Methods of Cryptococcal Polysaccharide Analysis Using ELISA.

One of the standard assays for the fungal pathogen Cryptococcus neoformans is the glucuronoxylomannan (GXM) ELISA. This assay utilizes monoclonal antibodies targeted against the critical virulence factor, the polysaccharide (PS) capsule. GXM ELISA is one of the most used assays in the field used for diagnosis of cryptococcal infection, quantification of PS content, and determination of binding specificity for antibodies. Here we present three variations of the GXM ELISA used by our group-indirect, capture, and competition ELISAs. We have also provided some history, perspective, and notes on these methods, which we hope will help the reader choose, and implement, the best assay for their research.While it has long been referred to as the GXM ELISA, we also suggest a name update to better reflect our updated understanding of the polysaccharide antigens targeted by this assay. The Cryptococcal PS ELISA is a more accurate description of this set of methodologies and the antigens they measure. Finally, we discuss the limitations of this assay and put forth future plans for expanding the antigens assayed by ELISA.

Identification and Quantification of Monosaccharides from Fungal Cell Walls and Exopolysaccharides by Gas Chromatography Coupled to Mass Spectrometry.

The fungal cell wall and secreted exopolysaccharides play an important role in the interactions between fungi and their environment. Despite their central role in fungal biology, ecology, and host-pathogen interactions, the composition of these polymers and their synthetic pathways are not well understood. The protocols presented in this article describe an approach to isolate fungal cell wall polysaccharides and to identify and quantify the monosaccharide composition of these polymers by gas chromatography-mass spectrometry (GC-MS). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: O-methyl trimethylsilyl monosaccharide derivatives composition analysis by GC-MS Support Protocol: Fungal cell wall extraction.

Ganoderma applanatum polysaccharides and ethanol extracts promote the recovery of colitis through intestinal barrier protection and gut microbiota modulations.

Inflammatory bowel disease is associated with intestinal homeostasis dysregulation and gut microbiota dysbiosis. This study aimed to investigate the protective effect of Ganoderma applanatum extracts (G. applanatum polysaccharides (GAP) and 75% ethanol extracts (GAE)) on colon inflammation and elucidate the therapeutic mechanism. GAP and GAE showed considerable protective effects against dextran sodium sulfate (DSS)-induced colitis, as demonstrated by reduced mortality, body weight, disease activity index score, colon length, and histological score. Through GAP and GAE administration, the destroyed intestinal barrier recovered to normal, as did intestinal inflammation. We also confirmed that GAP administration promoted the recovery of colitis in a gut microbiota-dependent manner. The similarity between GAP and GAE administration was that they both altered the disordered gut microbiota damaged by DSS, exhibiting reduced abundance of Escherichia_Shigella, Enterococcus, and Staphylococcus, but the modulation of the gut microbiota was distinct between GAP and GAE.

Selenium-Containing Exopolysaccharides Isolated from the Culture Medium of Lentinula edodes: Structure and Biological Activity.

In continuation of our research on the influence of selenium incorporation on the biosynthesis, structure, and immunomodulatory and antioxidant activities of polysaccharides of fungal origin, we have isolated from a post-culture medium of Lentinula edodes a selenium (Se)-containing exopolysaccharide fraction composed mainly of a highly branched 1-6-α-mannoprotein of molecular weight 4.5 × 106 Da, with 15% protein component. The structure of this fraction resembled mannoproteins isolated from yeast and other mushroom cultures, but it was characterized by a significantly higher molecular weight. X-ray absorption fine structure spectral analysis in the near edge region (XANES) suggested that selenium in the Se-exopolysaccharide structure was present mainly at the IV oxidation state. The simulation analysis in the EXAFS region suggested the presence of two oxygen atoms in the region surrounding the selenium. On the grounds of our previous studies, we hypothesized that selenium-enriched exopolysaccharides would possess higher biological activity than the non-Se-enriched reference fraction. To perform structure-activity studies, we conducted the same tests of biological activity as for previously obtained mycelial Se-polyglucans. The Se-enriched exopolysaccharide fraction significantly enhanced cell viability when incubated with normal (human umbilical vein endothelial cells (HUVEC)) cells (but this effect was absent for malignant human cervical HeLa cells) and this fraction also protected the cells from oxidative stress conditions. The results of tests on the proliferation of human peripheral blood mononuclear cells suggested a selective immunosuppressive activity, like previously tested Se-polyglucans isolated from L. edodes mycelium. The Se-exopolysaccharide fraction, in concentrations of 10-100 µg/mL, inhibited human T lymphocyte proliferation induced by mitogens, without significant effects on B lymphocytes. As with previously obtained Se-polyglucans, in the currently tested Se-polymannans, the selenium content increased the biological activity. However, the activity of selenium exopolysaccharides in all tests was significantly lower than that of previously tested mycelial isolates, most likely due to a different mode of selenium binding and its higher degree of oxidation.

