Calcofluor Staining Research Articles

Improvement of the Diagnostic Method for the Detection of Parasite Eggs Using the Formalin Ethyl Acetate Concentration Technique.

The formalin-ethyl acetate (FEA) concentration method is commonly used in routine clinical practice to detect parasite eggs in feces. This procedure involves extraction of oil with the organic solvent ethyl acetate (EA), which reduces fecal sediment and provides a cleaner background for microscopic analysis. However, clinically, some sediment failed to float after EA treatment. Hexane, commonly used in the food oil extraction from oilseeds did not float the feces. Gas chromatography-mass spectrometry (GC-MS) analysis showed that neither the amount of the oil nor the classes of the oil determined was differed whether hexane or EA was used to float the feces. Oil red, Bodipy and Calcofluor staining showed that the unabsorbed oil droplets in the fecal sediment were trapped within the leaf structure. HCl or acetic acid was added to see if the acid residue could dissolve the cellulose of the leaf to promote the bulk float. Our result showed that the fecal bulk contained the loosened mesophyll cell wall. The addition of acid residues improved fecal bulk float. The proximity of cellulose fiber to EA, but not hexane, may enhance the efficacy of oil extraction from cellulose. This is the first report that the interaction of cellulose with ethyl acetate in fecal solution has an effect on bulk float. This study improves the understanding of fecal bulk flotation and may assist in the visualization of parasite eggs in clinical practice with non-floating fecal samples in the FEA concentration method.

RNA Interference-Mediated Suppression of Ecdysone Signaling Inhibits Choriogenesis in Two Coleoptera Species.

During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the effect of chemical components on eggshell deserve further exploration. In two representative coleopterans, a coccinellid Henosepilachna vigintioctopunctata and a chrysomelid Leptinotarsa decemlineata, genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either HvEcR or Hvusp in H. vigintioctopunctata inhibited oviposition, suppressed the expression of HvChS1, and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of LdEcR or Ldusp in L. decemlineata constrained oviposition, decreased the expression of LdUAP1 and LdChS1, and reduced CLS contents in the resultant ovaries. Knockdown of LdUAP1 or LdChS1 caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the L. decemlineata ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two coleopteran species. Moreover, our findings suggest the deposition of a CLS on the chorions.

Immunodetection of Cell Wall Components in Studies on Cell Wall Rebuilding in Fagopyrum esculentum and Fagopyrum tataricum.

Immunocytochemical studies of the cell wall are used to visualize specific epitopes of pectins, arabinogalactan proteins, hemicelluloses, extensins, and other wall components using specific primary antibodies. This reaction, combined with calcofluor staining, allows to comprehend how the cell wall is rebuilt during the protoplast culture. In this protocol, the method of immunostaining using antibodies against cell wall components based on Fagopyrum esculentum and Fagopyrum tataricum protoplasts is described.

Early detection of grapevine graft incompatibility: Insights into translocated and virus-induced incompatibility

In vineyards to control phylloxera (Daktulosphaira vitifolia Ficth) attacks in Vitis vinifera L., heterografted vines are planted using American vines hybrids as rootstocks. However, graft incompatibilities can affect grape yield and plant longevity. Thus, to identify early graft incompatibility factors, we established in vitro micrografting protocols coupled with histology and histochemistry analysis in grapevine graft combinations of known compatibility behavior. The histochemical characterization of the graft union revealed irregular cell arrangement, slower vascular differentiation, persistence of the necrotic layer, accumulation of starch, and lower differentiation of phloem cells in hetero- compared to homografts, indicating the presence of translocated incompatibility symptoms. We highlight the utility of evaluating the graft interface cellular arrangement and starch content via calcofluor and I2KI staining, respectively, as allowed to identify the graft combinations with lower graft success. Wounded and grafted Syrah plantlets pointed out an impaired sucrose distribution in these plants and levels of Grapevine Rupestris Stem Pitting associated Virus (GRSPaV) infections correlated with graft (un)-success in two Syrah clones micrografted onto 110-Ritcher rootstock. Furthermore, silencing GRSPaV before grafting increased graft success rates. We propose that grapevine graft incompatibility is mainly a virus-induced phenomenon that can arise even in certified plants.

