Endophytic yeast diversity in leaf tissue of rice,corn and sugarcane cultivated in Thailand assessed by a culture-dependent approach

Endophytic yeasts are yeast that can colonize healthy plant tissues without causing any damage to the host plant. This work aimed to explore the diversity of endophytic yeasts in leaf tissue of main agricultural crops (rice, corn and sugarcane) in Thailand, by a culture-dependent approach. A total of 311 leaf samples, consisting of rice (n = 100), corn (n = 109) and sugarcane (n = 102). From the tissue of rice (n = 92), corn (n = 76) and sugarcane (n = 78) leaf samples, 117, 118 and 123 yeast strains were respectively isolated and identified based on the D1/D2 region of the large subunit (LSU) rRNA gene sequence analysis to be yeast species in both the phyla Basidiomycota and Ascomycota. Higher numbers of basidiomycetous yeast than ascomycetous yeast were detected in the leaf tissue of the three crops. Pseudozyma (Dirkmeia) churashimaensis (Ustilaginales) was the most prevalent yeast species in the rice and corn leaves with relative frequencies (RF) of 35.9 % and 17.8 %, respectively. Whereas the predominant species in the sugarcane leaves was Meyerozyma caribbica (Saccharomycetales) with an RF of 14.6 %. In addition, six new yeast species and one new yeast genus were proposed. Our findings suggest that these plant species are good sources from which new yeast species may be isolated.     

Manganese superoxide dismutase in pathogenic fungi: An issue with pathophysiological and phylogenetic involvements

Manganese-containing superoxide dismutases (MnSODs) are ubiquitous metalloenzymes involved in cell defence against endogenous and exogenous reactive oxygen species. In fungi, using this essential enzyme for phylogenetic analysis of Pneumocystis and Ganoderma genera, and of species selected among Ascomycota, Basidiomycota and Zygomycota, provided interesting results in taxonomy and evolution. The role of mitochondrial and cytosolic MnSODs was explored in some pathogenic Basidiomycota yeasts (Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. gattii, Malassezia sympodialis), Ascomycota filamentous fungi (Aspergillus fumigatus), and Ascomycota yeasts (Candida albicans). MnSOD-based phylogenetic and pathogenic data are confronted in order to evaluate the roles of fungal MnSODs in pathophysiological mechanisms.     

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast’ polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast’ polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes.     

Effect of synthetic surfactants and soapwort (Saponaria officinalis L.) extract on skin-mimetic model lipid monolayers

The effect of a saponin-rich extract from rhizomes of Soapwort (Saponaria officinalis L) and four synthetic surfactants: sodium lauryl sulphate (SLS), sodium laureth sulphate (SLES), ammonium lauryl sulphate (ALS) and cocamidopropyl betaine (CAPB) on two model lipid monolayers is analyzed using surface pressure, surface dilatational rheology and fluorescence microscopy. The following monolayers were employed: dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3 (DPPC/CHOL) and Ceramide [AP]/stearic acid/cholesterol in a molar ratio of 14:14:10 (CER/SA/CHOL). They mimicked a general bilayer structure and an intercellular lipid mixture, respectively. Both lipid mixtures on Milli-Q water were first compressed to the initial surface pressure, Π₀ = 30 mN/m and then the subphase was exchanged with the respective (bio)surfactant solution at 1% (w/w). All four synthetic surfactants behaved in a similar way: they increased surface pressure to about 40 mN/m and reduced the storage modulus of surface dilational surface rheology, E′, to the values close to zero. The corresponding fluorescence microscopy pictures confirmed that the lipids mimicking the stratum corneum components were almost completely removed by the synthetic surfactants under the present experimental conditions. The components of the Soapwort extract (SAP) increased surface pressure to significantly higher values than the synthetic surfactants, but even more spectacular increase was observed for the storage modulus of the SAP-penetrated lipid monolayers (up to E′= 715 mN/m).     

Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi

A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-β-d-glucopyranosyl-d-gluconic acid: phosphate α-d-glucosyltransferase as the systematic name and cellobionic acid phosphorylase as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into α-d-glucose 1-phosphate and d-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively.     

