Miniaturized Cultivation of Microbiota for Antimalarial Drug Discovery.

The ongoing search for effective antiplasmodial agents remains essential in the fight against malaria worldwide. Emerging parasitic drug resistance places an urgent need to explore chemotherapies with novel structures and mechanisms of action. Natural products have historically provided effective antimalarial drug scaffolds. In an effort to search nature's chemical potential for antiplasmodial agents, unconventionally sourced organisms coupled with innovative cultivation techniques were utilized. Approximately 60,000 niche microbes from various habitats (slow-growing terrestrial fungi, Antarctic microbes, and mangrove endophytes) were cultivated on a small-scale, extracted, and used in high-throughput screening to determine antimalarial activity. About 1% of crude extracts were considered active and 6% partially active (≥ 67% inhibition at 5 and 50 µg/mL, respectively). Active extracts (685) were cultivated on a large-scale, fractionated, and screened for both antimalarial activity and cytotoxicity. High interest fractions (397) with an IC50 < 1.11 µg/mL were identified and subjected to chromatographic separation for compound characterization and dereplication. Identifying active compounds with nanomolar antimalarial activity coupled with a selectivity index tenfold higher was accomplished with two of the 52 compounds isolated. This microscale, high-throughput screening project for antiplasmodial agents is discussed in the context of current natural product drug discovery efforts.

Screening mangrove endophytic fungi for antimalarial natural products.

We conducted a screening campaign to investigate fungi as a source for new antimalarial compounds. A subset of our fungal collection comprising Chinese mangrove endophytes provided over 5000 lipophilic extracts. We developed an accelerated discovery program based on small-scale cultivation for crude extract screening and a high-throughput malaria assay. Criteria for hits were developed and high priority hits were subjected to scale-up cultivation. Extracts from large scale cultivation were fractionated and these fractions subjected to both in vitro malaria and cytotoxicity screening. Criteria for advancing fractions to purification were developed, including the introduction of a selectivity index and by dereplication of known metabolites. From the Chinese mangrove endophytes, four new compounds (14-16, 18) were isolated including a new dimeric tetrahydroxanthone, dicerandrol D (14), which was found to display the most favorable bioactivity profile.

Differences in leaf construction cost between alien and native mangrove species in Futian, Shenzhen, China: implications for invasiveness of alien species.

Construction cost (CC) is a quantifiable measure of energy demand for biomass production, and low CC is hypothesized to give an alien plant growth advantages and increase its potential to be an invader. Comparison of leaf CC and growth traits between alien and native mangroves in Shenzhen Futian Nature Reserve showed CC per unit mass (CC(mass)), carbon concentration and gross and ash-free caloric values of alien mangroves were significantly lower than those of native species, while the height and chest circumference were just the opposite. Alien species Sonneratia apetala had the lowest CC(mass) while Sonneratia caseolaris had the lowest CC(area), and were 8.99% and 32.17% lower than those of native species, respectively. Conversely, specific leaf area (SLA) of these two Sonneratia species was significantly higher than native species. Lower CC and higher SLA make the two Sonneratia species grow and spread faster than other mangroves and enhance their invasive potential.

Comparison of initial hydrolysis of the three dimethyl phthalate esters (DMPEs) by a basidiomycetous yeast, Trichosporon DMI-5-1, from coastal sediment.

PURPOSE: Dimethyl phthalate esters (DMPEs) are a group of plasticizers commonly detected in the environment with potential adverse human health impact. The degradation of DMPEs by fungal systems has been studied to a limited extent, particularly by yeasts. In this study, a basidiomycetous yeast Trichosporon DMI-5-1 capable of degrading DMPEs was obtained and the degradation pathways were investigated. RESULTS: A DMPE-degrading yeast was isolated from costal sediment by enrichment culture technique and was identified as Trichosporon sp. DMI-5-1 based on microscopic morphology and 18S rDNA sequence. Comparative investigations on biodegradation of three isomers of DMPEs, namely dimethyl phthalate (DMP), dimethyl isophthalate (DMI), and dimethyl terephthalate (DMT), were carried out with this yeast strain. Trichosporon sp. DMI-5-1 could not mineralize DMPEs completely but transform them to respective monomethyl phthalate or phthalic acid. Biochemical degradation pathways for the three DMPE isomers by Trichosporon sp. DMI-5-1 were apparently different. The yeast carried out one-step ester hydrolysis of DMP and DMI to respective monoesters (monomethyl phthalate and monomethyl isophthalate, respectively) and no further metabolism of these two monoesters. Meanwhile, DMT was transformed by the yeast to monomethyl terephthalate and subsequently to terephthalic acid by stepwise hydrolysis of the two ester bonds. CONCLUSIONS: This study shows that different catalytic processes are involved in the transformation of DMPEs by the basidiomycetous yeast Trichosporon sp. DMI-5-1 and suggests that its esterases, responsible for the initial hydrolyzing the two ester bonds of DMPEs, are highly substrate specific.

Remispora spitsbergenensis sp. nov., a marine lignicolous ascomycete from Svalbard, Norway.