Simultaneous detection of fungal (1,3)-ß-d-glucan and procalcitonin using a dual-label time-resolved fluorescence immunoassay.

Neonatal infectious diseases are a serious threat to the health of newborns. The aim was to establish a new detection method for the simultaneous measurement of (1,3)-ß-d-glucan and procalcitonin in serum for the early screening and efficacy testing of neonatal infectious diseases. We established a sandwich dual-label time-resolved fluorescence immunoassay (TRFIA): anti-(1,3)-ß-d-glucan/procalcitonin antibodies immobilized on 96-well plates captured (1,3)-ß-d-glucan/procalcitonin antigens and then banded together with the detection antibodies labeled with europium(III) (Eu3+ )/samarium(III) (Sm3+ ) chelates. Finally, time-resolved fluorometry was used to measure the fluorescence intensity. The linear correlation coefficient (R2 ) of the (1,3)-ß-d-glucan standard curve was 0.9913, and the R2 of the procalcitonin standard curve was 0.9911. The detection sensitivity for (1,3)-ß-d-glucan was 0.4 pg/mL (dynamic range: 0.6-90 pg/mL), and the average recovery was 101.55%. The detection sensitivity for procalcitonin was 0.02 ng/mL (dynamic range: 0.05-95 ng/mL), and the average recovery was 104.61%. There was a high R2 between the present TRFIA method and a commercially available assay (R2  = 0.9829 for (1,3)-ß-d-glucan and R2  = 0.9704 for procalcitonin). Additionally, the cutoff values for (1,3)-ß-d-glucan and procalcitonin were 23.95 pg/mL and 0.055 ng/mL, respectively. The present TRFIA method has high sensitivity, accuracy, and specificity and is an effective method for early screening and efficient testing of neonatal invasive fungal infection.

Enzymatic hydrolysis of polysaccharide from Auricularia auricula and characterization of the degradation product.

An efficient enzymatic hydrolysis method was developed and optimized for the degradation of auricularia auricula polysaccharide (AAP) and the degradation product of AAP was characterized. Cellulase was used for the degradation of AAP. The yield of reducing sugar and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate were used as indices to optimize the enzymatic hydrolysis of AAP, based on response surface methodology (RSM). The resulting optimal enzymolysis conditions were as follows: enzyme dosage, 13,500 U/g; enzymolysis temperature, 50 °C; and pH, 4.2. Under these conditions, the actual yield of reducing sugar was 16.50 mg/mL and the DPPH radical scavenging rate was 87.97%. The degradation product of AAP (C-EAAP) was homogeneous and contained alpha and beta glycoside bonds, but did not contain protein or nucleic acid. The molecular weight of the degradation product was 5.94 × 105 Da. Monosaccharide composition analysis revealed that C-EAAP was composed of mannose (57.1%), glucuronic acid (10.0%), rhamnose (0.4%), glucose (22.5%), galactose (2.9%), xylose (6.0%), and fucose (1.1%). The antioxidant activity of the polysaccharide indicated that C-EAAP had better antioxidant activity than AAP. The scavenging rates of C-EAAP for hydroxyl radicals (·OH) and superoxide anion radicals (O2-·) were 1.65 and 1.90 times those of AAP.

Isolation, identification and screening of yeasts towards their ability to assimilate biodiesel-derived crude glycerol: microbial production of polyols, endopolysaccharides and lipid.