Microbial hitchhikers on intercontinental dust: catching a lift in Chad

Ancient mariners knew that dust whipped up from deserts by strong winds travelled long distances, including over oceans. Satellite remote sensing revealed major dust sources across the Sahara. Indeed, the Bodélé Depression in the Republic of Chad has been called the dustiest place on earth. We analysed desert sand from various locations in Chad and dust that had blown to the Cape Verde Islands. High throughput sequencing techniques combined with classical microbiological methods showed that the samples contained a large variety of microbes well adapted to the harsh desert conditions. The most abundant bacterial groupings in four different phyla included: (a) Firmicutes—Bacillaceae, (b) Actinobacteria—Geodermatophilaceae, Nocardiodaceae and Solirubrobacteraceae, (c) Proteobacteria—Oxalobacteraceae, Rhizobiales and Sphingomonadaceae, and (d) Bacteroidetes—Cytophagaceae. Ascomycota was the overwhelmingly dominant fungal group followed by Basidiomycota and traces of Chytridiomycota, Microsporidia and Glomeromycota. Two freshwater algae (Trebouxiophyceae) were isolated. Most predominant taxa are widely distributed land inhabitants that are common in soil and on the surfaces of plants. Examples include Bradyrhizobium spp. that nodulate and fix nitrogen in Acacia species, the predominant trees of the Sahara as well as Herbaspirillum (Oxalobacteraceae), a group of chemoorganotrophic free-living soil inhabitants that fix nitrogen in association with Gramineae roots. Few pathogenic strains were found, suggesting that African dust is not a large threat to public health.     

Pertactin-deficient Bordetella pertussis isolates in Poland—a country with whole-cell pertussis primary vaccination

The introduction of pertussis vaccination in the 1950s resulted in a significant decrease in the incidence of disease. However, since the 1990s many highly vaccinated countries have observed the re-emergence of the disease. One of the causes of this phenomenon might be related to the adaptation of Bordetella pertussis to vaccination. The purpose of the presented study was an investigation of the emergence and spread of vaccine antigen-deficient B. pertussis isolates in Poland and genomic characterization of the currently circulating pathogen population using PFGE, MLVA and MAST. The results revealed that all tested isolates expressed Ptx, FHA and ACT antigens but 15.4% (4/26) of isolates from 2010 to 2016 were Prn-deficient. Moreover, one TcfA-deficient isolate was collected in 2015. The genotyping showed a genetic distinction between the isolates circulating in 2010–2016 and isolates from previous periods. The majority of currently circulating isolates belonged to PFGE group IV (96.2%), type MT27 (73.1%), and carried ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-1 alleles (61.5%). The unique genetic structure of the B. pertussis population in Poland has changed since 2010 and became similar to that observed in countries with aP vaccination. This could be a result of increasing use of aP vaccines (60% of primary vaccination in 2013) over wP vaccines, which have been broadly used for primary vaccination in Poland for decades.     

Interferon Induced IFIT Family Genes in Host Antiviral Defense

Secretion of interferons (IFNs) from virus-infected cells is a hallmark of host antiviral immunity and in fact, IFNs exert their antiviral activities through the induction of antiviral proteins. The IFN-induced protein with tetratricopeptide repeats (IFITs) family is among hundreds of IFN-stimulated genes. This family contains a cluster of duplicated loci. Most mammals have IFIT1, IFIT2, IFIT3 and IFIT5; however, bird, marsupial, frog and fish have only IFIT5. Regardless of species, IFIT5 is always adjacent to SLC16A12. IFIT family genes are predominantly induced by type I and type III interferons and are regulated by the pattern recognition and the JAK-STAT signaling pathway. IFIT family proteins are involved in many processes in response to viral infection. However, some viruses can escape the antiviral functions of the IFIT family by suppressing IFIT family genes expression or methylation of 5'' cap of viral molecules. In addition, the variants of IFIT family genes could significantly influence the outcome of hepatitis C virus (HCV) therapy. We believe that our current review provides a comprehensive picture for the community to understand the structure and function of IFIT family genes in response to pathogens in human, as well as in animals.     

