risk for health and the environment. Phytoremediation may offer a viable solution to this problem, and the approach is gaining increasing interest. Improvement of plants by genetic engineering, i.e. by modifying characteristics like metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. So far, only a few cases have been reported where one or more of these characteristics have been successfully altered; e.g. mercuric ion reduction causing improved resistance and phytoextraction, and metallothionein causing enhanced cadmium tolerance. These, together with other approaches and potentially promising genes for transformation of target plants are discussed.

Journal Article↗

PubMed2000

Regulation of the cnr cobalt and nickel resistance determinant of Ralstonia eutropha (Alcaligenes eutrophus) CH34.

The linked resistance to nickel and cobalt of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) is encoded by the cnr operon, which is localized on the megaplasmid pMOL28. The regulatory genes cnrYXH have been cloned, overexpressed, and purified in Escherichia coli. CnrY fractionated as a 10.7-kDa protein in in vitro translation assays. CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in E. coli. His-CnrX was found to possess a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the presence of E. coli core RNA polymerase enzyme, could retard at least two different promoter DNA targets, cnrYp and cnrHp, localized within the cnrYXH locus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL M91650 description entry). To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of cnrYXH and the luxCDABE, luciferase reporter genes. Nickel and cobalt specifically induced the cnrYXH-luxCDABE fusion at optimal concentrations of 0.3 mM Ni(2+) and 2.0 mM Co(2+) in a noncomplexing medium for metals. The two promoter regions P(Y) (upstream cnrY) and P(H) (upstream cnrH) were probed and characterized using this vector and were found to control the nickel-inducible regulatory response of the cnr operon. The cnrHp promoter was responsible for full transcription of the cnrCBA structural resistance genes, while the cnrYp promoter was necessary to obtain metal-inducible transcription from the cnrHp promoter. The zinc resistance phenotype (ZinB) of a spontaneous cnr mutant strain, AE963, was investigated and could be attributed to an insertion of IS1087, a member of the IS2 family of insertion elements, within the cnrY gene.

Amino Acid Sequence↗

PubMed1999

Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance. An omega-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained. A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined. Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified. The predicted gene products of czrCBA show a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc. The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds. DNA-DNA hybridization indicated strong conservation of czr in other environmental P. aeruginosa isolates and in the P. aeruginosa type strain PAO1, a clinical isolate. This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1. A high sequence identity (till 99% at the nt level) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs. The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.

Bacterial Proteins↗

PubMed1999

Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs.

The level of genotypic relationship between czc+ soil bacteria mainly resistant to zinc (but also to various other metals), and related facultative hydrogenotrophs previously assigned to the genera Alcaligenes, Ralstonia, and Burkholderia was evaluated using ARDRA (Amplified Ribosomal DNA Restriction Analysis). The analysis included 44 strains isolated from harsh industrial environments in sediments, soils and wastes with high content of heavy metals. These strains were selected by their ability to grow in the presence of high concentrations of multiple heavy metals and to hybridise with czc or ncc probes. The czc operon confers resistance to cadmium, zinc and cobalt in strain Ralstonia eutropha CH34. The ncc operon confers resistance to nickel, cobalt and cadmium in strain 31A known as Alcaligenes xylosoxidans. The analysis showed a close phylogenetic clustering of the czc+ strains inside the Ralstonia genus despite of their different origins and that the Ralstonia genus contained also the hydrogenotrophs and some catabolic strains assigned to the genus Ralstonia eutropha, strains up to now registrated as CDC IV c-2 strains as well as reference strains belonging to Ralstonia solanacearum and Ralstonia pickettii. The ncc+ strains are phylogenetically less related to each other compared to the czc+ strains. This suggests that the tested czc+ strains and some of the ncc+ strains may be considered as belonging to the genus Ralstonia. Inside this major Ralstonia cluster, a subcluster gathers most of the czc+ isolates maybe giving a clue to define a new species. Besides, from 30 tested strains, 15 metal resistant strains of this subcluster proved to display the unusual mutator phenotype characteristic of the representative strain CH34.

Genotype↗

PubMed1999

Characterization of the bacterial community of a zinc-polluted soil.

The bacterial community of a zinc-contaminated soil (Maatheide soil in Lommel, Belgium) was studied using cultivation as well as cultivation-independent techniques. Colony-forming units (CFU) were determined by plating on media with or without metals. Dominant isolates were characterized by fatty acid methyl ester analysis (FAME analysis) and PCR fingerprinting using repetitive extragenic palindromic sequences as primers. DNA was directly extracted from soil samples and used as a template for the PCR amplification of the 16S rDNA (8-1511) or a 16S rDNA fragment (968-1401). Clones resulting from cloning the 16S rDNA from soil DNA were sequenced. Temperature gradient gel electrophoresis (TGGE analysis) was performed for 16S rDNA fragments (968-1401) amplified from the dominant isolates, the clones, and the total soil DNA extracted according to two protocols differing in strength of lysis. Total CFU ranged from 10(4) to 10(5)/g soil. The majority of the isolates were identified by FAME analysis as Arthrobacter spp. (18 out of 23). None of the isolates were identified as a Ralstonia eutropha like strain (formerly Alcaligenes eutrophus). Metalloresistant Rastomia eutropha like strains were previously shown to be dominant in the analyzed biotope. Most of the isolates were zinc tolerant but only seven could be considered zinc resistant. Sequences of the 16S rDNA clones obtained from total soil DNA were affiliated with genes of different bacteria such as alpha-proteobacteria, beta-proteobacteria, and the Cytophaga-Flexibacter-Bacteroides group. None of the sequenced clones aligned with the Ralstonia eutropha 16S rRNA gene. TGGE analysis of the 16S rDNA fragments (968-1401) amplified from the dominant strains, the clones, and the total soil DNA showed that isolates and clones represented only a part of the bands present in the TGGE pattern from total DNA. The 968-1401 fragment amplified from all Arthrobacter strains had a similar electrophoretic mobility. This band was seen as a major band in the pattern of DNA extracted from soil using a harsh cell lysis, whereas it did not appear, or appeared only as a weak band, in patterns obtained from soil DNA extracted using gentle lysis. The previously reported predominance of a Ralstonia eutropha like strain in this soil was no longer observed. This may suggest a population replacement by less resistant bacteria, concomitant with a progressive decrease of the zinc toxicity in the Maatheide soil.

