COX Inhibitory and Cytotoxic Naphthoketal-Bearing Polyketides from Sparticola junci.

Axenic fermentation on solid rice of the saprobic fungus Sparticola junci afforded two new highly oxidized naphthalenoid polyketide derivatives, sparticatechol A (1) and sparticolin H (2) along with sparticolin A (3). The structures of 1 and 2 were elucidated on the basis of their NMR and HR-ESIMS spectroscopic data. Assignment of absolute configurations was performed using electronic circular dichroism (ECD) experiments and Time-Dependent Density Functional Theory (TDDFT) calculations. Compounds 1-3 were evaluated for COX inhibitory, antiproliferative, cytotoxic and antimicrobial activities. Compounds 1 and 2 exhibited strong inhibitory activities against COX-1 and COX-2. Molecular docking analysis of 1 conferred favorable binding against COX-2. Sparticolin H (2) and A (3) showed a moderate antiproliferative effect against myelogenous leukemia K-562 cells and weak cytotoxicity against HeLa and mouse fibroblast cells.

A review of in vitro attempts to develop the axenic culture of Treponema pallidum and genomics-based suggestions to achieve this elusive goal.

To date, the axenic culture of Treponema pallidum remains a challenge in the field of microbiology despite countless attempts. Here, we conducted a comprehensive bibliographic analysis using several databases and search engines, namely Pubmed, Google scholar, Google, Web of Science and Scopus. Numerous unsuccessful empiric studies have been conducted and evaluated using as criteria dark-field microscopic observation of motile spiral shaped cells in the culture and virulence of the culture through rabbit infectivity. All of these studies failed to induce rabbit infectivity, even when deemed positive after microscopic observation leading to the misnomer of avirulent T. pallidum. In fact, this criterion was improperly chosen because not all spiral shaped cells are T. pallidum. However, these studies led to the formulation of culture media particularly favourable to the growth of several species of Treponema, including Oral Microbiology and Immunology, Zürich medium (OMIZ), Oral Treponeme Enrichment Broth (OTEB) and T-Raoult, thus allowing the increase in the number of cultivable strains of Treponema. The predicted metabolic capacities of T. pallidum show limited metabolism, also exhibited by other non-cultured and pathogenic Treponema species, in contrast to cultured Treponema species. The advent of next generation sequencing represents a turning point in this field, as the knowledge inferred from the genome can finally lead to the axenic culture of T. pallidum.

Make microalgal cultures axenic again - a fast and simple workflow utilizing fluorescence-activated cell sorting.

Since the removal of contaminations in microalgal cultures is extremely laborious and time-consuming, we developed a rapid workflow to obtain axenicity by a combination of fluorescence-activated cell sorting (FACS) and plate spreading. During method development, several cyanobacteria and green algae strains were successfully made axenic. At the end, method transferability to another FACS device was demonstrated. Our workflow offers great time-savings with less hands-on laboratory work compared to conventional isolation techniques.

Establish axenic cultures of armored and unarmored marine dinoflagellate species using density separation, antibacterial treatments and stepwise dilution selection.

Academic research on dinoflagellate, the primary causative agent of harmful algal blooms (HABs), is often hindered by the coexistence with bacteria in laboratory cultures. The development of axenic dinoflagellate cultures is challenging and no universally accepted method suit for different algal species. In this study, we demonstrated a promising approach combined density gradient centrifugation, antibiotic treatment, and serial dilution to generate axenic cultures of Karenia mikimotoi (KMHK). Density gradient centrifugation and antibiotic treatments reduced the bacterial population from 5.79 ± 0.22 log10 CFU/mL to 1.13 ± 0.07 log10 CFU/mL. The treated KMHK cells were rendered axenic through serial dilution, and algal cells in different dilutions with the absence of unculturable bacteria were isolated. Axenicity was verified through bacterial (16S) and fungal internal transcribed spacer (ITS) sequencing and DAPI epifluorescence microscopy. Axenic KMHK culture regrew from 1000 to 9408 cells/mL in 7 days, comparable with a normal culture. The established methodology was validated with other dinoflagellate, Alexandrium tamarense (AT6) and successfully obtained the axenic culture. The axenic status of both cultures was maintained more than 30 generations without antibiotics. This efficient, straightforward and inexpensive approach suits for both armored and unarmored dinoflagellate species.

