A guide to the use of bioassays in exploration of natural resources.

Bioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are critical for optimal bioprospecting. They are used both in the early stages of compounds isolation/purification/identification, and in later stages to evaluate their safety and efficacy. In this review, we provide a comprehensive overview of the most common bioassays used in the discovery and development of new bioactive compounds with a focus on marine bioresources. We present a comprehensive list of practical considerations for selecting appropriate bioassays and discuss in detail the bioassays typically used to explore antimicrobial, antibiofilm, cytotoxic, antiviral, antioxidant, and anti-ageing potential. The concept of quality control and bioassay validation are introduced, followed by safety considerations, which are critical to advancing bioactive compounds to a higher stage of development. We conclude by providing an application-oriented view focused on the development of pharmaceuticals, food supplements, and cosmetics, the industrial pipelines where currently known marine natural products hold most potential. We highlight the importance of gaining reliable bioassay results, as these serve as a starting point for application-based development and further testing, as well as for consideration by regulatory authorities.

Advanced Methods for Natural Products Discovery: Bioactivity Screening, Dereplication, Metabolomics Profiling, Genomic Sequencing, Databases and Informatic Tools, and Structure Elucidation.

Natural Products (NP) are essential for the discovery of novel drugs and products for numerous biotechnological applications. The NP discovery process is expensive and time-consuming, having as major hurdles dereplication (early identification of known compounds) and structure elucidation, particularly the determination of the absolute configuration of metabolites with stereogenic centers. This review comprehensively focuses on recent technological and instrumental advances, highlighting the development of methods that alleviate these obstacles, paving the way for accelerating NP discovery towards biotechnological applications. Herein, we emphasize the most innovative high-throughput tools and methods for advancing bioactivity screening, NP chemical analysis, dereplication, metabolite profiling, metabolomics, genome sequencing and/or genomics approaches, databases, bioinformatics, chemoinformatics, and three-dimensional NP structure elucidation.

Microfluidics for microalgal biotechnology.

Microalgae have expanded their roles as renewable and sustainable feedstocks for biofuel, smart nutrition, biopharmaceutical, cosmeceutical, biosensing, and space technologies. They accumulate valuable biochemical compounds from protein, carbohydrate, and lipid groups, including pigments and carotenoids. Microalgal biomass, which can be adopted for multivalorization under biorefinery settings, allows not only the production of various biofuels but also other value-added biotechnological products. However, state-of-the-art technologies are required to optimize yield, quality, and the economical aspects of both upstream and downstream processes. As such, the need to use microfluidic-based devices for both fundamental research and industrial applications of microalgae, arises due to their microscale sizes and dilute cultures. Microfluidics-based devices are superior to their competitors through their ability to perform multiple functions such as sorting and analyzing small amounts of samples (nanoliter to picoliter) with higher sensitivities. Here, we review emerging applications of microfluidic technologies on microalgal processes in cell sorting, cultivation, harvesting, and applications in biofuels, biosensing, drug delivery, and nutrition.

Association of nuisance filamentous algae Cladophora spp. with E. coli and Salmonella in public beach waters: impacts of UV protection on bacterial survival.

This study investigated whether filamentous algal species commonly found in nearshore public beach water systems provide protection from natural UV to bacteria present in the same environmental settings. To test this hypothesis, Cladophora spp., a filamentous nuisance algae group causing undesired water quality in the Great Lakes region was selected and its interactions with a non-pathogenic indicator organism Escherichia coli and a pathogenic strain of Salmonella enterica serovar Typhimurium were tested. In laboratory microcosms where the lake environment and natural sunlight conditions were simulated, a 7-log removal of E. coli was observed in only six hours of exposure to UV with an initial seed concentration of 10(3) CFU mL(-1). With the presence of algae, the same log removal was achieved in 16 hours. At higher seed concentrations of 10(5) CFU mL(-1), E. coli survived for two days with an extended survival up to 11 days in the presence of Cladophora spp. S. typhimurium has shown more resilient survival profiles, with the same log removals achieved in 14 and 20 days for low and high seed concentrations respectively, in the absence of algae. Cladophora spp. caused extended protection for S. typhimurium with much less log reductions reported. Algae-mediated protection from UV irradiation was attributed to certain organic carbon exuded from Cladophora spp. In addition, confocal microscopy images confirmed close interaction between bacteria and algae, more prominent with thin filamentous Cladophora spp.

Evaluating microalgal integrated biorefinery schemes: empirical controlled growth studies and life cycle assessment.

