Maclurin inhibits caspase-11 non-canonical inflammasome in macrophages and ameliorates acute lethal sepsis in mice.

Maclurin is a natural phenolic compound isolated from Morus alba(white mulberry) andGarcinia mangostana (purple mangosteen) and has been reported to regulate cancer progression, oxidative stress, and melanogenesis. The regulatory role of maclurin, however, has never been demonstrated. This study investigated in vitro and in vivo anti-inflammatory roles of maclurin and the underlying mechanism in caspase-11 non-canonical inflammasome-stimulated inflammatory responses in macrophages and an animal model of acute lethal sepsis. Maclurin protected J774A.1 macrophages from LPS-induced cytotoxicity and suppressed caspase-11 non-canonical inflammasome-stimulated pyroptosis. Maclurin decreased the secretion and mRNA expression of pro-inflammatory cytokines and inflammatory mediators, such as IL-1ß, IL-18, TNF-α, IL-6, nitric oxide (NO), and inducible NO synthase (iNOS) in caspase-11 non-canonical inflammasome-stimulated J774A.1 macrophages. Mechanistic studies revealed that maclurin markedly suppressed the proteolytic activation of caspase-11 and gasdermin D (GSDMD) in caspase-11 non-canonical inflammasome-stimulated J774A.1 macrophages, while it did not inhibit caspase-11-mediated direct sensing of LPS. In vivo study revealed that maclurin ameliorated acute lethal sepsis in mice by increasing the survival rate and decreasing the serum levels of IL-1ß and IL-18 without significant toxicity. In conclusion, this study suggests that maclurin is a novel anti-inflammatory agent in inflammatory responses and against acute lethal sepsis via the inhibition of the caspase-11 non-canonical inflammasome in macrophages, which justifies its potential as an anti-inflammatory therapeutic agent in traditional medicine.

Detection of Sialic Acid to Differentiate Cervical Cancer Cell Lines Using a Sambucus nigra Lectin Biosensor.

Pap smear screening is a widespread technique used to detect premalignant lesions of cervical cancer (CC); however, it lacks sensitivity, leading to identifying biomarkers that improve early diagnosis sensitivity. A characteristic of cancer is the aberrant sialylation that involves the abnormal expression of α2,6 sialic acid, a specific carbohydrate linked to glycoproteins and glycolipids on the cell surface, which has been reported in premalignant CC lesions. This work aimed to develop a method to differentiate CC cell lines and primary fibroblasts using a novel lectin-based biosensor to detect α2,6 sialic acid based on attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and chemometric. The biosensor was developed by conjugating gold nanoparticles (AuNPs) with 5 µg of Sambucus nigra (SNA) lectin as the biorecognition element. Sialic acid detection was associated with the signal amplification in the 1500-1350 cm-1 region observed by the surface-enhanced infrared absorption spectroscopy (SEIRA) effect from ATR-FTIR results. This region was further analyzed for the clustering of samples by applying principal component analysis (PCA) and confidence ellipses at a 95% interval. This work demonstrates the feasibility of employing SNA biosensors to discriminate between tumoral and non-tumoral cells, that have the potential for the early detection of premalignant lesions of CC.

Mannose-specific plant and microbial lectins as antiviral agents: A review.

Lectins are non-immunological carbohydrate-binding proteins classified on the basis of their structure, origin, and sugar specificity. The binding specificity of such proteins with the surface glycan moiety determines their activity and clinical applications. Thus, lectins hold great potential as diagnostic and drug discovery agents and as novel biopharmaceutical products. In recent years, significant advancements have been made in understanding plant and microbial lectins as therapeutic agents against various viral diseases. Among them, mannose-specific lectins have being proven as promising antiviral agents against a variety of viruses, such as HIV, Influenza, Herpes, Ebola, Hepatitis, Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) and most recent Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The binding of mannose-binding lectins (MBLs) from plants and microbes to high-mannose containing N-glycans (which may be simple or complex) of glycoproteins found on the surface of viruses has been found to be highly specific and mainly responsible for their antiviral activity. MBLs target various steps in the viral life cycle, including viral attachment, entry and replication. The present review discusses the brief classification and structure of lectins along with antiviral activity of various mannose-specific lectins from plants and microbial sources and their diagnostic and therapeutic applications against viral diseases.

