Identification of Pseudozyma graminicola CBS 10092 as a producer of glycolipid biosurfactants, mannosylerythritol lipids

A basidiomycetous yeast, Pseudozyma graminicola CBS 10092, was found to accumulate a large amount of glycolipids in the cultured medium when grown on soybean oil as the sole carbon source. Based on thin layer chromatography, the extracellular glycolipids gave spots corresponding to those of mannosylerythritol lipids (MELs), which are highly functional and promising biosurfactants. From the structural characterization by 1H and 13C NMR, the main product was identified as 4-O-[(4'-mono-O-acetyl-2', 3'-di-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol, which is a highly hydrophilic derivative of MELs known as MEL-C. According to high-performance liquid chromatography analysis, the main product, MEL-C, comprised approximately 85% of all the MELs, and the total amount reached approximately 10 g/L for 7 days. The fatty acids of the present MEL-C consisted of mainly C6, C8 and C14 acids, considerably different from those of MEL-C produced by other Pseudozyma strains such as P. antarctica and P. shanxiensis. The observed critical micelle concentration (CMC) and the surface-tension at CMC of the MEL-C were 4.0 x 10(-6) M and 24.2 mN/m, respectively, while those of MEL-A, the most intensively studied MEL, were 2.7 x 10(-6) M and 28.4 mN/m, respectively. This implied that the MEL-C has higher hydrophilicity than conventional MELs hitherto reported. In addition, on a water-penetration scan, the MEL-C efficiently formed the lamella phase (Lalpha) at a wide range of concentrations, indicating its excellent self-assembling properties. From these results, the newly identified MELs produced by P. graminicola are likely to have great potential for use in oil-in-water type emulsifiers and/or washing detergents, and would thus facilitate a broad range of applications for the promising yeast biosurfactants.     

Characterization of new types of mannosylerythritol lipids as biosurfactants produced from soybean oil by a basidiomycetous yeast, Pseudozyma shanxiensis

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These show not only the excellent surface-active properties but also versatile biochemical actions. In course of MEL production from soybean oil by P. shanxiensis, new extracellular glycolipids (more hydrophilic than the previously reported MELs) were found in the culture medium. As a result of the structural characterization, the glycolipids were identified as a mixture of 4-O-[(2', 4'-di-O-acetyl-3'-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol and 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol. Interestingly, the new MELs possessed a much shorter chain (C(2) or C(4)) at the C-2' position of the mannose moiety compared to the MELs hitherto reported, which mainly possess a medium-chain acid (C(10)) at the position. They would thus show higher hydrophilicity and/or water-solubility, and expand the development of the environmentally advanced yeast biosurfactants.     

Efficient production of di- and tri-acylated mannosylerythritol lipids as glycolipid biosurfactants by Pseudozyma parantarctica JCM 11752(T)

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known, because of their multifunctionality and biocompatibility. In order to attain an efficient production of MELs, Pseudozyma parantarctica JCM 11752(T), which is a newly identified strain of the genus, was examined for the productivity of MELs at different culture conditions. The yeast strain showed significant cell growth and production of di-acylated MELs even at 36 degrees C. In contrast, on conventional high-level MEL producers including P. rugulosa, the MEL yield considerably decreased with an increase of the cultivation temperature at over 30 degrees C. On P. parantarctica, soybean oil and sodium nitrate were the best carbon and nitrogen sources, respectively. Under the optimal conditions on a shake-flask culture at 34 degrees C, the amount of di-acylated MELs reached over 100 g/L by intermittent feeding of only soybean oil. Interestingly, the yeast strain produced tri-acylated MELs as well as di-acylated ones when grown on the medium containing higher soybean oil concentrations than 8% (vol/vol). The production of tri-acylated MELs was significantly accelerated at between 34 and 36 degrees C. With 20 % (vol/vol) of soybean oil at 34 degrees C, the yield of tri-acylated MELs reached 22.7 g/L. The extracellular lipase activity considerably depended on the culture temperature, and became the maximum at 34 degrees C; this would bring the accelerated production of tri-acylated MELs. Accordingly, the present strain of P. parantarctica provided high efficiency in MEL production at elevated temperatures compared to conventional MEL producers, and would thus be highly advantageous for the commercial production of the promising biosurfactants.     