Ethosomal Nanoformulations for Combinational Photothermal Therapy of Fungal Keratitis

AbstractFungal keratitis/mycotic keratitis or keratomycosis, is characterized by the inflammation of underlying stroma of corneal epithelium due to fungal infection. Multi‐drug resistance and subsequent recurrence of infection limits the clinical outcomes of anti‐fungal agents such as Amphotericin B and Natamycin; and therefore, demands research on newer anti‐microbial strategies. In this study, the authors have synthesized and characterized ethosomes (IAEs) encapsulating a NIR dye, Indocyanine green (ICG), and an anti‐fungal drug, Amphotericin B, for a combinational photothermal therapy of fungal keratitis. The in vitro anti‐fungal efficacy of the IAEs is validated against Candida albicans by colony formation assay, calcofluor staining, and SEM analysis. The results indicate that the IAEs, upon NIR irradiation, displays a synergistic anti‐fungal efficacy. The IAEs are highly biocompatible when tested using a human corneal epithelial (HCE) cell line model (in vitro) and a Draize eye test model in rabbits (in vivo). Further, it is observed that the IAEs,upon NIR laser exposure significantly eliminate the fungal infection in rat model of occular fungal keratitis. The research indicates that the IAEs nano‐formulation exhibits a synergistic and sustained anti‐fungal effect paving its way for further translation from bench to bedside for the efficient management of fungal keratitis.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens.

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

Raspberry Ketone-Mediated Inhibition of Biofilm Formation in Salmonella enterica Typhimurium-An Assessment of the Mechanisms of Action.

Salmonella enterica is an important foodborne pathogen that causes gastroenteritis and systemic infection in humans and livestock. Salmonella biofilms consist of two major components-amyloid curli and cellulose-which contribute to the prolonged persistence of Salmonella inside the host. Effective agents for inhibiting the formation of biofilms are urgently needed. We investigated the antibiofilm effect of Raspberry Ketone (RK) and its mechanism of action against Salmonella Typhimurium 14028 using the Congo red agar method, Calcofluor staining, crystal violet method, pellicle assay, and the TMT-labeled quantitative proteomic approach. RK suppressed the formation of different types of Salmonella biofilms, including pellicle formation, even at low concentrations (200 µg/mL). Furthermore, at higher concentrations (2 mg/mL), RK exhibited bacteriostatic effects. RK repressed cellulose deposition in Salmonella biofilm through an unknown mechanism. Swimming and swarming motility analyses demonstrated reduced motility in RK-treated S. typhimurium. Proteomics analysis revealed that pathways involved in amyloid curli production, bacterial invasion, flagellar motility, arginine biosynthesis, and carbohydrate metabolism, were targeted by RK to facilitate biofilm inhibition. Consistent with the proteomics data, the expressions of csgB and csgD genes were strongly down-regulated in RK-treated S. typhimurium. These findings clearly demonstrated the Salmonella biofilm inhibition capability of RK, justifying its further study for its efficacy assessment in clinical and industrial settings.

P328 An overexpression screen identifies Csa25 as a novel cellular morphogenesis regulator in the human fungal pathogen Candida albicans