Proteome Analysis of Serum Purified Using Solanum tuberosum and Lycopersicon esculentum Lectins.

Serum and plasma exhibit a broad dynamic range of protein concentrations, posing challenges for proteome analysis. Various technologies have been developed to reduce this complexity, including high-abundance depletion methods utilizing antibody columns, extracellular vesicle enrichment techniques, and trace protein enrichment using nanobead cocktails. Here, we employed lectins to address this, thereby extending the scope of biomarker discovery in serum or plasma using a novel approach. We enriched serum proteins using 37 different lectins and subjected them to LC-MS/MS analysis with data-independent acquisition. Solanum tuberosum lectin (STL) and Lycopersicon esculentum lectin (LEL) enabled the detection of more serum proteins than the other lectins. STL and LEL bind to N-acetylglucosamine oligomers, emphasizing the significance of capturing these oligomer-binding proteins when analyzing serum trace proteins. Combining STL and LEL proved more effective than using them separately, allowing us to identify over 3000 proteins from serum through single-shot proteome analysis. We applied the STL/LEL trace-protein enrichment method to the sera of systemic lupus erythematosus model mice. This revealed differences in >1300 proteins between the systemic lupus erythematosus model and control mouse sera, underscoring the utility of this method for biomarker discovery.

Positive effect of phasin in biohydrogen production of non polyhydroxybutyrate-producing Clostridium acetobutylicum ATCC 824.

In this study, the goal was to enhance the tolerance of Clostridium acetobutylicum ATCC 824 to biomass-based inhibitory compounds for biohydrogen production and evaluate various known genes that enhance the production of biochemicals in various hosts. The introduction of phaP, the major polyhydroxyalkanoate granule-associated protein that has been reported as a chaperone-like protein resulted in increased tolerance to inhibitors and leads to higher levels of hydrogen production, cell growth, and glucose consumption in the presence of these inhibitors. It was observed that the introduction of phaP led to an increase in the transcription of the hydrogenase gene, whereas transcription of the chaperone functional genes decreased compared to the wild type. Finally, the introduction of phaP could significantly enhance biohydrogen production by 2.6-fold from lignocellulosic hydrolysates compared to that of wild type. These findings suggested that the introduction of phaP could enhance growth and biohydrogen production, even in non-polyhydroxyalkanoate-producing strains.

Effect of temperature on structural configuration and immunoreactivity of pH-stressed soybean (Glycine max) agglutinin.

Soybean agglutinin (SBA) was purified using ammonium sulfate precipitation and liquid chromatography. Purified SBA was used to produce monoclonal antibodies through hybridoma technology. SBA secondary structure was studied using circular dichroism. pH-stressed (pHs 3.0, 7.2, 8.5, and 9.6) SBA physical properties (particle size, ζ-potential, and aggregation temperature) were investigated. Gel electrophoresis (non-native and native) was used to study heat-induced structural configuration changes in SBA. The effect of pH and temperature on the immunoreactivity of SBA was analyzed using enzyme-linked immunosorbent assay and immunoblots probed with two anti-SBA monoclonal antibodies with either linear or conformational epitopes. The hemagglutinating activity of heated SBA was measured by hemagglutination assay. Our results indicated that SBA had the least thermostability at pH 3.0 and the highest at pH 8.5. Temperature-induced structural configuration change on pH-stressed SBA led to immunoreactivity change. Heat-induced (70 and 80 °C) soluble SBA aggregation was proportionally related to hemagglutinating activity reduction.

Effect of temperature and pH on the structure and stability of tumor-specific lectin jacalin and insights into the location of its tryptophan residues: CD, DSC and fluorescence studies.