The Uptake of Trehalose Glycolipids by Macrophages Is Independent of Mincle

Trehalose glycolipids play an important role in the pathogenesis of Mycobacterium tuberculosis and are used as adjuvants for vaccines; however, much still remains unanswered about the mechanisms through which these glycolipids exert their immunomodulatory potential. Recently, the macrophage‐inducible C‐type lectin Mincle was determined to be the receptor for trehalose glycolipids, yet the role played by Mincle in glycolipid uptake is unknown. Accordingly, we developed several fluorescent trehalose glycolipid reporter systems that can be used to study the uptake of soluble trehalose glycolipids and glycolipid‐coated particles by macrophages. Our studies revealed that, although Mincle is essential for the activation of macrophages by trehalose glycolipids, the receptor does not play a role in the uptake of these glycolipids or of glycolipid‐coated particles.     

Mannopyranoside Glycolipids Inhibit Mycobacterial and Biofilm Growth and Potentiate Isoniazid Inhibition Activities in M. smegmatis

Lipomannan and lipoarabinomannan are integral components of the mycobacterial cell wall. Earlier studies demonstrated that synthetic arabinan and arabinomannan glycolipids acted as inhibitors of mycobacterial growth, in addition to exhibiting inhibitory activities of mycobacterial biofilm. Herein, it is demonstrated that synthetic mannan glycolipids are better inhibitors of mycobacterial growth, whereas lipoarabinomannan has a higher inhibition efficiency to biofilm. Syntheses of mannan glycolipids with a graded number of mannan moieties and an arabinomannan glycolipid are conducted by chemical methods and subsequent mycobacterial growth and biofilm inhibition studies are conducted on Mycobacterium smegmatis. Growth inhibition of (73±3) % is observed with a mannose trisaccharide containing a glycolipid, whereas this glycolipid did not promote biofilm inhibition activity better than that of arabinomannan glycolipid. The antibiotic supplementation activities of glycolipids on growth and biofilm inhibitions are evaluated. Increases in growth and biofilm inhibitions are observed if the antibiotic is supplemented with glycolipids, which leads to a significant reduction of inhibition concentrations of the antibiotic.     

Loading of the antigen-presenting protein CD1d with synthetic glycolipids

CD1 proteins present mammalian and microbial lipid and glycolipid antigens to different subsets of T cells. Few such antigens have been identified and the binding of these to CD1 molecules has mainly been studied by using responding T cells in cellular assays or recombinant solid-phase CD1 proteins. In the present study we use four different glycolipids, some of which contain tumor-associated carbohydrate antigens, to develop a procedure to easily detect binding of glycolipids to CD1 proteins on viable cells. Two of these glycolipids are novel glycoconjugates containing alpha-D-N-acetylgalactosamine (alpha-GalNAc) that were prepared by a combined solution and solid-phase approach. The key step, a Fischer glycosylation of 9-fluorenylmethoxycarbonylaminoethanol with GalNAc, furnished the alpha-glycoside 4 in 34% yield. Cells were incubated with glycolipids and stained with monoclonal antibodies specific for the carbohydrate part. The level of glycolipid bound to cells was then determined by flow cytometry with a secondary antibody labeled with fluorescein isothiocyanate. All four glycolipids were found to bind to CD1d but with different selectivity. The loading was dose dependent and could be inhibited by an established CD1d ligand, alpha-galactosylceramide. Through use of this procedure, glycolipids were selectively loaded onto CD1d expressed on professional antigen-presenting cells for future use as cellular vaccines. Moreover, the glycolipids described in this study represent novel CD1d-binding ligands that will be useful derivatives in the study of CD1d-dependent immune responses, for example, against tumors.     

Studies on glycolipids of kenaf,english walnut,myrobalan and manila tamarind seeds of the Vidarbha region (India)

Studies on the glycolipid compositions of Kenaf, English walnut, Myrobalan and Manila tamarind seeds found in the Vidarbha region of Central India have been carried out by silicic acid column chromatography and thin-layer chromatography (TLC). The total glycolipids have been separated into individual components such as monogalactosyldiglycerides 20–28%, digalactosyldiglycerides 40–41%, sterylgalactosides 15–18%, acylated sterylgalactosides 8–15% and unidentified components 0.8–2%. The fatty acid composition of total and component glycolipids as determined by gas-liquid chromatography (GLC) showed the predominant fatty acids to be palmitic, stearic and oleic acids. Sugar in the component glycolipids was found to be exclusively galactose.     

