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Lotus japonicus: a new model to study root-parasitic nematodes.

Sedentary plant-parasitic nematodes engage in complex interactions, and induce specialized feeding structures by redirecting plant developmental pathways, and parallels have been observed with rhizobial nodule development on legumes. A model legume would greatly facilitate a better understanding of the differences between parasitic (nematode) and mutualistic (rhizobia and mycorrhizae) symbioses, and we have developed Lotus japonicus as such a model. Conditions for efficient parasitism by root-knot nematode (Meloidogyne spp.) of the widely used Lotus "Gifu" ecotype were established. Features of Lotus biology, such as thin and translucent roots, proved ideal for monitoring the progress of nematode infection both on live specimens and post-staining. We examined L. japonicus mutants with nodulation phenotypes. One, har1, which is a hypernodulated mutant defective in a CLAVATA1-like receptor kinase gene, was found to be hyperinfected by M. incognita. However, another hypernodulated Lotus mutant exhibited the same level of M. incognita infection as wild-type plants. We also established conditions for infection of Lotus by soybean cyst nematode (Heterodera glycines). In contrast to the response to root-knot nematode, the Gifu ecotype is resistant to H. glycines, and elicits a hypersensitive response. This pattern of resistance recapitulates that seen on nematode-resistant soybean plants. We conclude that L. japonicus is a powerful model legume for studying compatible and incompatible plant-nematode interactions.

Animals↗

The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep. 1. Voluntary intake.

Voluntary intake was determined with vegetative Lotus pedunculatus cut and fed fresh to growing sheep of 42-46 kg live weight. Effects attributable to condensed tannins were assessed by growing the plant under high and low levels of soil fertility, inducing low and high concentrations of tannin (Expt 1), or by binding the tannins through spraying the herbage with polyethylene glycol (molecular weight 3350, PEG; Expt 2). Primary-growth lotus was used in Expt 1 and secondary-growth lotus in Expt 2. Concentrations of total and free condensed tannin were determined in fresh lotus, free tanning being defined as that not bound by mascerates of the plant. In Expt 1 the herbages fed contained respectively 46 and 106 g total condensed tannin/kg dry matter (DM) and 3 and 14 g free condensed tannin/kg DM. Mean metabolizable energy (ME) intakes were 0.89 and 0.77 MJ/kg live weight0 .75 per d (P less than 0.05) respectively. The lotus used in Expt 2 contained 63 and 5 g total reactive condensed tannin and free condensed tannin/kg DM respectively. After spraying with PEG at 2.4 g/g total condensed tannin, these values were reduced to 7 and 0.5 g/kg DM respectively. PEG addition increased apparent digestibility (proportion of each nutrient ingested) of cellulose, hemicellulose and nitrogen by 0.05, 0.08 and 0.26, and increased ME intake from 0.48 to 0.69 MJ/kg live weight0 .75 per d. It was concluded that high concentrations of condensed tannins depressed ME intake, due to depressions in both the voluntary intake and digestion of organic matter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗

Tribal delimitation and phylogenetic relationships of Loteae and Coronilleae (Faboideae: Fabaceae) with special reference to Lotus: evidence from nuclear ribosomal ITS sequences.

The temperate herbaceous tribes Loteae and Coronilleae have traditionally been regarded as taxonomically distinct entities. More recent morphological assessments, however, have challenged this view and suggest combining the two tribes under Loteae. Two key features used to distinguish the Coronilleae from Loteae include jointed fruits and branched root nodules. We evaluate the taxonomic utility of these characters using information derived from phylogenetic analyses of the internal transcribed spacers ITS1 + 2, and the intervening 5.8S region of nuclear ribosomal DNA. Results from this study show that neither the Loteae nor Coronilleae form individual monophyletic groups, and that key fruit and root nodule characters used to distinguish the Coronilleae are homoplastic. Given these data, we support the recognition of a single tribe, Loteae. We also find that Lotus, the largest and most morphologically complex genus in either tribe, is not monophyletic. Rather, it consists of two geographically distinct lineages, Old and New World, each of which are more closely related to other Loteae genera: Old World Lotus are more closely related to Old World Anthyllis, while New World Lotus show closer affinities to Old World Coronilla. These data also have important implications for the biogeography of New World Lotus: equally most parsimonious reconstructions suggest a complex scenario of intercontinental dispersals that involve not only Old World Lotus but Coronilla as well.