Jacalin, the jackfruit seed lectin, exhibits high specificity for the tumor-specific T-antigen and is used in various biomedical and biotechnological applications. Here, we report biophysical studies on the thermal unfolding of jacalin and the effect of pH and temperature on its secondary structure. Differential scanning calorimetric (DSC) studies revealed that native jacalin unfolds at ∼60 °C and that carbohydrate binding stabilizes the protein structure. Circular dichroism spectroscopic studies indicated that the secondary structure of jacalin remains mostly unaffected over pH 2.0-9.0, whereas considerable changes were observed in the tertiary structure. DSC experiments demonstrated that jacalin exhibits two overlapping transitions between pH 2 and 5, which could be attributed to dissociation of the tetrameric protein into subunits and their unfolding. Interestingly, only one transition between pH 6 and 9 was observed, suggesting that the subunit dissociation and unfolding occur simultaneously. While quenching of the protein intrinsic fluorescence by acrylamide increased significantly upon carbohydrate binding, quenching by succinimide is essentially unaffected. We attribute this difference to increased exposure of Trp-123 in the α-chain as it is involved in carbohydrate binding. Both acrylamide and succinimide gave biphasic Stern-Volmer plots, consistent with differential accessibility of the two tryptophan residues of jacalin to them.

Characterization of a Molecularly Engineered Banlec-Type Lectin (rBTL).

Lectins are proteins that reversibly bind to carbohydrates and are commonly found across many species. The Banana Lectin (BanLec) is a member of the Jacalin-related Lectins, heavily studied for its immunomodulatory, antiproliferative, and antiviral activity. In this study, a novel sequence was generated in silico considering the native BanLec amino acid sequence and 9 other lectins belonging to JRL. Based on multiple alignment of these proteins, 11 amino acids of the BanLec sequence were modified because of their potential for interference in active binding site properties resulting in a new lectin named recombinant BanLec-type Lectin (rBTL). rBTL was expressed in E. coli and was able to keep biological activity in hemagglutination assay (rat erythrocytes), maintaining similar structure with the native lectin. Antiproliferative activity was demonstrated on human melanoma lineage (A375), evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). rBTL was able to inhibit cellular growth in a concentration-dependent manner, in an 8-h incubation, 12 µg/mL of rBTL led to a 28.94% of cell survival compared to cell control with 100%. Through a nonlinear fit out log-concentration versus biological response, an IC50% of 3.649 µg/mL of rBTL was determined. In conclusion, it is possible to state that the changes made to the rBTL sequence maintained the structure of the carbohydrate-binding site without changing specificity. The new lectin is biologically active, with an improved carbohydrate recognition spectrum compared to nBanLec, and can also be considered cytotoxic for A375 cells.

A Comprehensive Review on Euphorbiaceae lectins: Structural and Biological Perspectives.

Euphorbiaceae, also known as the spurge family, is a large group of flowering plants. Despite being tropical natives, they are now widespread. Due to its medicinal and commercial importance, this family of plants attracted a lot of attention in the scientific community. The distinctive characteristic of the family is production of milky latex, which is a rich source of several lectins, the proteins that bind carbohydrates. Although their function is unclear, they are believed to defend plants against damaging phytopathogenic microorganisms, insects, and predatory animals. Additionally, they serve as crucial metabolic regulators under a variety of stressors. Detection, separation, purification, and characterization of lectins from the Euphorbiaceae family - mostly from the latex of plants - began over 40 years ago. This effort produced over 35 original research papers that were published. However, no systematic review that compiles these published data has been presented yet. This review summarizes and describes several procedures and protocols employed for extraction and purification of lectins belonging to this family. Physicochemical properties and biological activities of the lectins, along with their medicinal and pharmacological properties, have also been analyzed. Additionally, using examples of ricin and ricin agglutinin, we have structurally analyzed characteristics of the lectin known as Ribosome Inactivating Protein Type II (RIP-Type II) that belongs to this family. We anticipate that this review article will offer a useful compendium of information on this important family of lectins, show the scientists involved in lectin research the gaps in our knowledge, and offer insights for future research.

Fucose-binding lectins: purification, characterization and potential biomedical applications.