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectiveMorphological plasticity is one of the key attributes of microbial pathogens contributing to the successful establishment of infection in host tissues. Candida albicans, an opportunistic human fungal pathogen, lives as a commensal in the gut, skin, and genitourinary tracts of most healthy individuals. The budding yeast form helps it to disseminate easily in the host system, and the filamentous form (hypha and pseudohypha) is believed to invade the host tissue. Strikingly, alterations of gene expression that block cell cycle progression at different stages additionally lead to aberrant cellular morphology in C. albicans. While various morphological states of C. albicans have been well-studied, the search for key players bringing about these changes is far from complete. This is supported by the fact that ∼70% of the C. albicans proteome remains functionally uncharacterized. Thus, the primary objective of our study was to identify novel regulators contributing to cellular morphogenesis in C. albicans.MethodIn our current study, we screened an overexpression library of C. albicans ORFeome generated to identify novel regulators contributing to chromosome stability (CSA) in C. albicans. The screen involved overexpression of each gene using a tetracycline-inducible promoter for a duration of 12 h, followed by microscopy-based observations to identify associated aberrant cellular morphologies.ResultsScreening of overexpression library of the C. albicans ORFeome identified 14 unique Candidate genes from 1389 genes screened. While the functions of half of them have been verified in C. albicans, the remaining seven genes are not functionally characterized. Each of the seven uncharacterized genes was predicted to be non-essential for viability in C. albicans. Bioinformatic analysis predicts one of these proteins, Csa25, to be carrying a point centromere-specific kinetochore protein Ndc10-like DNA-binding domain at its N-terminus spanning over a region of 273 amino acids. Sub-cellular localization indicates this protein to be present throughout the nucleus at all stages of the cell cycle. Strikingly, overexpression of this protein led to yeast cells forming chains connected by septa, as visualized by calcofluor staining, without hampering nuclear segregation. In addition, a large proportion of cells overexpressing Csa25 were unable to exhibit hyphal morphology when subjected to hyphae-inducing conditions.ConclusionIn conclusion, our study identified Csa25 as a novel morphogenesis regulator involved in the negative regulation of yeast-hyphae transition in C. albicans. Further studies based on host-pathogen interaction will identify the critical role of Csa25 in the pathobiology of C. albicans and its survival in host-specific niches.

Trichoderma longibrachiatum-Associated Skin Inflammation and Atypical Hyperplasia in Mouse.

BackgroundThe relationship between infection and tumors has attracted increasing attention. Trichoderma spp. are often isolated from tumors. However, their potential role remains unclear. We recently reported the isolation of Trichoderma longibrachiatum from a patient with pulmonary spindle cell carcinoma that was confirmed as primary infection by application of laser capture microdissection and polymerase chain reaction. To explore whether the strain is pathogenic and whether it can cause atypical cell proliferation and infiltration of NK cells and T cells, we designed a mouse infection experiment.MethodsTwelve ICR mice were randomly separated into three groups. Cyclophosphamide was used to inhibit the immunity of mice. A mouse model of Trichoderma infection was successfully established by intracutaneous injection on the back skin with a suspension of strain PKUT180420015. The pathological manifestations of Trichoderma infection and the interaction between immune cells and fungi were observed by histopathology, immunohistochemistry and intensive fungal staining. Reisolation of the fungus was observed by infected tissue culture. The inoculated sites exhibited swelling 3 days after inoculation, and ulcers developed at approximately 14 days. Skin specimens were obtained and then cultured at 3, 7, and 14 days after inoculation. We selected mice 14 days after inoculation in Group 3, whose ulcers were the most typical, for histological analysis.ResultsInflammation, angioinvasion and necrosis were observed. Immunohistochemistry showed positive markers of Ki67, CD3, CD56, GZMB, and PRF. Periodic acid-Schiff staining, periodic acid-silver methenamine staining, and Calcofluor staining showed fungal spores in the vascular lumen, vascular walls and around the blood vessels.ConclusionsOur studies showed that a T. longibrachiatum strain (PKUT180420015) isolated from a biopsy specimen in a patient with pulmonary spindle cell carcinoma could induce atypical hyperplasia, with the expression of Ki67, CD3, CD56, GZMB, and PRF in mice. These data indicate that the fungus may be involved in inducing atypical hyperplasia or tumorigenesis.

COVID-19-Associated Pulmonary Aspergillosis in a Tertiary Hospital.