Journal Article↗

Aflatoxins and their biosynthetic precursors in lotus seeds: simultaneous UPLC-MS/MS determination, contamination profiling, and matrix-specific accumulation during Aspergillus flavus infection.

Aflatoxin (AF) contamination poses a severe global threat to food and medicinal material safety, yet existing research focuses on terminal AF metabolites while neglecting residual biosynthetic precursors, leading to potential underestimation of contamination risks. In this study, a UPLC-MS/MS method was established for the simultaneous quantification of six AFs and their five precursors in lotus seeds, with optimization of mass spectrum parameters, chromatographic separation conditions, and sample pretreatment. Method validation confirmed linearity (R2&#xa0;>&#xa0;0.99), LODs (0.03-0.36&#xa0;&#x3bc;g/kg), and recoveries (76.53%-120.0%, RSD&#xa0;<&#xa0;15%). Analysis of 41 natural lotus seed samples revealed a 63.4% AF contamination rate, dominated by B-group AFs, while O-methylsterigmatocystin (OMST) and versicolorin hemiacetal (VOH) were identified as the primary co-residual precursors with co-occurrence rates &#x2265; 50%. Notably, AFM1 was predominantly detected in natural samples with AFB1 concentrations exceeding 100&#xa0;&#x3bc;g/kg. Artificial inoculation experiments further demonstrated that sterilization and sealing conditions modulated AF biosynthesis in lotus seeds, with non-sterilized and non-sealed groups showing delayed fungal metabolism and lower toxin accumulation. A significant linear correlation was observed between AFM1 and AFB1 levels (r&#xa0;=&#xa0;0.94) in infected samples, demonstrating their accumulation levels are coupled with fungal overall metabolic flux. Given the high co-occurrence rate of OMST/VOH with AFB1 in natural samples, their individual and combined toxicities require in-depth investigation. This work deciphers matrix-specific AF dynamics in lotus seeds, supporting regulatory standard refinement (e.g., precursor inclusion) and targeted control (e.g., time-sensitive drying after harvest). Further studies will focus on exploring the molecular mechanisms of substrate-dependent AF synthesis.

Aflatoxins↗

Exploitation of colinear relationships between the genomes of Lotus japonicus, Pisum sativum and Arabidopsis thaliana, for positional cloning of a legume symbiosis gene.

The Lotus japonicus LjSYM2 gene, and the Pisum sativum orthologue PsSYM19, are required for the formation of nitrogen-fixing root nodules and arbuscular mycorrhiza. Here we describe the map-based cloning procedure leading to the isolation of both genes. Marker information from a classical AFLP marker-screen in Lotus was integrated with a comparative genomics approach, utilizing Arabidopsis genome sequence information and the pea genetic map. A network of gene-based markers linked in all three species was identified, suggesting local colinearity in the region around LjSYM2/PsSYM19. The closest AFLP marker was located just over 200 kb from the LjSYM2 gene, the marker SHMT, which was converted from a marker on the pea map, was only 7.9 kb away. The LjSYM2/PsSYM19 region corresponds to two duplicated segments of the Arabidopsis chromosomes AtII and AtIV. Lotus homologues of Arabidopsis genes within these segments were mapped to three clusters on LjI, LjII and LjVI, suggesting that during evolution the genomic segment surrounding LjSYM2 has been subjected to duplication events. However, one marker, AUX-1, was identified based on colinearity between Lotus and Arabidopsis that mapped in physical proximity of the LjSym2 gene.

Arabidopsis↗

Mesorhizobium bavaricum sp. nov. and Mesorhizobium monacense sp. nov., two novel Lotus-associated species harbouring symbiotic plasmids.