Packing density of glycolipid biosurfactant monolayers give a significant effect on their binding affinity toward immunoglobulin G

Mannosylerythritol lipid-A (MEL-A) is one of the most promising glycolipid biosurfactants, and abundantly produced by Pseudozyma yeasts. MEL-A gives not only excellent self-assembling properties but also a high binding affinity toward human immunoglobulin G (HIgG). In this study, three kinds of MEL-A were prepared from methyl myristate [MEL-A (m)], olive oil [MEL-A (o)], and soybean oil [MEL-A (s)], and the effect of interfacial properties of each MEL-A monolayer on the binding affinity toward HIgG was investigated using surface plasmon resonance (SPR) and the measurement of surface pressure (pi)-area (A) isotherms. Based on GC-MS analysis, the main fatty acids were C(8) and C(10) acids in all MEL-A, and the content of unsaturated fatty acids was 0% for MEL-A (m), 9.1% for MEL-A (o), 46.3% for MEL-A (s), respectively. Interestingly, the acid content significantly influenced on their binding affinity, and the monolayer of MEL-A (o) gave a higher binding affinity than that of MEL-A (m) and MEL-A (s). Moreover, the mixed MEL-A (o)/ MEL-A (s) monolayer prepared from 1/1 molar ratio, which comprised of 27.8% of unsaturated fatty acids, indicated the highest binding affinity. At the air/water interface, MEL-A (o) monolayer exhibited a phase transition at 13 degrees C from a liquid condensed monolayer to a liquid expanded monolayer, and the area per molecule significantly expanded above 13 degrees C, while the amount of HIgG bound to the liquid expanded monolayer was much higher than that bound to liquid condensed monolayer. The binding affinity of MEL-A toward HIgG is thus likely to closely relate to the monolayer packing density, and may be partly controlled by temperature.     

Species-specific activity of glycolipid ligands for invariant NKT cells

The immunomodulatory glycolipid α-galactosylceramide (α-GalCer) binds to CD1d and exhibits potent activity as a ligand for invariant CD1d-restricted natural killer-like T cells (iNKT cells). Structural analogues of α-GalCer have been synthesised to determine which components are required for CD1d presentation and iNKT cell activation, however, to date the importance of the phytosphingosine 4-hydroxyl for iNKT cell activation has been disputed. To clarify this, we synthesised two 4-deoxy α-GalCer analogues (sphinganine and sphingosine) and investigated their ability to activate murine and human iNKT cells. Analysis revealed that the analogues possessed comparable activity to α-GalCer in stimulating murine iNKT cells, but were severely compromised in their ability to stimulate human iNKT cells. Here we determined that species-specific glycolipid activity was due to a lack of recognition of the analogues by the T-cell receptors on human iNKT cells rather than insufficient presentation of the analogues on human CD1d molecules. From these results we suggest that glycolipids developed for potent iNKT cell activity in humans should contain a phytosphingosine base.     

Quantitative Determination of Natural Glycolipids from Oil Seed by Automated High‐Performance Thin‐Layer Chromatography (HPTLC)

The role of glycolipids in vegetable oil refining and production of bio‐based fuels has not been disclosed so far. Such investigations required a reliable and reproducible quantitative determination of these compounds. Fundamental data were therefore established on the quantitative determination of glycolipids in vegetable oil gums by means of high‐performance thin‐layer chromatography (HPTLC). Concentrating on five abundant natural glycolipid classes found in these oils, identification of a suitable separation method for the employed glycolipid mixture and those parameters relevant for successful detection were considered in detail. The special importance of sample volume when employing quantitative HPTLC was discussed. Acetone/chloroform/water 6:3:0.4 (v/v/v) was identified as a convenient mobile phase for the investigated issue. A derivatization reagent comprising methanol, copper(II)sulfate pentahydrate, sulfuric acid 98 %, and phosphoric acid 85 % was identified. Subsequent heating at 135 °C for 10 min finished the derivatization and enabled detection at λ = 370 nm. Calibration curves ranging from 1500 to 31.25 ng/mL, regarding both peak area and peak height, were determined. The good correlation of parameters enabled the application of the method to real oil gum samples from sunflower and soybean oil. This revealed that digalactosyldiglycerides in combination with either sterylglucosides or acylated sterylglucosides represented the major glycolipid classes in these oils.     