Influence of sequential inoculum of Starmerella bacillaris and Saccharomyces cerevisiae on flavonoid composition of monovarietal Sangiovese wines.

The selection of Starmerella bacillaris strains to be used with Saccharomyces cerevisiae as mixed cultures has been recently suggested in order to produce wines containing lower ethanol and higher glycerol concentrations and to promote fructose degradation due to their fructophilic character. However, studies about effects of such mixed starter cultures on phenolic compounds, which are responsible for the colour and health-enhancing properties in red wines, are currently lacking. Therefore, in this work, the influence of sequential inoculated fermentation (SIF) with Starm. bacillaris and S. cerevisiae on phenolic content of monovarietal Sangiovese wine was evaluated by fermentations at laboratory scale. Axenic fermentations (AXFs) with S. cerevisiae were performed as control. S. cerevisiae attained higher cell densities in AXF compared with SIF. The experimental wines obtained by SIF showed significant lower ethanol and higher glycerol concentrations, whereas no significant difference was detected in colour intensity. The total phenol index reached significantly lower values in SIF. Furthermore, the wines produced by SIF contained higher concentrations of vitisin A that has a greater colour stability than the anthocyanin monomer. Finally, a lower content of both free anthocyanins and flavan-3-ols, key compounds for wine quality possessing also health-enhancing properties, was found in wines obtained by SIF. On the contrary, no significant difference was detected on flavonol concentration between SIF and AXF. This study highlighted that the use of sequential inoculum of Starm. bacillaris and S. cerevisiae can contribute to increasing the colour stability of red wines, even if at the expense of compounds with health properties.

From neglected to dissected: How technological advances are leading the way to the study of Coxiella burnetii pathogenesis.

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for severe worldwide outbreaks of the zoonosis Q fever. The remarkable resistance to environmental stress, extremely low infectious dose and ease of dissemination, contributed to the classification of C. burnetii as a class B biothreat. Unique among intracellular pathogens, C. burnetii escapes immune surveillance and replicates within large autophagolysosome-like compartments called Coxiella-containing vacuoles (CCVs). The biogenesis of these compartments depends on the subversion of several host signalling pathways. For years, the obligate intracellular nature of C. burnetii imposed significant experimental obstacles to the study of its pathogenic traits. With the development of an axenic culture medium in 2009, C. burnetii became genetically tractable, thus allowing the implementation of mutagenesis tools and screening approaches to identify its virulence determinants and investigate its complex interaction with host cells. Here, we review the key advances that have contributed to our knowledge of C. burnetii pathogenesis, leading to the rise of this once-neglected pathogen to an exceptional organism to study the intravacuolar lifestyle.

Field Isolation and Cultivation of Trypanosomatids from Insects.

Monoxenous (one host) trypanosomatids from insects and other invertebrates can be introduced into axenic culture relatively easily and efficiently, allowing for their transfer from the field into the laboratory. Here we describe simple methods and alternative cultivation protocols, the wider application of which will allow substantial expansion of trypanosomatids available for research.

In Vitro Culture for Differentiation Simulation of Leishmania spp.

In the 1990s my laboratory discovered that Leishmania promastigotes can combine two environmental cues, typical to lysosomes, acidic pH (~5.5) and body temperature (37 °C) into a single signal that induced differentiation. Based on this concept, we modified EARLS-based medium 199 to become an amastigote-specific medium. Shifting promastigotes to this medium followed by incubation in a CO2 incubator induced differentiation. Axenic amastigotes reach maturation within 5 days, resembling the time it takes in vivo. This chapter provides a complete protocol we developed for L. donovani promastigote-to-amastigote differentiation. This protocol should be useful for both old-world and new-world species of Leishmania.

Development of a Coxiella burnetii culture method for high-throughput assay to identify host-directed therapeutics.