Two freshwater and two marine microalgae species were grown under nitrogen replete and deplete conditions evaluating the impact on total biomass yield and biomolecular fractions (i.e. starch, protein, and lipid). A life cycle assessment was performed to evaluate varying species/growth conditions considering each biomass fraction and final product substitution based on energy consumption, greenhouse gas emissions (GHG), and eutrophication potential. Lipid for biodiesel was assumed as the primary product. Protein and carbohydrate fractions were processed as co-products. Composition of the non-lipid fraction presented significant trade-offs among biogas production, animal feed substitution, nutrient recycling, and carbon sequestration. Maximizing total lipid productivity rather than lipid content yielded the least GHG emissions. A marine, N-deplete case with relatively low lipid productivity but effective nutrient recycling had the lowest eutrophication impacts. Tailoring algal species/growth conditions to optimize the mix of biomolecular fractions matched to desired products and co-products can enable a sustainable integrated microalgal biorefinery.

Transcriptomic analysis of the oleaginous microalga Neochloris oleoabundans reveals metabolic insights into triacylglyceride accumulation.

BACKGROUND: The lack of sequenced genomes for oleaginous microalgae limits our understanding of the mechanisms these organisms utilize to become enriched in triglycerides. Here we report the de novo transcriptome assembly and quantitative gene expression analysis of the oleaginous microalga Neochloris oleoabundans, with a focus on the complex interaction of pathways associated with the production of the triacylglycerol (TAG) biofuel precursor. RESULTS: After growth under nitrogen replete and nitrogen limiting conditions, we quantified the cellular content of major biomolecules including total lipids, triacylglycerides, starch, protein, and chlorophyll. Transcribed genes were sequenced, the transcriptome was assembled de novo, and the expression of major functional categories, relevant pathways, and important genes was quantified through the mapping of reads to the transcriptome. Over 87 million, 77 base pair high quality reads were produced on the Illumina HiSeq sequencing platform. Metabolite measurements supported by genes and pathway expression results indicated that under the nitrogen-limiting condition, carbon is partitioned toward triglyceride production, which increased fivefold over the nitrogen-replete control. In addition to the observed overexpression of the fatty acid synthesis pathway, TAG production during nitrogen limitation was bolstered by repression of the ß-oxidation pathway, up-regulation of genes encoding for the pyruvate dehydrogenase complex which funnels acetyl-CoA to lipid biosynthesis, activation of the pentose phosphate pathway to supply reducing equivalents to inorganic nitrogen assimilation and fatty acid biosynthesis, and the up-regulation of lipases-presumably to reconstruct cell membranes in order to supply additional fatty acids for TAG biosynthesis. CONCLUSIONS: Our quantitative transcriptome study reveals a broad overview of how nitrogen stress results in excess TAG production in N. oleoabundans, and provides a variety of genetic engineering targets and strategies for focused efforts to improve the production rate and cellular content of biofuel precursors in oleaginous microalgae.

Optimization of de novo transcriptome assembly from high-throughput short read sequencing data improves functional annotation for non-model organisms.

BACKGROUND: The k-mer hash length is a key factor affecting the output of de novo transcriptome assembly packages using de Bruijn graph algorithms. Assemblies constructed with varying single k-mer choices might result in the loss of unique contiguous sequences (contigs) and relevant biological information. A common solution to this problem is the clustering of single k-mer assemblies. Even though annotation is one of the primary goals of a transcriptome assembly, the success of assembly strategies does not consider the impact of k-mer selection on the annotation output. This study provides an in-depth k-mer selection analysis that is focused on the degree of functional annotation achieved for a non-model organism where no reference genome information is available. Individual k-mers and clustered assemblies (CA) were considered using three representative software packages. Pair-wise comparison analyses (between individual k-mers and CAs) were produced to reveal missing Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog identifiers (KOIs), and to determine a strategy that maximizes the recovery of biological information in a de novo transcriptome assembly. RESULTS: Analyses of single k-mer assemblies resulted in the generation of various quantities of contigs and functional annotations within the selection window of k-mers (k-19 to k-63). For each k-mer in this window, generated assemblies contained certain unique contigs and KOIs that were not present in the other k-mer assemblies. Producing a non-redundant CA of k-mers 19 to 63 resulted in a more complete functional annotation than any single k-mer assembly. However, a fraction of unique annotations remained (~0.19 to 0.27% of total KOIs) in the assemblies of individual k-mers (k-19 to k-63) that were not present in the non-redundant CA. A workflow to recover these unique annotations is presented. CONCLUSIONS: This study demonstrated that different k-mer choices result in various quantities of unique contigs per single k-mer assembly which affects biological information that is retrievable from the transcriptome. This undesirable effect can be minimized, but not eliminated, with clustering of multi-k assemblies with redundancy removal. The complete extraction of biological information in de novo transcriptomics studies requires both the production of a CA and efforts to identify unique contigs that are present in individual k-mer assemblies but not in the CA.