Arthrobacter↗

PubMed1998

Effect of drying on bioremediation bacteria properties.

Bioremediation bacteria with drought-resistance characteristics were selected and compared to a collection of 10 strains selected only for their bioremediation properties. Twenty-six strains were selected from dried diesel-polluted soil, and they exhibit a better level of survival during drying, compared to collection bioremediation strains (two orders of magnitude difference). The lyophilization process does not affect the strains' ability to grow on xenobiotic compound when measured immediately after drying. However, collection bioremediation strains selected only for their bioremediation properties lose up to 80% of their properties when stored at 25 degrees C for 15 d, but the strains selected for their drought resistance lose their properties to a lesser extent during the same period. The maximal growth rate and the rate of xenobiotic degradation of the still-active cells are not affected by the drying process.

Bacteria↗

PubMed1998

Effect of the siderophore alcaligin E on the bioavailability of Cd to Alcaligenes eutrophus CH34.

Alcaligin E, the siderophore of the heavy metal-resistant A. eutrophus strain CH34, was shown to interact with Cd and consequently affect its bioavailability and toxicity. The addition of alcaligin E markedly stimulated the growth in the presence of Cd of an alcaligin E-deficient CH34 derivative. Using bioluminescence assays, this effect could be assigned to a decrease in bioavailability of Cd in the presence of alcaligin E. However, Cd-uptake studies showed no influence of alcaligin E on the cellular concentration of Cd. Furthermore, by scanning electron microscopy, the morphology of precipitated Cd crystals was shown to be altered by alcaligin E. These data suggest that alcaligin E, besides its function in iron supply to the cell, provides a protection against heavy metal toxicity. A link between the A. eutrophus CH34 siderophore system and the czc-mediated Cd-efflux system is hypothesized.

Alcaligenes↗

PubMed1997

Organisation of the bph gene cluster of transposon Tn4371, encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds.

Tn4371 is a 55 kb transposon which encodes enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds into benzoate and 4-chlorobenzoate derivatives. We constructed a cosmid library of Tn4371 DNA. The bph genes involved in biphenyl/4-chlorobiphenyl degradation were found to be clustered in the middle of the transposon. Sequencing revealed an organisation of the bph genes similar to that previously found in Pseudomonas sp. KKS102, i.e. the bphEGF genes are located upstream of bphA1A2A3 and bphA4 is separated from bphA1A2A3 by bphBCD. Consensus sequences for sigma54-associated RNA polymerase were found upstream of bphA1 and bphEGF. Plasmid RP4::Tn4371 was transferred into a mutant of Alcaligenes eutrophus H16 lacking sigma54. In contrast to wild-type H16 exconjugants, the sigma54 mutant exconjugants could not grow on biphenyl, indicating the dependence of Tn4371 bph gene expression on sigma54. The Tn4371-encoded bph pathway was activated when biphenyl and various biphenyl-like compounds were present in the growth medium. Preliminary observations indicate the presence of a region outside the catabolic genes downstream of bphA4 which is involved in mediating at least the basal expression of BphC.

Alcaligenes↗

PubMed1997

Genetic and physical maps of the Alcaligenes eutrophus CH34 megaplasmid pMOL28 and its derivative pMOL50 obtained after temperature-induced mutagenesis and mortality.

We describe the construction of restriction and genetic maps of plasmid pMOL28, which has a size of approximately 180 kb. To do so, partial BamHI-digested DNA of pMOL28 was cloned into cosmid pLAFR3, which can package up to 20-30 kb inserted DNA. Subsequently, a cosmid walking strategy, combined with BamHI or EcoRI restriction analysis and hybridization, was used to construct the restriction maps for both enzymes. On these maps, 35 BamHI fragments and 29 EcoRI fragments were placed, accounting for a total size of approximately 180 kb. We also analyzed several rearranged derivatives of pMOL28 that were obtained after a process of temperature-induced mortality and mutagenesis (TIMM), which is characteristic for Alcaligenes eutrophus CH34 and related strains. The restriction and genetic maps of pMOL50 (222 kb), an enlarged derivative of pMOL28 obtained after TIMM, were constructed. By comparing the pMOL28 and pMOL50 maps, at least two transposable elements were identified which participated in the formation of pMOL50 from pMOL28 during TIMM. These transposable elements were IS1086, which was recently sequenced, and a new element named IS1089, which is located on the 44-kb inserted DNA fragment in pMOL50. Partial sequencing of IS1089 revealed similarity of this element with IS1071 of the chlorobenzoate catabolic transposon Tn5271 of Alcaligenes sp. BR60.

Alcaligenes↗