The property of lectins to specifically recognize and bind carbohydrates makes them an excellent candidate in biomedical research. Among them are fucose-binding lectins possessing the capacity to bind fucose are taxonomically, evolutionarily and ecologically significant class of lectins that are identified in a wide range of taxa. Purification of fucose-binding lectins dates back to 1967 when L-fucose binding protein from Lotus tetragonolobus was isolated using a dye that contained three α-L-fucopyranosyl residues. Beginning with that, several FBLs were purified from various animals as well as plant sources that were structurally and functionally characterised. This review focuses on fucose-binding lectins, their occurrence and purification with special emphasis on various strategies adopted to purify them followed by molecular and functional characterization. The exclusive ability to recognize and bind to fucose-containing glycans endows these lectins with the potential to act as anti-cancer agents, diagnostic markers and mitogens for immune cells. Though they have been in research focus for more than half a century with their occurrence reported in various taxa, they still need to be explored for their prospective functions to develop them as a biological tool in biomedical research.

Serum wisteria floribunda agglutinin-positive human Mac-2 binding protein is unsuitable as a diagnostic marker of occult hepatocellular carcinoma in end-stage liver cirrhosis.

BACKGROUND AND PURPOSE: Serum glycosylated Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+-M2BP) is a marker of liver fibrosis and hepatocellular carcinoma (HCC). In this study, we aimed to evaluate the diagnostic ability of WFA+-M2BP for occult HCC, which current diagnostic imaging tests fail to detect. METHODS: Patients who underwent hepatectomy for liver transplantation (LT) and whose whole liver could be sliced and subjected to histological examination between 2010 and 2018 were eligible for this study (n = 89). WFA+-M2BP levels were measured in samples collected before the LT. Comparison of the postoperative histological test results with the preoperative imaging data grouped the patients into histologically no group (N), histologically detected group (D), histologically increased group (I), and histologically decreased or same group (DS), and the results were compared with the WFA+-M2BP values. In addition, comparisons were made between each data with and without HCC, including occult HCC, and total tumor diameter. RESULTS: Irrespective of underlying hepatic disease conditions, there were 6 patients in the N group, 10 in the D group, 41 in the I group, and 32 in the DS group. The median of the serum WFA+-M2BP level for each group was as follows: N group, 8.05 (1.25-11.9); D group, 11.025 (1.01-18.21); I group, 9.67 (0.29-17.83); and DS group, 9.56 (0.28-19.44) confidence of interval. We found no significant differences between the pairings. Comparison of underlying hepatic diseases revealed that liver cirrhosis due to hepatitis B and C and non-B and -C liver cirrhosis had no significant differences. AFP levels, on the other hand, had significant relationships in comparison between the presence or absence of histological HCC, in correlation between total tumor diameter, and in the ROC analysis for the diagnosis of HCC including occult HCC. CONCLUSION: Serum WFA+-M2BP cannot help diagnose occult HCC that is already undetected using imaging tests in decompensated liver cirrhosis patients requiring LT.

Structural Analysis and Characterization of an Antiproliferative Lectin from Canavalia villosa Seeds.

Cells use glycans to encode information that modulates processes ranging from cell-cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin's specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways.

Genome-wide analysis of lectins in cyanobacteria: from evolutionary mode to motif patterns.

Lectins are glycoproteins that can bind to specific carbohydrates, and different lectin families exhibit different biological activities. They are also present in the cyanobacteria and many of them have shown excellent therapeutic effect, which deserve for bioprospecting. However, in comparison to those from terrestrial plants, the current knowledge on cyanobacterial lectins is very limited. To this end, genome-wide analyses were performed to find out their evolutionary mode and motif patterns in 316 genomes of representative taxa. In results, 196 putative cyanobacterial lectins were dig out and 105 of them were classified into known families. Seven lectins were found to be belonged to distinct two lectin families, and they may have the potential activities of both lectin families. Whereas no MFP-2, Chitin, and Nictaba family lectins were found. What's more, the Legume lectin-like lectin family was found to be the richest and most complex in cyanobacteria, which could be a main research direction for future cyanobacterial lectin bioprospecting and development. Our classification and prediction of cyanobacteria lectins is expected to provide assistance in the development of lectin-based medicine and provide solutions to the current thorny viral and tumor diseases in humans.