AIMS: To assess the ability of various newly isolated or belonging in official collections yeast strains to convert biodiesel-derived glycerol (Gly) into added-value compounds. METHODS AND RESULTS: Ten newly isolated yeast strains belonging to Debaryomyces sp., Naganishia uzbekistanensis, Rhodotorula sp. and Yarrowia lipolytica, isolated from fishes, metabolized Gly under nitrogen limitation. The aim of the study was to identify potential newly isolated microbial candidates that could produce single-cell oil (SCO), endopolysaccharides and polyols when these micro-organisms were grown on biodiesel-derived Gly. As controls producing SCO and endopolysaccharides were the strains Rhodotorula glutinis NRRL YB-252 and Cryptococcus curvatus NRRL Y-1511. At initial Gly (Gly0 ) ≈40 g l-1 , most strains presented remarkable dry cell weight (DCW) production, whereas Y. lipolytica and Debaryomyces sp. produced non-negligible quantities of mannitol and arabitol (Ara). Five strains were further cultivated at increasing Gly0 concentrations. Rhodotorula glutinis NRRL YB-252 produced 7·2 g l-1 of lipid (lipid in DCW value ≈38% w/w), whereas Debaryomyces sp. FMCC Y69 in batch-bioreactor experiment with Gly0 ≈80 g l-1 , produced 30-33 g l-1 of DCW and ~30 g l-1 of Ara. At shake-flasks with Gly0 ≈125 g l-1 , Ara of ~48 g l-1 (conversion yield of polyol on Gly consumed ≈0·62 g g-1 ) was achieved. Cellular lipids of all yeasts contained in variable concentrations oleic, palmitic, stearic and linoleic acids. CONCLUSIONS: Newly isolated, food-derived and non-previously studied yeast isolates converted biodiesel-derived Gly into several added-value metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Alternative ways of crude Gly valorization through yeast fermentations were provided and added-value compounds were synthesized.

Quantitative Analysis of Polysaccharide Composition in Polyporus umbellatus by HPLC-ESI-TOF-MS.

Polyporus umbellatus is a well-known and important medicinal fungus in Asia. Its polysaccharides possess interesting bioactivities such as antitumor, antioxidant, hepatoprotective and immunomodulatory effects. A qualitative and quantitative method has been established for the analysis of 12 monosaccharides comprising polysaccharides of Polyporus umbellatus based on high-performance liquid chromatography coupled with electrospray ionization-ion trap-time of flight-mass spectrometry. The hydrolysis conditions of the polysaccharides were optimized by orthogonal design. The results of optimized hydrolysis were as follows: neutral sugars and uronic acids 4 mol/L trifluoroacetic acid (TFA), 6 h, 120 °C; and amino sugars 3 mol/L TFA, 3 h, 100 °C. The resulting monosaccharides derivatized with 1-phenyl-3-methyl-5-pyrazolone have been well separated and analyzed by the established method. Identification of the monosaccharides was carried out by analyzing the mass spectral behaviors and chromatography characteristics of 1-phenyl-3-methyl-5-pyrazolone labeled monosaccharides. The results showed that polysaccharides in Polyporus umbellatus were composed of mannose, glucosamine, rhamnose, ribose, lyxose, erythrose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and fucose. Quantitative recoveries of these monosaccharides in the samples were in the range of 96.10-103.70%. This method is simple, accurate, and sensitive for the identification and quantification of monosaccharides, and can be applied to the quality control of Polyporusumbellatus as a natural medicine.

Antioxidant and antimicrobial activities of various extracts from Engleromyces sinensis fruiting body.