The intracellular Gram-negative bacterium, Coxiella burnetii, is a worldwide zoonotic pathogen and the causative agent of Q fever. The standard of care for C. burnetii infections involves extended periods of antibiotic treatment and the development of doxycycline-resistant strains stress the need for new treatment strategies. A previously developed axenic medium has facilitated in vitro growth of the organism. In this study, we have developed a simple culture method that is inexpensive, reliable and utilizes a modular hypoxic chamber system for either small or large scale production of bacteria without the need of a tri-gas incubator. This method provides consistent growth and yields sufficient viable bacteria within four days of culture and can be used for high-throughput screening. The viable bacteria were quantified by counting colony forming units and total bacteria were enumerated using a genomic equivalent method. The characterized bacterial inoculum was then used to optimize cell-based high-throughput immunofluorescence assays with a goal to quantify intracellular bacteria and then screen and identify compounds that inhibit early stages of C. burnetii infection in macrophages.

The Effect of pH on Antibiotic Efficacy against Coxiella burnetii in Axenic Media.

Coxiella burnetii, the etiologic agent of Q fever, replicates in an intracellular phagolysosome with pH between 4 and 5. The impact of this low pH environment on antimicrobial treatment is not well understood. An in vitro system for testing antibiotic susceptibility of C. burnetii in axenic media was set up to evaluate the impact of pH on C. burnetii growth and survival in the presence and absence of antimicrobial agents. The data show that C. burnetii does not grow in axenic media at pH 6.0 or higher, but the organisms remain viable. At pH of 4.75, 5.25, and 5.75 moxifloxacin, doxycycline, and rifampin are effective at preventing growth of C. burnetii in axenic media, with moxifloxacin and doxycycline being bacteriostatic and rifampin having bactericidal activity. The efficacy of doxycycline and moxifloxacin improved at higher pH, whereas rifampin activity was pH independent. Hydroxychloroquine is thought to inhibit growth of C. burnetii in vivo by raising the pH of typically acidic intracellular compartments. It had no direct bactericidal or bacteriostatic activity on C. burnetii in axenic media, suggesting that raising pH of acidic intracellular compartments is its primary mechanism of action in vivo. The data suggest that doxycycline and hydroxychloroquine are primarily independent bacteriostatic agents.

RNA-seq data of Ganoderma boninense at axenic culture condition and under in planta pathogen-oil palm (Elaeis guineensis Jacq.) interaction.

OBJECTIVE: Basal stem rot disease causes severe economic losses to oil palm production in South-east Asia and little is known on the pathogenicity of the pathogen, the basidiomyceteous Ganoderma boninense. Our data presented here aims to identify both the house-keeping and pathogenicity genes of G. boninense using Illumina sequencing reads. DESCRIPTION: The hemibiotroph G. boninense establishes via root contact during early stage of colonization and subsequently kills the host tissue as the disease progresses. Information on the pathogenicity factors/genes that causes BSR remain poorly understood. In addition, the molecular expressions corresponding to G. boninense growth and pathogenicity are not reported. Here, six transcriptome datasets of G. boninense from two contrasting conditions (three biological replicates per condition) are presented. The first datasets, collected from a 7-day-old axenic condition provide an insight onto genes responsible for sustenance, growth and development of G. boninense while datasets of the infecting G. boninense collected from oil palm-G. boninense pathosystem (in planta condition) at 1 month post-inoculation offer a comprehensive avenue to understand G. boninense pathogenesis and infection especially in regard to molecular mechanisms and pathways. Raw sequences deposited in Sequence Read Archive (SRA) are available at NCBI SRA portal with PRJNA514399, bioproject ID.

Concurrent biomineralization of silver ions into Ag0 and AgxO by Leptolyngbya strain JSC-1 and the establishment of its axenic culture.

Ionic silver is a potential hazard to aquatic life forms because of the increasing usage of silver based materials. The need for developing a sustainable and ecofriendly process to minimize the toxic effects of the free ions burden is now a scientific consensus. Therefore, we report the latest results in cyanobacterium Leptolyngbya JSC-1 investigating the tolerance towards toxic doses of silver, its extracellular biomineralization and silver nano-deposits formation inside the cells, and speculate about potential environmental impacts. In this study, scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDS) analysis reveal the extracellular biomineralization of soluble silver (1-100 µM) into corresponding nanoparticles (50-100 nm in diameter) by JSC-1, while X-ray photoelectron spectroscopy (XPS) examination divulged the presence of both Ag+ and Ag0 in extracellularly biomineralized silver, depicting a mixture of both AgxO and elemental Ag. The scanning transmission electron microscopy (STEM), EDS and elemental mapping visualized the formation of intracellular silver nanoparticles. Moreover, this feature of silver tolerance in JSC-1 was further exploited and a novel protocol was developed for isolation and maintenance of axenic culture of this filamentous cyanobacterium. Consequently, this capability of silver biomineralization by JSC-1, both extra- and intra-cellularly might be useful for modeling the Ag resistance mechanism in cyanobacteria and also might be a sustainable alternative for heavy metals bioremediation in aquatic environments.