Legumes establish a mutualistic interaction with nitrogen-fixing rhizobia. Lotus japonicus is a model for studying this symbiosis; however, only a limited number of rhizobial species nodulating this host have been taxonomically described. Here, we characterise four Mesorhizobium strains (DC-1.1T, Qj1B1, DC-1.5T, and Qj2B2) isolated from root nodules of Lotus japonicus and Lotus burttii. Multi-locus phylogeny and phylogenomic analyses resolved these isolates into two well-supported monophyletic clades. Genome-based comparisons supported their classification as distinct taxa, with strains DC-1.1T and Qj1B1 showing 95.2% average nucleotide identity (ANI) and 62.9-63.5% digital DNA-DNA hybridisation (dDDH) values relative to Mesorhizobium newzealandense ICMP 19545T, whereas DC-1.5T and Qj2B2 exhibited 92.5-92.8% ANI and 49.9-50.5% dDDH compared with Mesorhizobium waimense ICMP 19557T. Together with chemotaxonomic and physiological traits, these data support the proposal of two novel species, Mesorhizobium bavaricum sp. nov. (DC-1.1T and Qj1B1) and Mesorhizobium monacense sp. nov. (DC-1.5T and Qj2B2). Metagenomic analyses predicted high environmental prevalence for these novel taxa, particularly within soil habitats. Isolates DC-1.1T, Qj1B1, and DC-1.5T effectively nodulated Lotus burttii and significantly promoted plant growth, whereas Qj2B2 neither nodulated nor enhanced growth. Comparative genomic analysis revealed that the nodulating isolates harbour symbiotic genes (nod, fix, and nif) on symbiotic plasmids, a rare feature in Mesorhizobium strains, whereas Qj2B2 lacks essential nod and nif genes. Consistent with these genomic features, symbiotaxonomic analysis assigned the nodulating isolates to symbiovar loti. These results highlight the potential of these isolates as models for comparative analyses of symbiotic plasmid evolution and horizontal gene transfer.

Mesorhizobium↗

Legume genome evolution viewed through the Medicago truncatula and Lotus japonicus genomes.

Genome sequencing of the model legumes, Medicago truncatula and Lotus japonicus, provides an opportunity for large-scale sequence-based comparison of two genomes in the same plant family. Here we report synteny comparisons between these species, including details about chromosome relationships, large-scale synteny blocks, microsynteny within blocks, and genome regions lacking clear correspondence. The Lotus and Medicago genomes share a minimum of 10 large-scale synteny blocks, each with substantial collinearity and frequently extending the length of whole chromosome arms. The proportion of genes syntenic and collinear within each synteny block is relatively homogeneous. Medicago-Lotus comparisons also indicate similar and largely homogeneous gene densities, although gene-containing regions in Mt occupy 20-30% more space than Lj counterparts, primarily because of larger numbers of Mt retrotransposons. Because the interpretation of genome comparisons is complicated by large-scale genome duplications, we describe synteny, synonymous substitutions and phylogenetic analyses to identify and date a probable whole-genome duplication event. There is no direct evidence for any recent large-scale genome duplication in either Medicago or Lotus but instead a duplication predating speciation. Phylogenetic comparisons place this duplication within the Rosid I clade, clearly after the split between legumes and Salicaceae (poplar).

Chromosomes, Plant↗

Genome-wide analysis of ATP-binding cassette (ABC) proteins in a model legume plant, Lotus japonicus: comparison with Arabidopsis ABC protein family.

ATP-binding cassette (ABC) proteins constitute a large family in plants with more than 120 members each in Arabidopsis and rice, and have various functions including the transport of auxin and alkaloid, as well as the regulation of stomata movement. In this report, we carried out genome-wide analysis of ABC protein genes in a model legume plant, Lotus japonicus. For analysis of the Lotus genome sequence, we devised a new method 'domain-based clustering analysis', where domain structures like the nucleotide-binding domain (NBD) and transmembrane domain (TMD), instead of full-length amino acid sequences, are used to compare phylogenetically each other. This method enabled us to characterize fragments of ABC proteins, which frequently appear in a draft sequence of the Lotus genome. We identified 91 putative ABC proteins in L. japonicus, i.e. 43 'full-size', 40 'half-size' and 18 'soluble' putative ABC proteins. The characteristic feature of the composition is that Lotus has extraordinarily many paralogs similar to AtMRP14 and AtPDR12, which are at least six and five members, respectively. Expression analysis of the latter genes performed with real-time quantitative reverse transcription-PCR revealed their putative involvement in the nodulation process.

ATP-Binding Cassette Transporters↗

LORE1, an active low-copy-number TY3-gypsy retrotransposon family in the model legume Lotus japonicus.