Effects of residual antibiotics in groundwater on Salmonella typhimurium: changes in antibiotic resistance, in vivo and in vitro pathogenicity.

An outbreak-causing strain of Salmonella enterica serovar Typhimurium was exposed to groundwater with residual antibiotics for up to four weeks. Representative concentrations (0.05, 1, and 100 µg L(-1)) of amoxicillin, tetracycline, and a mixture of several other antibiotics (1 µg L(-1) each) were spiked into artificially prepared groundwater (AGW). Antibiotic susceptibility analysis and the virulence response of stressed Salmonella were determined on a weekly basis by using human epithelial cells (HEp2) and soil nematodes (C. elegans). Results have shown that Salmonella typhimurium remains viable for long periods of exposure to antibiotic-supplemented groundwater; however, they failed to cultivate as an indication of a viable but nonculturable state. Prolonged antibiotics exposure did not induce any changes in the antibiotic susceptibility profile of the S. typhimurium strain used in this study. S. typhimurium exposed to 0.05 and 1 µg L(-1) amoxicillin, and 1 µg L(-1) tetracycline showed hyper-virulent profiles in both in vitro and in vivo virulence assays with the HEp2 cells and C. elegans respectively, most evident following 2nd and 3rd weeks of exposure.

Pleiotropic cellular, hemostatic, and biological actions of Ankaferd hemostat.

Sustaining hemostasis in clinical hemorrhages is a challenging task and requires extensive effort to stabilize medically hard-to-treat traumatic injuries. Several hemostatic agents are preferred to control external and internal bleedings, yet commercially available products are not sufficiently effective or fast-acting to achieve hemostasis in extreme cases. Ankaferd Blood Stopper (ABS) is a herbal extract traditionally used as a hemostatic agent. Recent studies have shown that ABS could be utilized successfully as a hemostatic agent for the management of clinical hemorrhages when conventional methods were ineffective. This review serves as a basis to provide recent findings on several applications of ABS, specifically preclinical, biological, and clinical studies both in vitro and in vivo. Another section focuses on the ultrastructural morphology and protein network formation of ABS in an effort to understand the hemostatic mechanisms of this unique agent at tissue level.

Transcriptome sequencing and annotation of the microalgae Dunaliella tertiolecta: pathway description and gene discovery for production of next-generation biofuels.

BACKGROUND: Biodiesel or ethanol derived from lipids or starch produced by microalgae may overcome many of the sustainability challenges previously ascribed to petroleum-based fuels and first generation plant-based biofuels. The paucity of microalgae genome sequences, however, limits gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for the non-model microalgae species, Dunaliella tertiolecta, and identify pathways and genes of importance related to biofuel production. RESULTS: Next generation DNA pyrosequencing technology applied to D. tertiolecta transcripts produced 1,363,336 high quality reads with an average length of 400 bases. Following quality and size trimming, ~45% of the high quality reads were assembled into 33,307 isotigs with a 31-fold coverage and 376,482 singletons. Assembled sequences and singletons were subjected to BLAST similarity searches and annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO) identifiers. These analyses identified the majority of lipid and starch biosynthesis and catabolism pathways in D. tertiolecta. CONCLUSIONS: The construction of metabolic pathways involved in the biosynthesis and catabolism of fatty acids, triacylglycrols, and starch in D. tertiolecta as well as the assembled transcriptome provide a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock.

Correlating transport behavior with cell properties for eight porcine Escherichia coli isolates.

In this study we investigate how growth stage and depositional environment affect variability of cell properties and transport behavior of eight porcine E. coli isolates. We compared the surface properties for cells harvested during exponential and stationary growth phase and their transport behavior through columns packed with either uncoated or Fe-coated quartz sand. We then investigated correlations between measured cell properties and fitted bacterial attachment efficiencies. For both growth stages we found that bacterial attachment efficiencies in the uncoated quartz sand varied among the eight different isolates by over an order of magnitude whereas attachment efficiencies in the Fe-coated sands varied by a factor of less than two. With the exception of one isolate, growth condition had minimal impact on attachment efficiencies to the uncoated sands. A strong and statistically significant inverse relationship was observed between bacterial attachment efficiencies in the uncoated quartz sand columns and log-transformed zeta potential, whereas a mild yet statistically significant relationship between bacterial attachment efficiencies in the Fe-coated sands and cell width was observed. For the experimental conditions used in our study, we found that variability in E. coli transport was more dependent on the depositional environment than on growth conditions.

Ultrastructural and morphological analyses of the in vitro and in vivo hemostatic effects of Ankaferd Blood Stopper.