Abstract: Remispora was established for R. maritima, a fungus with globose/subglobose, lightly colored and coriaceous ascomata; deliquescing asci; ellipsoidal ascospores; and bipolar, pleomorphic ascospore appendages. Seven species currently are included in Remispora: R crispa, R. galerita, R maritima, R. minuta, R. pilleata, R. quadriremis and R stellata. Variations on ascospore appendages can be observed in Remispora. In general the appendage is exosporic in nature and comprises an amorphous, electron-transparent matrix, and a fibrous, electron-dense component. An eighth Remispora species, R. spitsbergenensis sp. nov., is described here, discovered from washed-up wood collected at the shore of Longyearbyen, Svalbard, Norway. Ascospore appendages of R. spitsbergenensis appear as fibrous strands and amorphic material under the scanning electron microscope, which are characteristic of a Remispora species. Remispora spitsbergenensis resembles R. quadriremis and R. stellata because all possess four or more ascospore appendages at one end. Remispora spitsbergenensis possesses consistently four polar appendages at each end in contrast to six in R. stellata. Also ascospore appendages of R. spitsbergenensis are ribbon-like, compared with the obclavate, curved and attenuate appendages in R. quadriremis and R. stellata. A key for the identification of the eight Remispora species is provided.

Havispora longyearbyenensis gen. et sp. nov.: an arctic marine fungus from Svalbard, Norway.

Information on the diversity and ecology of arctic marine fungi is lacking. During a short visit to Longyearbyen (78 degrees 13'N 15 degrees 33'E), Svalbard, Norway, a new marine fungus growing on driftwood collected at the shore was encountered. This taxon belongs to the Halosphaeriales (Ascomycota), a fungal order of mostly marine species. Havispora longyearbyenensis gen. et sp. nov. is morphologically similar to Nautosphaeria and Nereiospora, all with tufts of appendages at polar and equatorial positions of the ascospore but differing in color and septation of the ascospore and morphology and ontogeny of the ascospore appendage.

A new hTopo I isomerase inhibitor produced by a mangrove endophytic fungus no. 2240.

A new hTopo I isomerase inhibitor, (+)-3,3',7,7',8,8'-hexahydroxy-5,5'-dimethylbianthraquinone (2240A), was isolated from the mangrove endophytic fungus no. 2240 collected from an estuarine mangrove at the South China Sea coast. Its structure was elucidated by spectral analyses including two-dimensional NMR, HR-EI-MS, IR, and UV. The hTopo I isomerase inhibition experiment showed that 2240A (1) possessed strong inhibiting activity. When its inhibition concentration was 4.65 micromol/l, its percent inhibition rate was 59.1%, while the lowest inhibition concentration of the positive control camptothecin was 1.00 x 10(3) micromol/l.

Effect of culture conditions on antifouling compound production of a sponge-associated fungus.

Microorganisms associated with invertebrate hosts have long been suggested to be a source for bioactive metabolites. In this study, we reported that a sponge-associated fungus, Letendraea helminthicola, produced two antifouling compounds: 3-methyl-N-(2-phenylethyl) butanamide and cyclo(D-Pro-D-Phe). To optimize the production of these antifouling compounds, we then examined the production of compounds under different culture conditions (temperature, salinity, pH, and carbon and nitrogen sources). This fungus grew well and produced more compounds at temperatures between 18 and 30 degrees C; the fungus grew well at 75 parts per thousand (ppt) salinity but produced the highest amount of antifouling compounds at 30 and 45 ppt. The optimal initial pH value for mycelial growth was 5.5 to 6.5, whereas the production of the antifouling compounds was maximized at pH 3.5 and 4.5. Glucose and xylose (as carbon sources) increased the production of antifouling compounds. Yeast extract and peptone (as nitrogen sources) maximized the production of mycelial biomass and antifouling compounds. Our results indicate that culture conditions greatly affect the production of bioactive compounds from mycelial fungal cultures as exemplified by strain L. helminthicola and that the conditions favorable for fungal growth may not be the best conditions for bioactive compound production.

Dioxiranes generated in situ from pyruvates and oxone as environmentally friendly oxidizing agents for disinfection.

Dioxiranes generated in situ from pyruvates (alpha-keto esters) and Oxone have been found to be environmentally friendly oxidizing agents for disinfection. These oxidizing agents were highly effective for destruction of various strains of bacteria, fungi, and bacterial endospores in a wide temperature range with exceptional stability. Notably, by using an aqueous solution of methyl pyruvate (1a) and Oxone/NaHCO3, complete destruction of bacteria such as Staphylococcus aureus and fungus Penicillium corylophilum was achieved within 5 min at 20 degrees C at neutral pH. Highly chemical-resistant bacterial endospores of Bacillus cereus could also be destroyed. The high antibacterial activity of 1a could be attributed to its strong electron-withdrawing alpha-ester group.

Survey of coastal mangrove fungi for xylanase production and optimized culture and assay conditions.

Xylanase activity was detected among 34 of 77 fungal isolates derived from decaying wood, debris and soil samples collected in coastal mangrove environment of Hong Kong. Of those, three isolates CY2809 (Staganospora sp.), CY4786 and CY5040 were chosen for comparison of xylanase production in parallel to HU5048 (Aspergillus awamori), a terrestrial, highly productive isolate. Based on the assessment of mycelial biomass, xylanase activity and content of xylose-equivalent reducing sugars in their liquid cultures, the isolate CY4786 was best for xylanase production in a basal medium containing birchwood xylan (10.0 g/L) as a sole carbon source, yeast extract (2.5 g/L) and sea salts (15.0 g/L) with initial pH 7.8. When assayed at the optimized regime of 50 degrees C and pH 4.6, the activity of xylanase produced by CY4786 in 7d liquid culture at 25 degrees C reached 1.07 x 10(4) unit/mL. The results indicate that the mangrove fungi act as hemicellulose decomposers in the mangrove environment where highly xylanase-productive isolates can be searched for exploitation. A discussion is given on the possible use of the content of xylose-equivalent reducing sugars as an index to simplify conventional xylanase activity assay method for fungal isolate survey.