We have identified a low-copy-number retrotransposon family present in nine to 10 copies in the Lotus japonicus model legume genome, and characterized its activity. LORE1 (Lotus retrotransposon 1) belongs to the Ty3-gypsy group of elements, and is a long terminal repeat (LTR) retrotransposon. Genetic mapping located LORE1 elements in gene-rich regions of Lotus chromosomes, and analysis of native as well as new insertion sites revealed integration outside the highly repetitive sequences of centromeres and telomeres. Sequencing of individual LORE1 family members identified several intact elements, and analysis of new insertions showed that at least one member is active and reinserts into functional genes, creating gene-disruption mutations. Southern blot analysis and SSAP on a selection of symbiotic mutants revealed up to 12 new insertion sites in individual mutant lines and a Mendelian segregation of new inserts. Expression analysis showed that LORE1 elements are transcribed in all organs analysed and, in contrast to other active retrotransposons, LORE1 appears not to be transcriptionally upregulated during in vitro tissue culture. Activity of LORE1 in callus and whole plants suggests that a simple insertion mutagenesis based on endogenous LORE1 elements can be established for Lotus.

Base Sequence↗

Plasma renin assay calculation using Lotus 1-2-3.

The calculation of results from a Plasma Renin Activity (PRA) assay requires many intermediate steps that are both tedious and prone to error. Using Lotus 1-2-3C and the IBM PCT, these assay calculations can be reduced to simply entering the raw counts obtained from the gamma counter. This calculation method is for use with Clinical Assay's Gamma-CoatT PRA kit, one of the most widely used commercial kits. A template that utilizes a logarithmic curve fit equation to calculate a point to point standard curve for the assay is set up on the Lotus spreadsheet. By using a point to point curve fit, a standard curve that is a very close approximation to a manually drawn plot is generated by the computer. Using Lotus's macro feature, a routine is built into the template to automatically move the cursor from cell to cell for the entry of the raw counts. All data can be entered using only the numeric key pad and RETURN key. When all data has been entered, the macro calculates the assay results and prints out a hardcopy report. In 52 samples, comparing the PRA results obtained from the manually plotted standard curve and those calculated by the template yielded a coefficient of correlation of 0.997, p less than 0.001. We conclude that the computer generated results are comparable to results obtained from a manually drawn curve. All calculations can be quickly obtained simply by entering the raw counts from the gamma counter using this application of Lotus 1-2-3C with the Clinical Assay's GammaCoatT PRA kit.

Humans↗

CARDIO--a Lotus 1-2-3 based computer program for rapid calculation of cardiac output from dye or thermal dilution curves.

We have developed a menu-driven computer program (CARDIO), based on a Lotus 1-2-3 template and a series of macrocommands, that rapidly and semiautomatically calculates cardiac output from dye or thermal dilution curves. CARDIO works with any dye or thermal dilution recorder with an analog output, any analog to digital (A-to-D) conversion system, and any computer capable of running Lotus 1-2-3 version 2. No prior experience with Lotus 1-2-3 is needed to operate CARDIO, but experienced users can take full advantage of Lotus 1-2-3's graphics, data manipulation, and data retrieval capabilities.

Cardiac Output↗

Versatile lotus petal flap for vulvoperineal reconstruction after gynecological ablative surgery.

OBJECTIVE: The objective of the present work was to assess the efficacy and complications of the use of the lotus petal flap in the vulvoperineal reconstruction among female patients treated for vulvar malignancies. METHODS: Between December 2000 and April 2003, five patients underwent vulvoperineal reconstructions with the fasciocutaneous skin flaps elevated from gluteal folds immediately after vulvoperineal ablative surgeries at National Cancer Center Hospital, Tokyo, Japan. RESULTS: The mean surface area of vulvoperineal tissue defects was 157.9 cm(2) (64.0-195.0 cm(2)), which could be filled completely by bilateral lotus petal flaps. The mean length of follow-up was 18 months (7-32 months). All flaps successfully survived without fatal necrosis. In postoperative follow-up, all patients had no complaint of pain and no abnormal sensation at the site of flap or at the donor site, and the lotus petal flap caused no severe damage to excretion, mobility of the hip, or the sensation in the vulvoperineal area. The gluteal fold could make the donor-site scar stand out in all patients. CONCLUSION: The lotus petal flap is thought to be one of the most ideal reconstructive procedures for vulvoperineal region from various viewpoints of oncology, function, wound healing, and cosmetic surgery.