Anti-Tumor Effect of Orally Administered Spinach Glycolipid Fraction on Implanted Cancer Cells, Colon-26, in Mice

We succeeded in purifying a major glycolipid fraction from a green vegetable, spinach. This fraction consists mainly of three glycolipids: monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG). In a previous study, we found that the glycolipid fraction inhibited DNA polymerase activity, cancer cell growth and tumor growth with subcutaneous injection. We aimed to clarify oral administration of the glycolipid fraction, suppressing colon adenocarcinoma (colon-26) tumor growth in mice. A tumor graft study showed that oral administration of 20 mg/kg glycolipid fraction for 2 weeks induced a 56.1% decrease in the solid tumor volume (P < 0.05) without any side-effects, such as loss of body weight or major organ failure, in mice. The glycolipid fraction induced the suppression of colon-26 tumor growth with inhibition of angiogenesis and the expression of cell proliferation marker proteins such as Ki-67, proliferating cell nuclear antigen (PCNA), and Cyclin E in the tumor tissue. These results suggest that the orally administered glycolipid fraction from spinach could suppress colon tumor growth in mice by inhibiting the activities of neovascularization and cancer cellular proliferation in tumor tissue.     

Role of glycolipids in breadmaking

In wheat flour used for breadmaking, glycolipids are essential “endogenous surfactants” (1). Bread volume and crumb structure of wheat flour‐based bakery products are influenced by glycolipids, without the decisive mode of action being conclusively known. Little information on how glycolipids from other plant sources perform as wheat flour improvers is available. In this paper we review our work done on revealing the baking performance of the four major glycolipid classes present in commercial lecithins (sterol glucosides (SG), acylated sterol glucosides (ASG), cerebrosides and digalactosyl diacylglycerides (DGDG). Our purpose is to better understand the role of these substances in the baking process. Our results suggest that there are two groups of glycolipids with excellent baking performance. Both groups were found to have an impact on the dough liquor rather than on the gluten‐starch matrix. The cerebrosides and DGDG most likely influenced the overall baking result by directly stabilizing the dough liquor/gas cell interface, while ASG and SG did this indirectly through a synergistic effect with a dough liquor constituent, most probably the endogenous flour lipids.     

A model for cell-surface-exposed carbohydrate moieties suitable for structural studies by NMR spectroscopy

In the present study a synthetic glycolipid system is presented that can be readily incorporated into phospholipid micelles and that allows the study of cell-surface-exposed carbohydrate units by high-resolution NMR techniques. Here, we present an efficient route for the synthesis of glycolipid compounds that contain mannose, mannobiose, or mannotriose coupled either directly to an alkyl chain or through a poly(ethylene glycol) linker. Furthermore, we have validated our model system by measuring the binding of cyanovirin N (CV-N), a cyanobacterial protein that binds with nanomolar affinity to the terminal arms of high-mannose structures of the HIV surface-envelope glycoprotein gp120, to glycolipids the carbohydrate portions of which comprise the corresponding high-mannose moieties. From the results of chemical-shift mapping with uniformly (15)N-labelled CV-N, we conclude that binding to the protein occurs at sites similar to those involved in binding the nonconjugated carbohydrates. We characterized the insertion of the glycolipids into dodecylphosphocholine (DPC) micelles by measuring translational diffusion, and we observed that the diffusion constants of the glycolipids were very similar to those of the DPC micelles themselves, but significantly deviated from those of the free glycolipids. We also present experimental proof that the glycolipids remain inserted in the micelles while binding to CV-N. Finally, by addition of a ligand that had a higher affinity to CV-N but which was not attached did not couple to a lipid anchor, CV-N could be released from the glycolipid and, hence, from the micelle-associated state.     