Unified field studies of the algae testbed public-private partnership as the benchmark for algae agronomics.

National scale agronomic projections are an important input for assessing potential benefits of algae cultivation on the future of innovative agriculture. The Algae Testbed Public-Private Partnership was established with the goal of investigating open pond algae cultivation across different geographic, climatic, seasonal, and operational conditions while setting the benchmark for quality data collection, analysis, and dissemination. Identical algae cultivation systems and data analysis methodologies were established at testbed sites across the continental United States and Hawaii. Within this framework, the Unified Field Studies were designed for algae cultivation during all 4 seasons across the testbed network. With increasingly diverse algae research and development, and field deployment strategies, the challenges associated with data collection, quality, and dissemination increase dramatically. The dataset presented here is the complete, curated, climatic, cultivation, harvest, and biomass composition data for each season at each site. These data enable others to do in-depth cultivation, harvest, techno-economic, life cycle, resource, and predictive growth modelling analysis, as well as development of crop protection strategies throughout the algae cultivation industry.

Efficient and reproducible experimental infections of rats with Blastocystis spp.

Although Blastocystis spp. infect probably more than 1 billion people worldwide, their clinical significance is still controversial and their pathophysiology remains poorly understood. In this study, we describe a protocol for an efficient and reproducible model of chronic infection in rats, laying the groundwork for future work to evaluate the pathogenic potential of this parasite. In our experimental conditions, we were unable to infect rats using vacuolar forms of an axenically cultivated ST4 isolate, but we successfully established chronic infections of 4 week-old rats after oral administration of both ST3 and ST4 purified cysts isolated from human stool samples. The infection protocol was also applied to 4 week-old C57BL/9, BALB/C and C3H mice, but any mouse was found to be infected by Blastocystis. Minimal cyst inoculum required for rat infection was higher with ST3 (105) than with ST4 (102). These results were confirmed by co-housing experiments highlighting a higher contagious potential of ST4 in rats compared to ST3. Finally, experiments mimicking fecal microbiota transfer from infected to healthy animals showed that Blastocystis spp. could easily infect a new host, even though its intestinal microbiota is not disturbed. In conclusion, our results provide a well-documented and robust rat model of Blastocystis chronic infection, reproducing "natural" infection. This model will be of great interest to study host parasite interactions and to better evaluate clinical significance of Blastocystis.

Biological study of Trypanosoma caninum under co-culture with different feeder layer cells.

Trypanosoma caninum is a parasite isolated from domestic dogs, of which several biological aspects remain unknown, including evolutive forms found in vertebrate hosts. The objective of this study was to evaluate co-cultures of T. caninum with different cell lines as feeder layers to monitor the differentiation process and investigate infective potential. The study was performed using DH-82, MDCK, and Lulo cell lines. T. caninum from axenic culture was added to the cultured adherent cells. At intervals over 30 days, aliquots of the supernatant were collected for quantification and assessment of differentiation. Infectivity assays were performed on the aforementioned cell lines seeded on glass coverslips and evaluated after 6, 24, and 72 h. In the supernatant of the feeder layer, T. caninum presented similar growth profiles, with epimastigote and trypomastigote forms in binary and multiple divisions. During co-culture with DH-82 and MDCK cells, a higher level of differentiation to trypomastigotes was observed. This study shows that the differentiation process of this parasite can vary according to culture conditions and that DH-82 and MDCK lineages could be applied to the study of trypomastigote forms. All forms of T. caninum described until now (aflagellar epimastigotes, typical epimastigotes, or trypomastigotes) were unable to infect the cell line Finally, this study provides additional data about morphobiological aspects. Although the biological cycle of T. caninum has not been established, the present data suggest the importance of feeder layers in promoting the growth and differentiation of this new parasite.