ClTI,a Kunitz trypsin inhibitor purified from Cassia leiandra Benth. seeds,exerts a candidicidal effect on Candida albicans by inducing oxidative stress and necrosis

Cassia leiandra is an Amazonian plant species that is used popularly for the treatment of mycoses. Recently, a protease inhibitor, named ClTI, with insecticidal activity against Aedes aegypti, was purified from the mature seeds of C. leiandra. In this work, we show that ClTI has antifungal activity against Candida species and describe its mode of action towards Candida albicans. This study is relevant because the nosocomial infections caused by Candida species are a global public health problem that, together with the growing resistance to current drugs, has increased the urgency of the search for new antifungal compounds. ClTI inhibited the growth of Candida albicans, Candida tropicalis, Candida parapsilosis, and Candida krusei. However, ClTI was more potent against C. albicans. The candidicidal mode of action of ClTI on C. albicans involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+ -ATPase), induction of oxidative stress, and DNA damage. ClTI also exhibited antibiofilm activity and non-cytotoxicity to mammalian cells. These results indicate that ClTI is a promising candidate for the future development of a new, natural, and safe agent for the treatment of infections caused by C. albicans.     

Detection of a root-associated group of Hyaloscyphaceae (Helotiales) species that commonly colonizes Fagaceae roots and description of three new species in genus Glutinomyces

We used a culture-dependent approach to investigate root-associated fungal communities in Fagaceae roots at four fagaceous species-dominant forests in Japan. In total 1029 isolates were collected and classified, based on colony morphological features and molecular information. Species of order Helotiales (Ascomycota) were dominantly isolated at all four sites, in which a globally-distributed putative endophytic group in Hyaloscyphaceae predominated. This group of fungi was morphologically and phylogenetically investigated using these isolates as well as additional isolates collected from 8 different sites in Japan. Among the Hyaloscyphaceae, Glutinomyces species were frequently detected, and three novel species, G. inflatus, G. vulgaris, and G. takaragaikensis spp. nov. were identified and described according to their morphology and genealogical concordance.     

Host age determines parasite load of Laboulbeniales fungi infecting ants: Implications for host-parasite relationship and fungal life history

Arthropod-parasitic fungi of the order Laboulbeniales are known to exhibit specialization to individual host taxa in most cases. Some species exhibit ecological specificity to multiple, often unrelated hosts in certain microhabitats; and often position specificity to different host body parts. The myrmecophilous Rickia wasmannii (Ascomycota: Laboulbeniales) infects Myrmica species (Hymenoptera: Formicidae) (host specificity), and occasionally other arthropod inquilines inside the ant nest (ecological specificity). An effect of the position of infection on the thallus densities has also been reported. Another determinative factor that may also exist in the Rickia-Myrmica host-parasite system, the chronological age of ant worker hosts, has also been linked to parasite load. Comprehensive studies on the age-related infection intensity, however, are still lacking. Here we investigated whether the level of infection correlates with the age of the M. scabrinodis host consistently. We found that older hosts exhibited higher parasite load, even though the infection level of the different colonies varied widely. The results highlight that the level of R. wasmannii infections are strongly influenced by host individual and host colony factors.     

Comparison of the SuperARMS and Droplet Digital PCR for Detecting EGFR Mutation in ctDNA From NSCLC Patients

BACKGROUND: Liquid biopsy is emerging as an important approach for tumor genotyping in non-small cell lung cancer, ddPCR and SuperARMS are both methods with high sensitivity and specificity for detecting EGFR mutation in plasma. We aimed to compare ddPCR and SuperARMS to detect plasma EGFR status in a cohort of advanced NSCLC patients. METHOD: A total of 79 tumor tissues and paired plasma samples were collected. The EGFR mutation status in tissue was tested by ADx-ARMS, matched plasma was detected by ddPCR and SuperARMS, respectively. RESULTS: The EGFR mutation rates were identified as 64.6% (tissue, ARMS), 55.7% (plasma, ddPCR), and 49.4% (plasma, Super ARMS), respectively. The sensitivity of ddPCR was similar with Super-ARMS in plasma EGFR detection (80.4% vs 76.5%), as well as the specificity (89.3% vs 100%). And the McNemar’s test showed there was no significant difference (P = .125). The concordance rate between SuperARMS and ddPCR was 91.1%. A significant interaction was observed between cfDNA EGFR mutation status and EGFR-TKIs treatment tested by both methods. CONCLUSION: Super-ARMS and ddPCR share the similar accuracy for EGFR mutation detection in plasma biopsy; both methods predicted well the efficacy of EGFR-TKIs by detecting plasma EGFR status.     