Our study aims to assess the prevalence of CAPA (COVID-19-associated pulmonary aspergillosis) and describe the associated risk factors and their impact on mortality. A prospective study was conducted. We included patients with COVID-19 disease who were admitted to the ICU with a diagnosis of respiratory failur. Mycological culture and other biomarkers (calcofluor staining, LFD, LFA, PCR, GM, and B-D-glucan) were performed. A total of 300 patients were included in the study. Thirty-five patients were diagnosed with CAPA (prevalence 11.7%). During admission, 57 patients died (19%), and, in the group of CAPA patients, mortality was 31.4%. In multivariate analysis, independent risk factors associated with CAPA diagnosis were age (OR: 1.05; 95% CI 1.01–1.09; p = 0.037), chronic lung disease (OR: 3.85; 95% CI 1.02–14.9; p = 0.049) and treatment with tocilizumab during admission (OR: 14.5; 95% 6.1–34.9; p = 0.001). Factors independently associated with mortality were age (OR: 1.06; 95% CI 1.01–1.11; p = 0.014) and CAPA diagnosis during admission (OR: 3.34; 95% CI 1.38–8.08; p = 0.007). CAPA is an infection that appears in many patients with COVID-19 disease. CAPA is associated with high mortality rates, which may be reduced by early diagnosis and initiation of appropriate antifungal therapy, so screening of COVID-19 ARDS (acute respiratory distress syndrome) patients for CAPA is essential.

Invasive aspergillosis a complication severe respiratory viral infections (influenza and COVID-19)

Invasive aspergillosis is a life-threatening complication in patients with severe influenza and COVID-19 in intensive care units. Risk factors for the invasive aspergillosis development are transitory immunosuppression associated with severe influenza and COVID-19, as well as the use of glucocorticosteroids and immunosuppressive therapy. In the presence of risk factors, suspected clinical and radiological signs of invasive aspergillosis, bronchoscopy and examination of material from the lower respiratory tract are necessary: test for galactomannan, microscopy with white calcofluor staining and inoculation on Sabouraud agar medium. Voriconazole or are recommended as first-line treatment for invasive aspergillosis in patients with severe influenza and COVID-19. Amphotericin B Liposomal, Amphotericin B Lipid Complex, and Caspofungin are the alternative options for the invasive aspergillosis treatment. Combination therapy is possible. It is necessary to control the underlying disease with eliminate or reduce the severity of risk factors.

Biomacromolecules in recent phosphate-shelled brachiopods: identification and characterization of chitin matrix

Phosphate-shelled brachiopods differ in filter-feeding lifestyle, with Lingula anatina an active infaunal burrower, and Discinisca tenuis a shallow marine epibenthic animal. The shells of these animals are built of organophosphatic constituents, the organic fibres/sheets reinforced with calcium phosphate to provide a sophisticated ultrastructural robustness. This investigation examined the nature of the organic fibres in order to improve understanding of how living organisms produce hierarchically structured biomaterials. Unlike powdered samples commonly used in previous studies, organic fibres were isolated for the first time and the shell fractions were purified, in order to study the content and nature of the biopolymer fibres. Biochemical methods including Calcofluor staining revealed a chitin matrix. Ultrastructural analysis, thermal gravimetric analysis, and spectroscopic analyses show that the core polysaccharide framework is composed of layers of β-chitin sheets and/or fibrils that are coated with a fibrous organic matrix. There is more chitin matrix in the L. anatina shells (26.6 wt.%) compared to the D. tenuis shells (12.9 wt.%). Taken together, the data show that the chitin matrix contributes to increased skeletal strength, making L. anatina highly adapted for life as an active burrower. In comparison, D. tenuis contains less chitin and lives as attached epibenthos in a shallow marine environment.Graphical abstractFirst spectroscopic evidence of β-chitin sheets in recent organophosphatic brachiopods

Effects of homeopathic medicines on macrophage - E. cuniculi interaction in vitro

Encephalitozoon cuniculi (E. cuniculi) is a fungus that behaves as an intracellular parasite infecting different types of cells. In rabbits and immunosuppressed animals of other species, including humans, it parasites neural tissues causing a highly characteristic neurological syndrome, for which repertory analysis indicates Phosphorus. Successful treatment of 7 naturally infected rabbits with Phosphorus confirmed that this medicine probably is the remedy of the ‘epidemic genius’ of microsporidiosis. In the present study, an in vitro model was used to evidence the intracellular relationship between macrophages and E. cuniculi after treatment of co-cultures with different potencies of Phosphorus. RAW 254.7 macrophages were co-cultivated with E. cuniculi and treated with Phosphorus 6cH, 30cH and 200cH. Controls were untreated co-cultures and cultures treated with vehicle (0.06% final alcohol concentration). Phagocytosis and lysosome activity were after after 1 and 24 hours of incubation by means of the calcofluor and acridine orange staining methods, followed by automatic image analysis (Metamorph®). Cytokine production was assessed with the MAGPIX®-Luminex system. The vehicle increased IL-6, MCP-1 and MIP1 production (p