Lipid A Has Significance for Optimal Growth of Coxiella burnetii in Macrophage-Like THP-1 Cells and to a Lesser Extent in Axenic Media and Non-phagocytic Cells.

Lipid A is an essential basal component of lipopolysaccharide of most Gram-negative bacteria. Inhibitors targeting LpxC, a conserved enzyme in lipid A biosynthesis, are antibiotic candidates against Gram-negative pathogens. Here we report the characterization of the role of lipid A in Coxiella burnetii growth in axenic media, monkey kidney cells (BGMK and Vero), and macrophage-like THP-1 cells by using a potent LpxC inhibitor -LPC-011. We first determined the susceptibility of C. burnetii LpxC to LPC-011 in a surrogate E. coli model. In E. coli, the minimum inhibitory concentration (MIC) of LPC-011 against C. burnetii LpxC is < 0.05 µg/mL, a value lower than the inhibitor's MIC against E. coli LpxC. Considering the inhibitor's problematic pharmacokinetic properties in vivo and Coxiella's culturing time up to 7 days, the stability of LPC-011 in cell cultures was assessed. We found that regularly changing inhibitor-containing media was required for sustained inhibition of C. burnetii LpxC in cells. Under inhibitor treatment, Coxiella has reduced growth yields in axenic media and during replication in non-phagocytic cells, and has a reduced number of productive vacuoles in such cells. Inhibiting lipid A biosynthesis in C. burnetii by the inhibitor was shown in a phase II strain transformed with chlamydial kdtA. This exogenous KdtA enzyme modifies Coxiella lipid A with an α-Kdo-(2 → 8)-α-Kdo epitope that can be detected by anti-chlamydia genus antibodies. In inhibitor-treated THP-1 cells, Coxiella shows severe growth defects characterized by poor vacuole formation and low growth yields. Coxiella progenies prepared from inhibitor-treated cells retain the capability of normally infecting all tested cells in the absence of the inhibitor, which suggests a dispensable role of lipid A for infection and early vacuole development. In conclusion, our data suggest that lipid A has significance for optimal development of Coxiella-containing vacuoles, and for robust multiplication of C. burnetii in macrophage-like THP-1 cells. Unlike many bacteria, C. burnetii replication in axenic media and non-phagocytic cells was less dependent on normal lipid A biosynthesis.

Use of Axenic Culture Tools to Study Coxiella burnetii.

Coxiella burnetii is a highly infectious obligate intracellular bacterium and the etiological agent of the zoonosis Query (Q) fever. This Gram-negative gamma-proteobacterium has adapted to replicate within a specialized compartment in mammalian phagocytic cells, known as the Coxiella-containing vacuole (CCV). Knowledge of critical characteristics of the CCV microenvironment (e.g., luminal pH), analysis of the C. burnetii genome sequence, and strategic metabolic profiling have provided the basis for determining the physicochemical and nutritional conditions necessary to support axenic replication of C. burnetii. In this unit, the media currently utilized for axenic culture of C. burnetii are described, with emphasis on application. To aid in experimental reproducibility and interpretation of results, considerations and limitations are discussed. Lastly, expected results for C. burnetii axenic growth under control conditions are provided as a reference. © 2018 by John Wiley & Sons, Inc.

RNA-seq analysis reveals differences in transcript abundance between cultured and sand fly-derived Leishmania infantum promastigotes.

Leishmania infantum is responsible for human and canine leishmaniasis in the Mediterranean basin, where the major vector is Phlebotomus perniciosus. Because isolation of sufficient parasites from the sand fly gut is technically challenging, axenic cultivation of promastigotes is routinely used to obtain material for biochemical and genetic analyses. Here, we report the use of Spliced Leader RNA-seq (SL-seq) to compare transcript abundance in cultured promastigotes and those obtained from the whole midgut of the sand fly 5 days after infection. SL-seq allows for amplification of RNA from the parasite avoiding contamination with RNA from the gut of the insect. The study has been performed by means of a single technical replicate comparing pools of samples obtained from sand fly-derived (sfPro) and axenic culture promastigotes (acPro). Although there was a moderate correlation (R2 = 0.83) in gene expression, 793 genes showed significantly different (≥2-fold, p <0.05) mRNA levels in sand fly-derived promastigotes and in culture, of which 31 were up-regulated ≥8-fold (p < 10-8 in most cases). These included several genes that are typically up-regulated during metacyclogenesis, suggesting that sand fly-derived promastigotes contain a substantial number of metacyclics, and/or that their differentiation status as metacyclics is more advanced in these populations. Infection experiments and studies evaluating the proportion of metacyclic promastigotes in culture and within the sand fly gut, previously reported by us, support the last hypothesis.