Aged↗

The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep. 4. Sites of carbohydrate and protein digestion as influenced by dietary reactive tannin concentration.

1. Vegetative secondary growth Lotus pedunculatus was cut daily, and fed fresh at hourly intervals (600 g dry matter (DM)/d) to three groups each of three sheep fitted with permanent cannulas into the rumen and duodenum. Lotus fed to two of the groups was sprayed with low and high rates of polyethylene glycol (PEG; molecular weight 3350), which specifically binds the condensed tannins (CT). Nutrient intake and faecal excretion were measured directly, duodenal flows estimated from continuous intraruminal infusion of inert ruthenium phenanthroline (Ru-P) and CrEDTA markers, and rumen pool sizes measured at slaughter. 2. Dietary concentrations of total reactive CT (i.e. that not bound to PEG) were 95, 45 and 14 g/kg DM, whilst the corresponding values for free CT were 15, 5 and 2 g/kg DM. 3. Increasing dietary reactive CT concentration linearly increased duodenal flows of non-ammonia nitrogen, but linearly decreased the apparent digestibility of energy and organic matter, and rumen digestion of hemicellulose but not of cellulose. Rumen digestion as a proportion of total digestion was increased by the higher PEG rate for organic matter, energy, pectin and lignin. 4. High dietary CT concentration was associated with increased N retention. Rumen ammonia concentration and pool size showed only a slight decline on this diet, indicating that there must have been increased recycling of N into the rumen. 5. Increasing dietary reactive CT concentration had no effect on the rate at which carbohydrate constituents were degraded in the rumen per unit time (FDR), but increased the rate at which their undegraded residues (FOR) left the rumen per unit time. The latter appeared to be the principal mechanism by which rumen digestion as a proportion of total digestion was reduced at high dietary CT concentrations. From a comparison of FDR and FOR of carbohydrate components in lotus and Brassica oleracea diets, it was concluded that hemicellulose digestion was rate-limiting for rumen cell-wall digestion, probably due to bonding with lignin. However, the considerable post-rumen digestion of hemicellulose was not associated with post-rumen lignin digestion. 6. It was concluded that a desired concentration of CT in Lotus sp. should represent a balance between the positive effect of CT in improving the efficiency of N digestion and their negative effect in depressing rumen carbohydrate digestion. A recommended concentration is 30-40 g/kg DM.

Animals↗

Immunochemical studies on blood groups LXII. Fractionation of hog and human A, H, and AH blood group active substance on insoluble immunoadsorbents of Dolichos and Lotus lectins.

The purified lectins from Lotus tetragonolobus and Dolichos biflorus were coupled to Sepharose 2B to make insoluble adsorbents for purification and fractionation of blood group A and H active glycoproteins. With both adsorbents, hog gastric mucin A + H blood substance (HGM), purified by phenol-ethanol precipitation, yielded fractions showing only A, only H, or AH activities. The AH fraction was obtained when the adsorbent column was overloaded with HGM and its A and H specificities seem to be carried on the same molecules since they were not separable by chromatography on either column. However A and H specificities of blood group substance from the stomach of a presumably heterozygous individual hog were both on the same molecules as they too could not be fractionated on either column. Analytical properties of the isolated fractions were generally similar to those of the unfractionated material, the purfied A substances had a higher galactosamine/fucose ratio than did the H substances. Although the original A + H showed very little specific optical rotation, the separated A and H substances rotated positively and negatively, respectively. The lectin-Sepharose adsorbents have also proven useful in isolating A or H substances directly from the crude commercial hog gastric mucin. Blood group A2 substance from a human ovarian cyst yielded two fractions on the Lotus-Sepharose column; the effluent did not interact with the Lotus lectin but precipitated the Ulex and Dolichos lectins and anti-A, and appears to contain type 1 H determinants. The other fraction reacted with Lotus and Ulex lectin as well as with Dolichos and anti-A.

ABO Blood-Group System↗

Contribution of the alternative pathway to respiration during thermogenesis in flowers of the sacred lotus.