Microbiological diagnosis of Acanthamoebic keratitis: experience from tertiary care center of North India

Acanthamoeba keratitis (AK) is a painful vision-threatening infection caused by pathogenic free-living Acanthamoeba. Due to the non-specific clinical presentation, this condition tends to be misdiagnosed by clinicians. A timely diagnosis is crucial for favorable visual outcome. Three hundred patients with suspected microbial keratitis presenting to the Advanced Eye Center at our tertiary care center in North India during the period from 2014 to 2018 were included. Patient's corneal scrapings, contact lens, lens solution, lens case, and tears were processed for microscopic examination by Giemsa and Calcofluor staining, non-nutrient agar (NNA) culture and molecular diagnosis by conventional PCR (cPCR) and Real-time PCR (qPCR). 18S rDNA gene sequencing was done to assess phylogenetic relationship. AK was found in 3.6% (11/300) of non-bacterial non-fungal keratitis patients. Among microbiological techniques, microscopy for Acanthamoeba was positive in 7 cases, NNA culture was positive in 9 cases and 11 cases were detected both by cPCR and qPCR. The sensitivity of microscopy, culture, cPCR and qPCR was 63.64%, 81.82 %, 100%, and 100% respectively whereas specificity was 100% for all the tests. 18S rDNA sequencing revealed that A. castellanii was the predominant species and isolates were genetically distinct. AK should be considered in the differential diagnosis of infectious keratitis. Molecular tests are useful for rapid, sensitive and specific diagnosis and must be included in workup of keratitis.

Determination of Chitin Content in Insects: An Alternate Method Based on Calcofluor Staining.

Chitin is an aminopolysaccharide present in yeast cells and arthropod cuticle and is one of the most abundant biopolymers. The conventional methods for the quantitation of chitin content in biological samples are based on its hydrolysis (acid or enzymatic), and the assessment of the byproduct, glucosamine. However, previously described methodologies are time-consuming, laborious, low throughput, and not applicable to insect samples in many cases. Here we describe a new approach to chitin content quantitation based on calcofluor fluorescent brightener staining of samples, followed by microplate fluorescence readings. Calcofluor is a specific chitin stain commonly used for topological localization of the polymer. The protocol was tested in three important disease vector species, namely Lutzomyia longipalpis, Aedes aegypti, and Rhodnius prolixus, and then compared to a classic colorimetric chitin assessment method. Results show that chitin content in the tested insects can vary largely in a range of 8–4600 micrograms of chitin per insect, depending on species, sex, and instar. Comparisons between measurements from the previous protocol and calcofluor method showed statistically significant differences in some samples. However, the difference might be due to interference in the classic method from non-chitin sources of glucosamine and reducing agents. Furthermore, chitinase hydrolysis reduces the total chitin mass estimated between 36 and 74%, consolidating the fluorescent measurements as actual stained chitin in the same extent that was observed with the standard protocol. Therefore, the calcofluor staining method revealed to be a fast and reliable technique for chitin quantitation in homogenized insect samples.

Efficiency of vanillin in impeding metabolic adaptability and virulence of Candida albicans by inhibiting glyoxylate cycle, morphogenesis, and biofilm formation.