Ultrastructural and morphological analyses of a novel hemostatic agent, Ankaferd Blood Stopper (ABS), in comparison to its in vitro and in vivo hemostatic effects were investigated. High-resolution scanning electron microscopy (SEM) images accompanied with morphological analysis after topical application of ABS revealed a very rapid (<1 second) protein network formation within concurrent vital erythroid aggregation covering the classical coagulation cascade. Histopathological examination revealed similar in vivo ABS-induced hemostatic network at the porcine hepatic tissue injury model. Instantaneous control of bleeding was achieved in human surgery-induced dental tissue injury associated with primary and secondary hemostatic abnormalities. Ankaferd Blood Stopper could hold a great premise for clinical management of surgery bleedings as well as immediate cessation of bleeding on external injuries based on upcoming clinical trials.

Coupled factors influencing concentration-dependent colloid transport and retention in saturated porous media.

The coupled influence of input suspension concentration (Ci), ionic strength (IS), and hydrodynamics on the transport and retention of 1.1 microm carboxyl-modified latex colloids in saturated quartz sand (150 microm) under unfavorable attachment conditions (pH 10) was investigated. The percentage of retained colloids in column experiments decreased with Ci at intermediate IS conditions (31 or 56 mM) when colloids were weakly associated with the solid phase by a shallow secondary energy minima. In contrast, the effects of Ci on colloid retention were absent when IS was too low (6 mM) or too high (106 mM). The concentration effects under intermediate IS conditions were dependent on the system hydrodynamics, magnitude of Ci, and injection order of Ci, but they were largely independent of the input colloid mass. These observations were explained in part by time- and concentration-dependent filling of retention sites. Only a small fraction of the solid surface area was found to contribute to retention when IS was 31 mM, and micromodel observations indicated that colloid retention was enhanced in lower velocity regions of the pore space that occurred near grain-grain contacts. Consequently, retention profiles for IS = 31 mM conditions were increasingly nonexponential at lower values of Ci (during filling), whereas the observed concentration effect was largely eliminated as retention locations became filled. In addition, micromodel observations indicated that liquid and solid phase mass transfer of colloids to retention locations was influenced by Ci under intermediate IS conditions. Higher values of Ci are expected to produce less relative mass transfer to retention locations due to increased numbers of collisions that knock weakly associated colloids off the solid phase. Hence, the concentration effects were found to be largely independent of input colloid mass during filling of retention sites.

Phenotypic characterization of Escherichia coli through whole-cell fatty acid profiling to investigate host specificity.

The objective of the study was to investigate whole-cell fatty acid methyl ester (FAME) profiles of 605 Escherichia coli isolates to determine their host specificity. The isolates were cultured from six possible sources of fecal pollution; 180 isolates from sewage, 85 from dairy cow, 98 from chicken, 76 from swine, 94 from deer, and 72 from waterfowl, mostly geese and ducks. The FAME profiles were presented as the relative masses of 12 FAMEs identified in the isolates and it was found that none of the six hosts carried a "signature" FAME, a FAME that is uniquely associated with a particular host category. However, two-sample t-test analyses indicated that the mean relative masses of seven FAMEs out of the 12 identified showed statistically significant differences (95% confidence interval) between isolates of human and non-human origins. In addition, a linear discriminant function based on mean relative mass variations in individual FAMEs classified the known-source isolates into their respective host categories with a 47.6% average rate of correct classification (ARCC) in a six-way discriminant analysis. The ARCC increased to 61.3% when the individual hosts were pooled into larger categories of human, livestock, and wildlife. The accuracy was 75.5% when isolates of human origin were discriminated against those of non-human origins. Random cluster formation analysis indicated that the library size was sufficient to prevent random grouping among the isolates.

Microbial source tracking using host specific FAME profiles of fecal coliforms.

The objective of this study was to investigate the host-specific differences in fatty acid methyl ester (FAME) profiles of fecal coliforms (FC). A known-source library was constructed with 314 FC isolates cultured from 6 possible sources of fecal pollution; 99 isolates from sewage; 29 from bovine; 29 from poultry; 50 from swine; 46 from waterfowl; and 61 from deer. It was found that the hydroxy FAMEs 12:0 2 OH, 12:03 OH, and 14:02 OH were exclusively associated with isolates of human origin. On the other hand, 3 saturated FAMEs, 10:0, 15:0, and 18:0 were found only in isolates from non-human sources, 15:0 being associated with livestock samples only. In addition to the presence of these signature FAMEs, the mean relative masses of 16:1 omega7c and 16:1 ISO/14:03 OH were significantly different between the isolates of human and non-human origins. A linear discriminant function differentiated FC isolates of human origin from those of livestock and wildlife origin at 99% accuracy. These results strongly suggest that the FAME profiles of FC show statistically significant host specificity and may have the potential to be used as a phenotypic microbial source tracking tool.