The Role of Plant Lectins in the Cellular and Molecular Processes of Skin Wound Repair: An Overview.

There is increasing pressure for innovative methods to treat compromised and difficult-to-heal wounds. Consequently, new strategies are needed for faster healing, reducing infection, hydrating the wound, stimulating healing mechanisms, accelerating wound closure, and reducing scar formation. In this scenario, lectins present as good candidates for healing agents. Lectins are a structurally heterogeneous group of glycosylated or non-glycosylated proteins of non-immune origin, which can recognize at least one specific monosaccharide or oligosaccharide specific for the reversible binding site. Cell surfaces are rich in glycoproteins (glycosidic receptors) that potentially interact with lectins through the number of carbohydrates reached. This lectin-cell interaction is the molecular basis for triggering various changes in biological organisms, including healing mechanisms. In this context, this review aimed to (i) provide a comprehensive overview of relevant research on the potential of vegetable lectins for wound healing and tissue regeneration processes and (ii) discuss future perspectives.

Jackfruit waste: an invented anticancer therapy using Jacalin lectin from jackfruit seed.

Every food source contains both edible and inedible waste components. Millions of tonnes of trash from the food business are made from fruits, and these wastes are containing higher-value medicinal components, such as alkaloids, flavonoids, phenolic contents, a huge amount of proteins and secondary metabolites. These bioactive phytoconstituents are being used for the treatment of many serious fatal diseases. So, utilizing the recovered bioactive molecules from food wastes as functional ingredients offers a long-term alternative source of therapeutically active components that will lead to the discovery of novel phytoconstituents or novel treatment approaches. The goal of this systematic study is to provide an overview of the jackfruit (Artocarpus heterophyllus Lam, Moraceae) edible byproducts, such as jackfruit seeds that are largely neglected. This seed contains numerous bioactive lead molecules, such as carbohydrate-binding protein jacalin, which exhibits potent anticancer activity against colon cancer, blood cancer and breast cancer as well as can enlighten the new possible treatment approaches in targeted therapy and photodynamic chemotherapy. Moreover, jackfruit waste seed can be taken as a dietary food, which is having property to prevent and treat cancer and other lifestyle diseases. The works that have been carried out to utilize jackfruit waste other than the juicy edible bulbs have been reviewed in this article.

A systematic analysis of the phloem protein 2 (PP2) proteins in Gossypium hirsutum reveals that GhPP2-33 regulates salt tolerance.

BACKGROUND: Phloem protein 2 (PP2) proteins play a vital role in the Phloem-based defense (PBD) and participate in many abiotic and biotic stress. However, research on PP2 proteins in cotton is still lacking. RESULTS: A total of 25, 23, 43, and 47 PP2 genes were comprehensively identified and characterized in G.arboretum, G.raimondii, G.barbadense, and G.hirsutum. The whole genome duplication (WGD) and allopolyploidization events play essential roles in the expansion of PP2 genes. The promoter regions of GhPP2 genes contain many cis-acting elements related to abiotic stress and the weighted gene co-expression network analysis (WGCNA) analysis displayed that GhPP2s could be related to salt stress. The qRT-PCR assays further confirmed that GhPP2-33 could be dramatically upregulated during the salt treatment. And the virus-induced gene silencing (VIGS) experiment proved that the silencing of GhPP2-33 could decrease salt tolerance. CONCLUSIONS: The results in this study not only offer new perspectives for understanding the evolution of PP2 genes in cotton but also further explore their function under salt stress.

Selective targeting of lectins and their macropinocytosis in urothelial tumours: translation from in vitro to ex vivo.