Background and Purpose: Candida albicans is the fourth most common cause of nosocomial fungal infections across the world. The current drug regimens are suffering from such drawbacks as drug resistance, toxicity, and costliness; accordingly, they highlight the need for the discovery of novel drug agents. The metabolic adaptability under low-carbon conditions and expression of functional virulence traits mark the success of pathogens to cause infection. The metabolic pathways, such as glyoxylate cycle (GC), enable C. albicans to survive under glucose-deficient conditions prevalent in the hostile niche. Therefore, the key enzymes, namely isocitrate lyase (ICL) and malate synthase (MLS), represent attractive agents against C. albicans. Similarly, virulence traits, such as morphogenesis and biofilm formation, are the crucial determinants of C. albicans pathogenicity. Regarding this, the present study was conducted to uncover the role of vanillin (Van), a natural food flavoring agent, in inhibiting GC, yeast-to-hyphal transition, and biofilm formation in human fungal pathogen C. albicans.Materials and Methods:For the determination of hypersensitivity under low-glucose conditions, phenotypic susceptibility assay was utilized. In addition, enzyme activities were estimated based on crude extracts while in-silico binding was confirmed by molecular docking. The assessment of morphogenesis was accomplished using hyphal-inducing media, and biofilm formation was estimated using calcofluor staining, MTT assay, and biomass measurement. Additionally, the in vivo efficacy of Van was demonstrated using Caenorhabditis elegans nematode model.Results:Based on the results, Van was found to be a potent GC inhibitor that phenocopied ICL1 deletion mutant and displayed hypersensitivity under low-carbon conditions. Accordingly, Van facilitated the inhibition of ICL and MLS activities in vitro. Molecular docking analyses revealed the in-silico binding affinity of Van with Icl1p and Mls1p. Those analyses were also confirmative of the binding of Van to the active sites of both proteins with better binding energy in comparison to their known inhibitors. Furthermore, Van led to the attenuation of such virulence traits as morphogenesis, biofilm formation, and cell adherence. Finally, the antifungal efficacy of Van was demonstrated by the enhanced survival of C. elegans with Candida infection. The results also confirmed negligible hemolytic activity on erythrocytes.Conclusion:As the findings of the present study indicated, Van is a persuasive natural compound that warrants further attention to exploit its anticandidal potential.

Characterization of the main steps in first shell formation in Mytilus galloprovincialis: possible role of tyrosinase.

Bivalve biomineralization is a highly complex and organized process, involving several molecular components identified in adults and larval stages. However, information is still scarce on the ontogeny of the organic matrix before calcification occurs. In this work, first shell formation was investigated in the mussel Mytilus galloprovincialis. The time course of organic matrix and CaCO3 deposition were followed at close times post fertilization (24, 26, 29, 32, 48 h) by calcofluor and calcein staining, respectively. Both components showed an exponential trend in growth, with a delay between organic matrix and CaCO3 deposition. mRNA levels of genes involved in matrix deposition (chitin synthase; tyrosinase- TYR) and calcification (carbonic anhydrase; extrapallial protein) were quantified by qPCR at 24 and 48 hours post fertilization (hpf) with respect to eggs. All transcripts were upregulated across early development, with TYR showing highest mRNA levels from 24 hpf. TYR transcripts were closely associated with matrix deposition as shown by in situ hybridization. The involvement of tyrosinase activity was supported by data obtained with the enzyme inhibitor N-phenylthiourea. Our results underline the pivotal role of shell matrix in driving first CaCO3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis.

Assessment of the in vitro and in vivo activity of atorvastatin against Candida albicans.

Aim. The aim of this work was to characterize the response of Candida albicans to atorvastatin, and to assess its in vivo antifungal capability.Methodology. The effect of atorvastatin on the growth and viability of C. albicans was assessed. The ability of the statin to alter cell permeability was quantified by measuring amino acid and protein leakage. The response of C. albicans to atorvastatin was assessed using label-free quantitative proteomics. The in vivo antifungal activity of atorvastatin was assessed using Galleria mellonella larvae infected with C. albicans.Results. Atorvastatin inhibited the growth of C. albicans. The atorvastatin-treated cells showed lower ergosterol levels than the controls, demonstrated increased calcofluor staining and released elevated quantities of amino acids and protein. Larvae infected with C. albicans showed a survival rate of 18.1±4.2 % at 144 h. In contrast, larvae administered atorvastatin (9.09 mg kg-1) displayed a survival rate of 60.2±6.4 % (P<0.05). Label-free quantitative proteomics identified 1575 proteins with 2 or more peptides and 465 proteins were differentially abundant (P<0.05). There was an increase in the abundance of enzymes with oxidoreductase and hydrolase activity in atorvastatin-treated cells, and squalene monooxygenase (4.52-fold increase) and lanosterol synthase (2.84-fold increase) were increased in abundance. Proteins such as small heat shock protein 21 (-6.33-fold) and glutathione peroxidase (-2.05-fold) were reduced in abundance.Conclusion. The results presented here indicate that atorvastatin inhibits the growth of C. albicans and is capable of increasing the survival of G. mellonella larvae infected with C. albicans.