Engleromyces sinensis, as rare macro-ascomycetes and traditional ethnomedicine in the southeast part of China, have been applied in anti-infection, anti-inflammatory and anti-tumor for a long time. In this study, the antioxidant activities of ethyl acetate crude extract (EACE), acetone crude extract (ACE), 95% ethanol crude extract (ECE), methanol crude extract (MCE) and water crude extract (WCE) from E. sinensis fruiting body were investigated using conventional antioxidant assays in vitro for the first time. As results, it was noteworthy that WCE showed the greatest 2,2-diphenyl-1-picrylhydrazil (DPPH) radicals-scavenging activity and reducing power, with EC50 values of 3.56 and 19.28mg/mL. MCE and EACE exhibited higher hydroxyl radicals-scavenging activity and ferrous ion-chelating activity significantly, with EC50 values of 2.16 and 0.47mg/mL. The total phenolics and total polysaccharides content results revealed that WCE had the highest phenolics and polysaccharides contents with 1.19 mg GAEs/g extracts and 40.07 mg D-glucose/g extracts. The antimicrobial activity of the WCE, ECE, ACE, EACE was assessed in final and two of them, ACE and EACE showed a strong ability to inhibit the microbial growth. The research work demonstrated that E. sinensis fruiting body can present a promising source of antioxidant and antimicrobial agents.

Utility of Serum and Bronchoalveolar Lavage Fluid Galactomannan in Diagnosis of Chronic Pulmonary Aspergillosis.

The value of serum and bronchoalveolar lavage fluid galactomannan (BALF-GM) in diagnosing chronic pulmonary aspergillosis (CPA) remains unclear. Here, we study the diagnostic efficacy of GM in the diagnosis of CPA. We included consecutive treatment-naive subjects with CPA. For calculating the specificity of serum GM, we enrolled diseased controls (minimally symptomatic subjects previously treated for pulmonary tuberculosis, not meeting the criteria for CPA). To calculate the specificity of BALF-GM, subjects with pulmonary disorders other than CPA who underwent bronchoscopy were included. We determined the cutoff of serum and BALF-GM levels using receiver operating characteristic (ROC) curve analysis. We enrolled 243 consecutive treatment-naive subjects (53.5% males) of CPA with a mean (standard deviation) age of 43.6 (14.7) years. Forty-five (60% males; age, 46.7 [15.7] years) and 27 (59.3% males; age, 52.6 [12.8] years) subjects served as controls for serum and BALF-GM, respectively. The best cutoff value for serum and BALF-GM was 0.55 (area under the ROC curve [AUROC], 0.605; sensitivity, 38%; specificity, 87%) and 1.375 (AUROC, 0.836; sensitivity, 68%; specificity, 93%), respectively. At a cutoff value of 2.5, BALF-GM had a sensitivity and specificity of 50% and 100%, respectively. BALF-GM performs better than serum GM and may be helpful in the diagnosis of CPA in selected patients. More studies are required to confirm our findings.

Anti-hydroxyl radical activity, redox potential and anti-AChE activity of Amanita strobiliformis polysaccharide extract.

This study outlines antioxidant and anti-AChE activities of the polysaccharide (PSH) extract from the mushroom species Amanita strobiliformis. Both the presence of α and ß glucans within the aforementioned extract was recorded. PSH extract displayed a profound scavenging activity of OH radicals (IC50 value, 11.86 ± 0.59 µg/mL) and high potential for reduction of Fe3+ ions (174.11 ± 8.70 mg eq. AA/g d.w.) being almost 48- and 5-fold more effective than mannitol and butylated hydroxytoluene used as a positive control, respectively. Compared with galanthamine (0.001 µg), the same extract exhibited a moderate anti-AChE activity (10 µg) in solid. Since purified PSH extract exhibited higher bioactivity (IC50 value 7.27 ± 0.31 µg/mL, 197.68 ± 9.47 mg eq. AA/g d.w. and 0.1 µg, respectively), it can be predominantly ascribed to the polysaccharide compounds. A. strobiliformis PSH extract may be considered as a promising resource of potent bioactive polysaccharides of natural origin successfully addressing both oxidative stress and lack of acetylcholine.

Design and Quantitative Analysis of Cancer Detection System Based on Fluorescence Immune Analysis.