Production of new types of sophorolipids by Candida batistae

Sophorolipids (SLs) are glycolipid biosurfactants abundantly produced from different feedstocks by yeasts, and have been widely developed for various applications. In this study, we searched for novel SLs, aiming to broaden the functions and application range. As a result of screening based on the phylogenetic information of a known SL producer, we found that Candida batistae CBS 8550 produces new types of SLs. Interestingly, the present product mainly constituted acid-form SLs (more than 60% of the total SLs), considerably different from conventional SLs that mainly constitute lactone-form ones. In the shake-flask culture with glucose and olive oil as the carbon sources, the yeast produced 6 g/L of SLs after 3 days cultivation. The critical micelle concentrations of the present SL product and isolated acid-form SL (GL-A) were 366 and 138 mg/L, respectively, while those of conventional SLs and isolated acid-form SL were 17 and 95 mg/L, respectively. From these results, the phylogenetic approach should lead to the discovery of new biosurfactant producers, and the yeast product possessing high hydrophilicity may facilitate a broad range of applications for SLs.     

4“-O-Alkylated α-Galactosylceramide Analogues as iNKT-Cell Antigens: Synthetic,Biological, and Structural Studies

Invariant natural killer T-cells (iNKT) are a glycolipid-responsive subset of T-lymphocytes that fulfill a pivotal role in the immune system. The archetypical synthetic glycolipid, α-galactosylceramide (α-GalCer), whose molecular framework is inspired by a group of amphiphilic natural products, remains the most studied antigen for iNKT-cells. Nonetheless, the potential of α-GalCer as an immunostimulating agent is compromised by the fact that this glycolipid elicits simultaneous secretion of Th1- and Th2-cytokines. This has incited medicinal chemistry efforts to identify analogues that are able to perturb the Th1/Th2 balance. In this work, we present the synthesis of an extensive set of 4“-O-alkylated α-GalCer analogues, which were evaluated in vivo for their cytokine induction. We have found that conversion of the 4”-OH group to ether moieties decreases the immunogenic potential in mice relative to α-GalCer. Yet, the benzyl-modified glycolipids are able to produce a distinct pro-inflammatory immune response. The crystal structures suggest an extra hydrophobic interaction between the benzyl moiety and the α2-helix of CD1d.     

Self-Assembling Properties and Recovery Effects on Damaged Skin Cells of Chemically Synthesized Mannosylerythritol Lipids

Mannosylerythritol lipids (MELs), which are one of the representative sugar-based biosurfactants (BSs) produced by microorganisms, have attracted much attention in various fields in the sustainable development goals (SDGs) era. However, they are inseparable mixtures with respect to the chain length of the fatty acids. In this study, self-assembling properties and structure-activity relationship (SAR) studies of recovery effects on damaged skin cells using chemically synthesized MELs were investigated. It was revealed, for the first time, that synthetic and homogeneous MELs exhibited significant self-assembling properties to form droplets or giant vesicles. In addition, a small difference in the length of the fatty acid chains of the MELs significantly affected their recovery effects on the damaged skin cells. MELs with medium or longer length alkyl chains exhibited much higher recovery effects than that of C18-ceramide NP.     

Chemical characterization and physicochemical behavior of biosurfactants

Microbes have been isolated from soil and water samples after an enrichment culture process with kerosene and tested for biosurfactant production by measuring the surface and interfacial tensions and emulsification power of culture broths. The isolation and characterization of extracellular surface-active agents from the culture broth of Pseudomonas aeruginosa 44T1 strain have been made. Preliminary structure identification with specific TLC reagents of the CHCl₃MeOH (2:1) extracts showed two spots with a glycolipidic structure and Rf values of 0.70 and 0.45, respectively, using the solvent system CHCl₃:MeOH:H₂O (65:25:4). Separation of surface active agents by Chromatographic absorption column in Florisil or silica gel and further spectroscopic study (IR, 1H-NMR, 13C-NMR) and chemical degradation techniques (acid and alkaline hydrolysis) gave a structure of β[β(2-0-α-L-rhamnopyranosyloxy)decanoyl] decanoic acid and β[β (2-0-α-L-rhamnopyranosyl-α-L-rhamnopyranosyloxy) decanoyloxy] decanoic acid (glycolipid A and glycolipid B, respectively) for the two glycolipids detected. A new method of mass spectrometry, fast atom bombardment mass spectrometry (FABMS), was used to probe molecular structure. The mass spectra obtained contain molecular weight recognition and sequence information signals, and they are in agreement with the proposed structures. Physicochemical evaluations of the two isolated glycolipids were made. The minimum surface tension obtained was 25 mN/m in water solutions. At pH 7 the CMC value was 11 ppm. In both cases, at pH 3 the CMC was displaced to lower and at pH 9 it was displaced to higher concentration values. Glycolipid B showed a lower value of interfacial tension (0.2 mN/m) than glycolipid A (1.0 mN/m). Glycolipid A showed a lower CMC value at alkaline pH, whereas glycolipid B had a lower CMC at acidic pH than at alkaline pH.     