Use of Green Fluorescent Protein Expressing Colletotrichum falcatum, the Red Rot Pathogen for Precise Host–Pathogen Interaction Studies in Sugarcane

Productivity in sugarcane, a vegetatively propagated crop, is affected by various diseases. Red rot caused by the fungal pathogen Colletotrichum falcatum Went is a devastating disease of sugarcane causing serious yield losses in India and in many other countries. Understanding the pathogenesis of this hemibiotrophic fungal pathogen within host tissue will deepen our knowledge on host–pathogen interaction and helps to develop effective disease management strategies. Green fluorescent protein (GFP) is an effective tool for exploring fungal biology during host–pathogen interactions. In the present study we have conducted detailed investigations using GFP transformed strain of C. falcatum to establish the pathogenesis, colonization and spread of this fungus inside host tissues. We transformed C. falcatum with eGFP using Agrobacterium-mediated transformation using hygromycin B as a selectable marker and the transgene was stably integrated with mitotic stability. The C. falcatum transformants maintained morphological characters and growth parameters as like the wild type and the virulence nature was not altered as compared to the wild-type C. falcatum. GFP-tagged C. falcatum pathotypes clearly demonstrated the variations in C. falcatum colonization during compatible and incompatible interactions with sugarcane. These variations were comparable to in planta studies conducted using non GFP-tagged C. falcatum and using calcofluor staining. This was further validated with histological assays with calcofluor staining. The study is first of its kind in sugarcane and C. falcatum and will give a better understanding of pathogenesis inside the host in a spatio-temporal manner.

Effects of Homeopathic Phosphorus on Encephalitozoon cuniculi-Infected Macrophages In-Vitro.

Encephalitozoon cuniculi (E. cuniculi), a fungus that acts as an intracellular pathogen, causes a marked neurological syndrome in many host species and is a zoonotic concern. Although no well-established treatment for this syndrome is known, previous successful clinical experience using homeopathic phosphorus has been described in which symptom remission with no mortality occurred in 40/42 animals by means of unknown immunological mechanisms. The latter observation was the main motivation for this study. To verify, in an in-vitro model, if macrophages infected with E. cuniculi can change in function after treatment with different potencies of phosphorus. RAW 264.7 macrophages were infected with E. cuniculi in-vitro and treated with various homeopathic potencies of phosphorus. The vehicle was used as a control solution (0.06% succussed ethanol). After 1 and 24 hours, the following parameters were analyzed: parasite internalization (by the Calcofluor staining method), lysosome activity (by the acridine orange method), cytokine/chemokine production (by the MAGPIX system), and cell ultrastructure. Automatic image analysis was used when applicable, and the experiments were performed in triplicate. Treatment with vehicle alone increased interleukin (IL)-6, tumor necrosis factor alpha and monocyte chemotactic protein -1 production (p ≤ 0.05) and reduced the number of internalized parasites (p ≤ 0.001). A progressive and time-dependent increase in RANTES (regulated on activation, normal T-cell expressed and secreted) and lysosome activity (p ≤ 0.002) was observed only after treatment with the highest potency of phosphorus (Phos 200cH), together with decreased apoptosis rate, intense parasite digestion, and the presence of non-internalized spores. Phos 200 cH has a modulatory action on the activity of infected macrophages, especially a specific increase in RANTES, a key element in the prognosis of E. cuniculi-infected and of immunosuppressed patients bearing infections.