Human blood is an important medical detection index. With the development in clinical medical detection instruments and detection technology, the requirements for detection accuracy and efficiency have been gradually improved. Fluorescent immunochromatography is a new detection technique. It has the characteristics of high efficiency, convenience, no pollution, and wide detection range. Human blood can be detected quickly using fluorescent immunochromatography. At present, it has received great attention from the field of clinical testing. In this paper, a set of fluorescent immunochromatographic analyzer has been designed. It is mainly based on the principle of fluorescence immunochromatography. A new method of signal analysis and system design for fluorescent immunochromatography analyzer is proposed. By using the improved threshold function denoising algorithm, the quantitative detection of fluorescent immunochromatographic strip is realized. The concentration of pathogenic factors (cancer cells) in human serum can be measured conveniently and accurately. The system integrates many peripheral modules, including fluorescence signal acquisition, fluorescence signal processing, quantitative curve fitting, and test results. In this paper, the quantitative detection experiments of the system are carried out in three aspects: linearity, repeatability, and sensitivity. The experimental results show that the linear correlation coefficient is up to 0.9976, and the limit of detection is up to 0.05 ng/ml. The requirements of the system are satisfied. The system performance is good, and the quantitative result is accurate. Therefore, the establishment of a fluorescence analysis system is of great significance.

Application of near infrared spectroscopy for rapid determination the geographical regions and polysaccharides contents of Lentinula edodes.

In this study, a calibration model based on Near-infrared spectroscopy (NIR) technique and chemometrics method was developed for rapid and non-destructive detecting the polysaccharide contents of lentinula edodes samples collected from different regions. The polysaccharide contents of these samples were firstly determined by standard phenol-sulphruic acid method. Then, NIR spectra of these samples were collected by using Fourier transform infrared spectrometry. Based on these experimental data, a random forest method was further used to distinguish the regions of these samples, with a classification accuracy of 96.6%. After that, a rapid, accurate, and quantitative model was established for predicting the polysaccharide contents of these samples. In the model establishing process, some signal pre-treatment methods were optimized, and the validation results with highest determination coefficient (R2) and low root mean square errors of prediction (RMSEP) were, 0.925 and 0.720, respectively. These results showed that combined NIR technique with chemometrics was an effective and green method for lentinula edodes quality control.

Spatial Distribution of Glucan Type and Content between Caps and Stalks in Pleurotus eryngii: Impact on the Anti-inflammatory Functionality.

: Pleurotus eryngii is recognized for its prominent nutritional and medicinal value. In our study, we tested the effect of glucans on lipopolysaccharide (LPS)-induced production of TNF-α. We demonstrated that glucan extracts are more effective than mill mushroom preparations. Additionally, the effectiveness of stalk-derived glucans were slightly more pronounced than of caps. Cap and stalk glucans from mill or isolated glucan competed dose-dependently with anti-Dectin-and anti-CR-3 antibodies, indicating that they contain ß-glucans recognized by these receptors. Using the dextran sulfate sodium (DSS)-inflammatory bowel disease mice model, intestinal inflammatory response to the mill preparations was measured and compared to extracted glucan fractions from caps and stalks. We found that mill and glucan extracts were very effective in downregulating IFN-γ and MIP-2 levels and that stalk-derived preparations were more effective than from caps. The tested glucans were equally effective in regulating the number of CD14/CD16 monocytes and upregulating the levels of fecal-released IgA to almost normal levels. In conclusion, the most effective glucans in ameliorating some IBD-inflammatory associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii. These spatial distinctions may be helpful in selecting more effective specific anti-inflammatory mushrooms-derived glucans.

[Rapid identification of geographical origins and determination of polysaccharides contents in Ganoderma lucidum based on near infrared spectroscopy and chemometrics].

Near infrared spectroscopy combined with chemometrics methods was used to distinguish Ganoderma lucidum samples collected from different origins, and a prediction model was established for rapid determine polysaccharides contents in these samples. The classification accuracy for training dataset was 96.87%, while for independent dataset was 93.33%; as for the prediction model, 5-fold cross-validation was used to optimize the parameters, and different signal processing methods were also optimized to improve the prediction ability of the model. The best square of correlation coefficients for training dataset was 0.965 4, and 0.851 6 for validation dataset; while the root-mean-square deviation values for training dataset and validation dataset were 0.018 5 and 0.023 6, respectively. These results showed that combining near infrared spectroscopy with suitable chemometrics approaches could accuracy distinguish different origins of G. lucidum samples; the established prediction model could precious predict polysaccharides contents, the proposed method can help determine the activity compounds and quality evaluation of G. lucidum.