Ochrobactrum sp. MPV1 from a dump of roasted pyrites can be exploited as bacterial catalyst for the biogenesis of selenium and tellurium nanoparticles.

BACKGROUND: Bacteria have developed different mechanisms for the transformation of metalloid oxyanions to non-toxic chemical forms. A number of bacterial isolates so far obtained in axenic culture has shown the ability to bioreduce selenite and tellurite to the elemental state in different conditions along with the formation of nanoparticles-both inside and outside the cells-characterized by a variety of morphological features. This reductive process can be considered of major importance for two reasons: firstly, toxic and soluble (i.e. bioavailable) compounds such as selenite and tellurite are converted to a less toxic chemical forms (i.e. zero valent state); secondly, chalcogen nanoparticles have attracted great interest due to their photoelectric and semiconducting properties. In addition, their exploitation as antimicrobial agents is currently becoming an area of intensive research in medical sciences. RESULTS: In the present study, the bacterial strain Ochrobactrum sp. MPV1, isolated from a dump of roasted arsenopyrites as residues of a formerly sulfuric acid production near Scarlino (Tuscany, Italy) was analyzed for its capability of efficaciously bioreducing the chalcogen oxyanions selenite (SeO32-) and tellurite (TeO32-) to their respective elemental forms (Se0 and Te0) in aerobic conditions, with generation of Se- and Te-nanoparticles (Se- and TeNPs). The isolate could bioconvert 2 mM SeO32- and 0.5 mM TeO32- to the corresponding Se0 and Te0 in 48 and 120 h, respectively. The intracellular accumulation of nanomaterials was demonstrated through electron microscopy. Moreover, several analyses were performed to shed light on the mechanisms involved in SeO32- and TeO32- bioreduction to their elemental states. Results obtained suggested that these oxyanions are bioconverted through two different mechanisms in Ochrobactrum sp. MPV1. Glutathione (GSH) seemed to play a key role in SeO32- bioreduction, while TeO32- bioconversion could be ascribed to the catalytic activity of intracellular NADH-dependent oxidoreductases. The organic coating surrounding biogenic Se- and TeNPs was also characterized through Fourier-transform infrared spectroscopy. This analysis revealed interesting differences among the NPs produced by Ochrobactrum sp. MPV1 and suggested a possible different role of phospholipids and proteins in both biosynthesis and stabilization of such chalcogen-NPs. CONCLUSIONS: In conclusion, Ochrobactrum sp. MPV1 has demonstrated to be an ideal candidate for the bioconversion of toxic oxyanions such as selenite and tellurite to their respective elemental forms, producing intracellular Se- and TeNPs possibly exploitable in biomedical and industrial applications.

Selection of strains for shiitake production in axenic substrate.

Shiitake mushroom consumption is increasing in Brazil. In addition to the implementation of new production methods, it is also important to increase productivity, quality and reduce production costs. In this study, six commercial Lentinula edodes strains were characterized for genetic diversity (rep-PCR analysis) and mushroom production (yield, number and weight of individual mushrooms) using different substrates and cultural conditions. All strains showed genetic differences by repetitive element palindromic based-polymerase chain reaction (rep-PCR). The richest substrate resulted in the greatest production under both environmental conditions. Strains LE4 and LE6 produced the majority of their mushrooms earlier than the other strains. The highest number of mushrooms was observed in the LE6 strain while the highest weights of individual mushrooms were observed in the LE4 strain. Controlled environmental conditions resulted in superior production for all strains, except for LE4, which had empirically greater yield in the semi-controlled environmental condition.