Urinary bladder cancer can be treated by intravesical application of therapeutic agents, but the specific targeting of cancer urothelial cells and the endocytotic pathways of the agents are not known. During carcinogenesis, the superficial urothelial cells exhibit changes in sugar residues on the apical plasma membranes. This can be exploited for selective targeting from the luminal side of the bladder. Here we show that the plant lectins Jacalin (from Artocarpus integrifolia), ACA (from Amaranthus caudatus) and DSA (from Datura stramonium) selectively bind to the apical plasma membrane of low- (RT4) and high-grade (T24) cancer urothelial cells in vitro and urothelial tumours ex vivo. The amount of lectin binding was significantly different between RT4 and T24 cells. Endocytosis of lectins was observed only in cancer urothelial cells and not in normal urothelial cells. Transmission electron microscopy analysis showed macropinosomes, endosome-like vesicles and multivesicular bodies filled with lectins in RT4 and T24 cells and also in cells of urothelial tumours ex vivo. Endocytosis of Jacalin and ACA in cancer cells was decreased in vitro after addition of inhibitor of macropinocytosis 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and increased after stimulation of macropinocytosis with epidermal growth factor (EGF). Clathrin, caveolin and flotillin did not colocalise with lectins. These results confirm that the predominant mechanism of lectin endocytosis in cancer urothelial cells is macropinocytosis. Therefore, we propose that lectins in combination with conjugated therapeutic agents are promising tools for improved intravesical therapy by targeting cancer cells.

DVL, lectin from Dioclea violacea seeds, has multiples mechanisms of action against Candida spp via carbohydrate recognition domain.

Lectins are proteins of non-immunological origin with the ability to bind to carbohydrates reversibly. They emerge as an alternative to conventional antifungals, given the ability to interact with carbohydrates in the fungal cell wall inhibiting fungal growth. The lectin from D. violacea (DVL) already has its activity described as anti-candida in some species. Here, we observed the anti-candida effect of DVL on C. albicans, C. krusei and C. parapsilosis and its multiple mechanisms of action toward the yeasts. Additionally, it was observed that DVL induces membrane and cell wall damage and ROS overproduction. DVL was also able to cause an imbalance in the redox system of the cells, interact with ergosterol, inhibit ergosterol biosynthesis, and induce cytochrome c release from the mitochondrial membrane. These results endorse the potential application of DVL in developing a new antifungal drug to fight back against fungal resistance.

Recognition factors of Dolichos biflorus agglutinin (DBA) and their accommodation sites.

Dolichos biflorus agglutinin (DBA) is one of the well known plant lectins that are widely used in clinical serology to differentiate human blood group A1 and A2 erythrocytes and also applied to glycobiology. However, the knowledge of recognition factors of polyvalent (super) glycotopes in glycans and the roles of functional group and epimer in monosaccharide (sub-monosaccharide recognition factor) have not been well established. The size and shape of the recognition (combining) site of DBA has not been clearly defined. In this study, many importnat recognition factors of DBA-glycan binding were characterized by our established enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results of these assays showed that the intensity profile of the recognition factors for the major combining site of DBA was expressed by Mass relative potency (Mass R.P.) and shown by decreasing order of high density of polyvalent GalNAcα1 → (super glycotopes, 3.7 × 103) >> the corresponding ß anomers >> monomeric GalNAcα1 → related glycotopes (GalNAc as 1.0) >> their GalNAc ß-anomers >> Gal (absence of NHCH3CO at carbon-2 of GAlNAc) and GlcNAc (different epimer of Carbon-4 in GalNAc). From the all data available, it is proposed that the combining site of DBA should consist of a small cavity shape as major site and most complementary to monomeric GalNAcα → located at both terminal reducing end (Tn) and nonreducing end of glycan chains, and with a wide and broad area as subsite to accomodate from mono- to tetra-saccharides (GalNAcß, Galß1 → 3/4GlcNAc, lFuc1 → 2Galß1 → 3/4GlcNAc, GalNAcß1 → 3Galα1 → 4Galß1 → 4Glc) at the nonreducing side. In this study, it has provided the most (comprehensive) recognition knowledge of DBA-glycan interactions at the factors of glycotope, super glycotope/sub-monosaccharide levels. Thus, it should expand and upgrade the conventional concept of the combining (recognition) site of DBA since 1980s.