Novel Treatment of Cryptococcal Meningitis via Neurapheresis Therapy.

Cryptococcal meningitis (CM) has emerged as the most common life-threatening fungal meningitis worldwide. Current management involves a sequential, longitudinal regimen of antifungals; despite a significant improvement in survival compared with uniform mortality without treatment, this drug paradigm has not led to a consistent cure. Neurapheresis therapy, extracorporeal filtration of yeasts from cerebrospinal fluid (CSF) in infected hosts, is presented here as a novel, one-time therapy for CM. In vitro filtration of CSF through this platform yielded a 5-log reduction in concentration of the yeast and a 1-log reduction in its polysaccharide antigen over 24 hours. Additionally, an analogous closed-loop system achieved 97% clearance of yeasts from the subarachnoid space in a rabbit model over 4-6 hours. This is the first publication demonstrating the direct ability to rapidly clear, both in vitro and in vivo, the otherwise slowly removed fungal pathogen that directly contributes to the morbidity and mortality seen in CM.

Evaluation on quality consistency of Ganoderma lucidum dietary supplements collected in the United States.

Ganoderma lucidum is a well-known medicinal mushroom. At present, numerous G. lucidum products have emerged in the form of dietary supplements in the United States due to its various benefits. However, the quality consistency of these products based on their label ingredients has seldom been evaluated due to the lack of a suitable toolkit. In this study, 19 batches of products of G. lucidum (Red Reishi, Reishi), herbal/mushroom supplements purchased in the United States, were evaluated based on their bioactive components including triterpenes and polysaccharides by using chromatographic methods and saccharide mapping. The results showed that the measured ingredients of only 5 tested samples (26.3%) were in accordance with their labels, which suggested the quality consistency of G. lucidum dietary supplements in the U.S. market was poor, which should be carefully investigated.

Current Challenges in the Diagnosis of Fungal Infections.

Diagnosing fungal infections is a challenge, particularly in the immunocompromised host. Signs and symptoms are nonspecific, colonization is difficult to distinguish from invasive disease, blood cultures are commonly negative, and patients are often unable to undergo invasive diagnostic procedures. Culture and microscopic examination remain the "gold standard" but are insensitive. Antigen assays such as the galactomannan and glucan detection systems are frequently used, yet these tests vary in sensitivity and specificity, depending on the patient population involved. Molecular-based assays are not yet clinically validated.

Current Algorithms in Fungal Diagnosis in the Immunocompromised Host.

Invasive fungal diseases (IFDs) are a major cause of morbidity and mortality in immunocompromised patients such as patients with hematological malignancies or allogeneic hematopoietic stem cell transplant recipients. Whereas the definite diagnosis of IFD requires invasive diagnostic procedures, imaging and noninvasive diagnostic assays may help in decision making with regard to the institution and the choice of antifungal agents, the duration of therapy, surgical intervention, and monitoring of fungal manifestations.Unfortunately, signs and symptoms of IFD are often nonspecific in the immunocompromised patient. Therefore, in immunocompromised patients with suspected IFD, all samples collected need to be cultured for fungi, and, in the case of specimens obtained by invasive diagnostic procedures, also microscopically examined. For high sensitivity of the cultural and microscopic approaches, specific media and stains, respectively, are crucial. Non-culture based method such as the detection of galactomannan or ß-d-glucan and molecular tools such as polymerase chain reaction may help in the early diagnosis of IFD. Imaging studies may be indicative for IFD, but invasive diagnostics such as bronchoalveolar lavage and/or biopsy should be pursued in order to identify the causative pathogen. This chapter summarizes the current knowledge on diagnosing IFD and proposes practical help in the use of diagnostic tools.

[ON DEVELOPMENT OF TOOLS OF IMMUNE DIAGNOSTIC OF INFECTIOUS DISEASES: PROBLEMS OF DIAGNOSTIC IN VIVO AND IN VITRO].

The article considers a number of problematic issues concerning development of effective means of immune diagnostic of infectious diseases of bacterial and mycotic etiology related to approaches of choosing appropriate diagnostic targets.