Chemical analysis of the female sex pheromone in Palpita nigropunctalis (Lepidoptera: Crambidae)

The lilac pyralid, Palpita nigropunctalis Bremer (Lepidoptera: Crambidae), is a common pest of Oleaceae plants. A crude extract of the female sex pheromone glands was examined by gas chromatography-electroantennogram detection (GC-EAD) and GC coupled to a mass spectrometer (GC/MS). The GC-EAD analysis revealed three EAG-active components (I–III) in a ratio of 1:0.2:0.01 (I: II: III). GC/MS analysis successfully recorded the mass spectra of I and II. For I, ions at m/z 238 (M⁺) and 220 ([M-18]⁺) indicated the structure of a monoenyl aldehyde with a 16-carbon chain. For II, M⁺ was not detected, but ions at m/z 222 ([M-60]⁺) and 61 ([AcOH+1]⁺) suggested that II was a monoenyl acetate with a 16-carbon chain. Further GC/MS analysis of the extract treated with dimethyl disulfide revealed that the double bonds in both I and II are located at the same position of 11th-carbon. In addition, the pheromone extract was examined by GC/Fourier transform-infrared spectrophotometer (GC/FT-IR). An IR spectrum of I showed characteristic absorption at 1716 and 966?cm^?1, indicating a formyl group and E configuration of the double bond, respectively. In the case of II, absorption at 1745 and 968?cm^?1 indicated an ester carbonyl and E configuration, respectively. Taken together and by comparison with authentic standards, I and II were confirmed as (E)-11-hexadecenal and (E)-11-hexadecenyl acetate, respectively; while III was speculated as (E)-11-hexadecen-1-ol. The synthetic I, II and III all coincided well with those of the natural components in chemical data, and elicited strong electroantennographic activity in male P. nigropunctalis.     

Design,synthesis, and evaluation of novel inhibitors for wild-type human serine racemase

Most of the endogenous free d-serine (about 90%) in the brain is produced by serine racemase (SR). d-Serine in the brain is involved in neurodegenerative disorders and epileptic states as an endogenous co-agonist of the NMDA-type glutamate receptor. Thus, SR inhibitors are expected to be novel therapeutic candidates for the treatment of these disorders. In this study, we solved the crystal structure of wild-type SR, and tried to identify a new inhibitor of SR by in silico screening using the structural information. As a result, we identified two hit compounds by their in vitro evaluations using wild-type SR.Based on the structure of the more potent hit compound 1, we synthesized 15 derivatives and evaluated their inhibitory activities against wild-type SR. Among them, the compound 9C showed relatively high inhibitory potency for wild-type SR. Compound 9C was a more potent inhibitor than compound 24, which was synthesized by our group based upon the structural information of the mutant-type SR.     

Triterpenoids from ten Lithocarpus species of Hong Kong

From the petrol extracts of the leaves and stems of ten Lithocarpus species (L. attenuata, L. cornea, L. elizabethae, L. glabra, L. haipinii, L. hancei, L. harlandi, L. irwinii, L. litchioides, and L. polystachya) of the Fagaceae family, were isolated 23 different triterpenoids, and sitosterol and stigmasterol. Of the triterpenoids, 11 belonged to the oleanane and rearranged oleanane group [β-amyrin, friedelin, friedelan-3β-ol, glutinol, taraxerone, taraxerol and its acetate, canophyllol (28-hydroxyfriedelan-3-one), friedelan-2,3-dione (3-hydroxyfriedel-3-en-2-one), pachysandiol-A (2α,3β-dihydroxyfriedelane) and a new compound lithocarpic lactone C₃₀H₅₀O₂]. Four compounds were from the lupane and rearranged lupane group (lupenone, lupeol, betulin and taraxasterol), 2 from the hopane group (22-hydroxyhopan-3-one and 3β,22-dihydroxyhopane), and 6 were probably new compounds.     

Subunits of the vacuolar H+-ATPase complex,Vma4 and Vma10, are essential for virulence and represent potential drug targets in Candida albicans

Hyphal morphogenesis of Candida albicans is important for its pathogenesis. Here, we showed that the filamentous growth of C. albicans requires vacuolar H⁺-ATPase function. Results showed that levels of Vma4 and Vma10 increased in cells undergoing hyphal growth compared to those undergoing yeast growth. Deleting VMA4 or VMA10 abolished vacuolar functions and hyphal morphogenesis. These deletion mutants were also characterized as avirulent in a mouse model of systemic infection. Furthermore, VMA4 and VMA10 deletion strains showed hypersensitivity to fluconazole, terbinafine, and amphotericin B. Based on these findings, Vma4 and Vma10 are not only involved in vacuole biogenesis and hyphal formation, but also are good targets for antifungal drug development in C. albicans.     