We report results from in vivo measurements, using oxygen isotope discrimination techniques, of fluxes through the alternative and cytochrome respiratory pathways in thermogenic plant tissue, the floral receptacle of the sacred lotus (Nelumbo nucifera). Fluxes through both pathways were measured in thermoregulating flowers undergoing varying degrees of thermogenesis in response to ambient temperature. Significant increases in alternative pathway flux were found in lotus receptacles with temperatures 16 degrees C to 20 degrees C above ambient, but not in those with lesser amounts of heating. Alternative pathway flux in the hottest receptacles was 75% of the total respiratory flux. In contrast, fluxes through the cytochrome pathway did not change significantly during thermogenesis. These data support the hypothesis that increased flux through the alternative pathway is responsible for heating in the lotus and that it is unlikely that uncoupling proteins, which would have produced increased fluxes through the cytochrome pathway, contribute significantly to heating in this tissue. Comparisons of actual flux, with capacity determined using inhibitors, suggested that the alternative pathway was operating at close to maximum capacity in heating tissues of lotus. However, in nonheating tissues the inhibitor data significantly overestimated the alternative pathway flux. This confirms that isotopic measurements are necessary for accurate determination of fluxes through the two pathways.

Cytochromes↗

Refinements in the lotus petal flap repair of the vulvo-perineum.

BACKGROUND: Many reconstructive techniques have been used to repair the vulval, vaginal and perianal areas after excision. These range from grafts to various types of flap repair. The authors have modified a procedure called the lotus petal flap repair to provide a simpler, equally reliable, yet aesthetically enhanced technique for vulvo-perineal reconstruction. METHODS: Where primary closure was not possible the modified lotus petal flap was employed. The present flap design was based on the dense perforators supplying the perineum. The flap resembles the petals of a lotus flower as in the design of Yii and Niranjan. However, the present modification uses a thinned cutaneous flap without identification of the blood supply. The flap is sited over an area of dense perforators. Transposition with this thinner design allows for easy coverage of the defects. RESULTS: In the present series of eight patients and 12 flaps no cases of partial or complete flap failure were recorded. The only complications encountered in the series were one case of cellulitis after discharge from hospital, and a single case of urethral incontinence. CONCLUSIONS: The modified lotus flap repair is a reliable, simple, and aesthetically appealing alternative to those already available to the plastic surgeon for vulval repair.

Aged↗

[Utilization of Lotus 1-2-3 for on-line data logging system from clinical monitors].

We contrived to obtain digital output data from clinical monitors using "Lotus 1-2-3" and its on-line data logging add-in software "1-2-3 DataLogger". Easy macroprogramming of Lotus 1-2-3 enabled us to obtain digital data automatically into the worksheet of Lotus 1-2-3 from the monitors. Data taken up into the worksheet could be analysed readily using graphic function or calculation of Lotus 1-2-3.

Anesthesiology↗

Evolution of NIN-like proteins in Arabidopsis, rice, and Lotus japonicus.

Genetic studies in Lotus japonicus and pea have identified Nin as a core symbiotic gene required for establishing symbiosis between legumes and nitrogen fixing bacteria collectively called Rhizobium. Sequencing of additional Lotus cDNAs combined with analysis of genome sequences from Arabidopsis and rice reveals that Nin homologues in all three species constitute small gene families. In total, the Arabidopsis and rice genomes encode nine and three NIN-like proteins (NLPs), respectively. We present here a bioinformatics analysis and prediction of NLP evolution. On a genome scale we show that in Arabidopsis, this family has evolved through segmental duplication rather than through tandem amplification. Alignment of all predicted NLP protein sequences shows a composition with six conserved modules. In addition, Lotus and pea NLPs contain segments that might characterize NIN proteins of legumes and be of importance for their function in symbiosis. The most conserved region in NLPs, the RWP-RK domain, has secondary structure predictions consistent with DNA binding properties. This motif is shared by several other small proteins in both Arabidopsis and rice. In rice, the RWP-RK domain sequences have diversified significantly more than in Arabidopsis. Database searches reveal that, apart from its presence in Arabidopsis and rice, the motif is also found in the algae Chlamydomonas and in the slime mold Dictyostelium discoideum. Thus, the origin of this putative DNA binding region seems to predate the fungus-plant divide.

Amino Acid Sequence↗