Synthetic Arabinomannan Heptasaccharide Glycolipids Inhibit Biofilm Growth and Augment Isoniazid Effects in Mycobacterium smegmatis

Biofilm formation, involving attachment to an adherent surface, is a critical survival strategy of mycobacterial colonies in hostile environmental conditions. Here we report the synthesis of heptasaccharide glycolipids based on mannopyranoside units anchored on to a branched arabinofuranoside core. Two types of glycolipids—2,3‐branched and 2,5‐branched—were synthesized and evaluated for their efficacies in inhibiting biofilm growth by the non‐pathogenic mycobacterium variant Mycobacterium smegmatis. Biofilm formation was inhibited at a minimum biofilm growth inhibition concentration (MBIC) of 100 μg mL^?1 in the case of the 2,5‐branched heptasaccharide glycolipid. Further, we were able to ascertain that a combination of the drug isoniazid with the branched heptasaccharide glycolipid (50 μg mL^?1) potentiates the drug, making it three times more effective, with an improved MBIC of 30 μg mL^?1. These studies establish that synthetic glycolipids not only act as inhibitors of biofilm growth, but also provide a synergistic effect when combined with significantly lowered concentrations of isoniazid to disrupt the biofilm structures of the mycobacteria.     

Synthesis and Biological Activity of the Lipoteichoic Acid Anchor from Streptococcus sp. DSM 8747

In this study, the role of lipoteichoic acid (LTA) anchors in the activation of the innate immune response was investigated through the chemical synthesis of a series of LTA derivatives and the determination of their ability to induce NO production in bone marrow‐derived macrophages (BMM). To this end, an efficient synthesis of the sn‐3‐O‐(α‐D ‐galactofuranosyl)‐1,2‐di‐O‐acylglycerol LTA core was developed, which was then used as a key structure to produce both phosphate and glycerylphosphate‐funtionalised LTA anchors, as well as galactofuranosyldiglycerides with different fatty acid chain lengths. With a series of LTA anchors in hand, we then determined the effect of these glycolipids on the innate immune response by exploring their capacity to activate macrophages. Here, we report that several of the LTA‐derivatives were able to induce NO production by BMMs. In general, the unnatural (sn‐1) core glycolipid anchors showed lower levels of activity than the corresponding natural (sn‐3) analogues, and the activity of the glycolipids also appears to be dependent on the length of lipid present, with an optimum lipid length of C20 for the sn‐3 derivatives. Interestingly, a triacylated anchor and the 6‐O‐phosphorylated anchor, showed only modest activity, while the 6‐O‐glycerophosphorylated derivative was unable to induce NO production. Taken as a whole, our results highlight the subtle effects that glycolipid length can have on the ability to activate BMMs.     

Sex-and age-related differences in ceramide dihexosides of primary human brain tumors

Neutral glycolipids (NGL) are promising diagnostic markers of human gliomas, but differences in NGL with age and sex have not been examined. Previous work demonstrated that ceramide dihexosides (CDH) levels in mouse kidney are age- and sex-dependent, probably due to levels of sex hormones. We quantitated CDH in 181 human gliomas and found significant differences with sex and age, particularly menopause and male puberty. This emphasizes the importance of assessing results of studies on glycolipids in disease states with respect to age and sex in order to avoid erroneous conclusions concerning the relationship of glycolipid composition with diagnosis and pathogenesis.