Identifying airborne fungi in Seoul,Korea using metagenomics

Fungal spores are widespread and common in the atmosphere. In this study, we use a metagenomic approach to study the fungal diversity in six total air samples collected from April to May 2012 in Seoul, Korea. This springtime period is important in Korea because of the peak in fungal spore concentration and Asian dust storms, although the year of this study (2012) was unique in that were no major Asian dust events. Clustering sequences for operational taxonomic unit (OTU) identification recovered 1,266 unique OTUs in the combined dataset, with between 223?96 OTUs present in individual samples. OTUs from three fungal phyla were identified. For Ascomycota, Davidiella (anamorph: Cladosporium) was the most common genus in all samples, often accounting for more than 50% of all sequences in a sample. Other common Ascomycota genera identified were Alternaria, Didymella, Khuskia, Geosmitha, Penicillium, and Aspergillus. While several Basidiomycota genera were observed, Chytridiomycota OTUs were only present in one sample. Consistency was observed within sampling days, but there was a large shift in species composition from Ascomycota dominant to Basidiomycota dominant in the middle of the sampling period. This marked change may have been caused by meteorological events. A potential set of 40 allergyinducing genera were identified, accounting for a large proportion of the diversity present (22.5?7.2%). Our study identifies high fungal diversity and potentially high levels of fungal allergens in springtime air of Korea, and provides a good baseline for future comparisons with Asian dust storms.     

Diverse Mesorhizobium bacteria nodulate native Astragalus and Oxytropis in arctic and subarctic areas in Eurasia

Rhizobia nodulating native Astragalus and Oxytropis spp. in Northern Europe are not well-studied. In this study, we isolated bacteria from nodules of four Astragalus spp. and two Oxytropis spp. from the arctic and subarctic regions of Sweden and Russia. The phylogenetic analyses were performed by using sequences of three housekeeping genes (16S rRNA, rpoB and recA) and two accessory genes (nodC and nifH). The results of our multilocus sequence analysis (MLSA) of the three housekeeping genes tree showed that all the 13 isolates belonged to the genus Mesorhizobium and were positioned in six clades. Our concatenated housekeeping gene tree also suggested that the isolates nodulating Astragalus inopinatus, Astragalus frigidus, Astragalus alpinus ssp. alpinus and Oxytropis revoluta might be designated as four new Mesorhizobium species. The 13 isolates were grouped in three clades in the nodC and nifH trees. ¹⁵N analysis suggested that the legumes in association with these isolates were actively fixing nitrogen.     

Distinct Patterns of Stromal and Tumor Expression of ROR1 and ROR2 in Histological Subtypes of Epithelial Ovarian Cancer

OBJECTIVE: The ROR1 and ROR2 receptor tyrosine kinases have both been implicated in ovarian cancer progression and have been shown to drive migration and invasion. There is an increasing importance of the role of stroma in ovarian cancer metastasis; however, neither ROR1 nor ROR2 expression in tumor or stromal cells has been analyzed in the same clinical cohort. AIM: To determine ROR1 and ROR2 expression in ovarian cancer and surrounding microenvironment and examine associations with clinicopathological characteristics. METHODS: Immunohistochemistry for ROR1 and ROR2 was used to assess receptor expression in a cohort of epithelial ovarian cancer patients (n = 178). Results were analyzed in relation to clinical and histopathological characteristics and survival. Matched patient sample case studies of normal, primary, and metastatic lesions were used to examine ROR expression in relation to ovarian cancer progression. RESULTS: ROR1 and ROR2 are abnormally expressed in malignant ovarian epithelium and stroma. Higher ROR2 tumor expression was found in early-stage, low-grade endometrioid carcinomas. ROR2 stromal expression was highest in the serous subtype. In matched patient case studies, metastatic samples had higher expression of ROR2 in the stroma, and a recurrent sample had the highest expression of ROR2 in both tumor and stroma. CONCLUSION: ROR1 and ROR2 are expressed in tumor-associated stroma in all histological subtypes of ovarian cancer and hold potential as therapeutic targets which may disrupt tumor and stroma interactions.