Testing an Ethnobiological Evolutionary Hypothesis on Plant-Based Remedies to Treat Malaria in Africa

The malaria hypothesis, which addresses a strong selective pressure on human genes resulting from a chain of processes that originated with the practice of agriculture, is an example of an evolutionary consequence of niche construction. This scenario has led us to formulate the following questions: Are the genetic adaptations of populations with a history of contact with malaria reflected in the local medical systems? Likewise, could environmental changes (deforestation) and the incidence of malaria result in an adaptive response in these local health care systems? We collected secondary data for the entire African continent from different databases and secondary sources and measured the response of health care systems as the variation in the richness of antimalarial medicinal plants. Our results did not indicate a cause-and-effect relationship between the tested variables and the medical systems, but a subsequent analysis of variance showed an increase in the mean of medicinal plants in regions with a higher incidence of malaria prior to disease control measures. We suggest that this response had a greater impact on local medical knowledge than other variables, such as genetic frequency and deforestation.     

Knowledge and use of medicinal plants by local specialists in an region of Atlantic Forest in the state of Pernambuco (Northeastern Brazil)

This paper investigates the wealth of medicinal plants used by the Apatani tribe of Arunachal Pradesh. Apatani have traditionally settled in seven villages in the Ziro valley of Lower Subansiri district of Arunachal Pradesh in the Eastern Himalayan region of India. The present study has resulted in the documentation of 158 medicinal plant species used by the Apatani group of villages. These medicinal plant species were distributed across 73 families and 124 genera. Asteraceae was the most dominant family (19 species, 11 genera) of medicinal plants, followed by Zingiberaceae, Solanaceae, Lamiaceae and Araceae. For curing ailments, the use of aboveground plant parts was higher (80%) than the belowground plant parts in the Apatani group of villages. Of the aboveground plant parts, leaf was used in the majority of cases (56 species), followed by fruit. Different belowground plant forms such as root, tuber, rhizome, bulb and pseudo-bulb were used by Apatani as a medicine. About 52 types of ailments were cured by using these 158 medicinal plant species. The results of this study are further discussed in the changing socio-economic contexts.     

Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo- and thermotolerance can be addressed for the definition of culture conditions.     

Identification and Validation of Specific Markers of Bacillus anthracis Spores by Proteomics and Genomics Approaches

Bacillus anthracis is the causative bacteria of anthrax, an acute and often fatal disease in humans. The infectious agent, the spore, represents a real bioterrorism threat and its specific identification is crucial. However, because of the high genomic relatedness within the Bacillus cereus group, it is still a real challenge to identify B. anthracis spores confidently. Mass spectrometry-based tools represent a powerful approach to the efficient discovery and identification of such protein markers. Here we undertook comparative proteomics analyses of Bacillus anthracis, cereus and thuringiensis spores to identify proteoforms unique to B. anthracis. The marker discovery pipeline developed combined peptide- and protein-centric approaches using liquid chromatography coupled to tandem mass spectrometry experiments using a high resolution/high mass accuracy LTQ-Orbitrap instrument. By combining these data with those from complementary bioinformatics approaches, we were able to highlight a dozen novel proteins consistently observed across all the investigated B. anthracis spores while being absent in B. cereus/thuringiensis spores. To further demonstrate the relevance of these markers and their strict specificity to B. anthracis, the number of strains studied was extended to 55, by including closely related strains such as B. thuringiensis 9727, and above all the B. cereus biovar anthracis CI, CA strains that possess pXO1- and pXO2-like plasmids. Under these conditions, the combination of proteomics and genomics approaches confirms the pertinence of 11 markers. Genes encoding these 11 markers are located on the chromosome, which provides additional targets complementary to the commonly used plasmid-encoded markers. Last but not least, we also report the development of a targeted liquid chromatography coupled to tandem mass spectrometry method involving the selection reaction monitoring mode for the monitoring of the 4 most suitable protein markers. Within a proof-of-concept study, we demonstrate the value of this approach for the further high throughput and specific detection of B. anthracis spores within complex samples.Bacillus anthracis is a highly virulent bacterium, which is the etiologic agent of anthrax, an acute and often lethal disease of animals and humans (1). According to the Centers for Disease Control and Prevention, B. anthracis is classified as a category A agent, the highest rank of potential bioterrorism agents (http://www.bt.cdc.gov/agent/agentlist-category.asp). The infectious agent of anthrax, the spore, was used as a bioterrorism weapon in 2001 in the United States when mailed letters containing B. anthracis spores caused 22 cases of inhalational and/or cutaneous anthrax, five of which were lethal (2). These events have emphasized the need for rapid and accurate detection of B. anthracis spores.Bacillus anthracis is a member of the genus Bacillus, Gram-positive, rod-shaped bacteria characterized by the ability to form endospores under aerobic or facultative anaerobic conditions (3). The genus Bacillus is a widely heterogeneous group encompassing 268 validly described species to date (http://www.bacterio.net/b/bacillus.html, last accessed on August 9^th 2013). B. anthracis is part of the B. cereus group which consists of six distinct species: B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4, 5). The latter three species are generally regarded as nonpathogenic whereas B. cereus and B. thuringiensis could be opportunistic or pathogenic to mammals or insects (5, 6). B. cereus is a ubiquitous species that lives in soil but is also found in foods of plant and animal origin, such as dairy products (7). Its occurrence has also been linked to food poisoning and it can cause diarrhea and vomiting (6, 8). B. thuringiensis is primarily an insect pathogen, also present in soil, and often used as a biopesticide (9).B. anthracis is highly monomorphic, that is, shows little genetic variation (10), and primarily exists in the environment as a highly stable, dormant spore in the soil (1). Specific identification of B. anthracis is challenging because of its high genetic similarity (sequence similarity >99%) with B. cereus and B. thuringiensis (5, 11). The fact that these closely related species are rather omnipresent in the environment further complicates identification of B. anthracis. The main difference among these three species is the presence in B. anthracis of the two virulence plasmids pXO1 and pXO2 (1), which are responsible for its pathogenicity. pXO1 encodes a tripartite toxin (protective antigen (PA), lethal factor (LF), and edema factor (EF)) which causes edema and death (1), whereas pXO2 encodes a poly-γ-d-glutamate capsule which protects the organism from phagocytosis (1). B. anthracis identification often relies on the detection of the genes encoded by these two plasmids via nucleic acid-based assays (12–14). Nevertheless, the occasionally observed loss of the pXO2 plasmid within environmental species may impair the robustness of detection (1). In addition, in recent years a series of findings has shown that the presence of pXO1 and pXO2 is not a unique feature of B. anthracis. Indeed, Hu et al. have demonstrated that ∼7% of B. cereus/B. thuringiensis species can have a pXO1-like plasmid and ∼1.5% a pXO2-like plasmid (15). This was particularly underlined for some virulent B. cereus strains (i.e. B. cereus strains G9241, B. cereus biovar anthracis strains CA and CI) (16–20).Because of these potential drawbacks, the use of chromosome-encoded genes would be preferable for the specific detection of B. anthracis. Such genes (rpoB, gyrA, gyrB, plcR, BA5345, and BA813) have been reported as potential markers (21–25), but concerns have also been raised about their ability to discriminate B. anthracis efficiently from closely related B. cereus strains (26). Ahmod et al. have recently pointed out, by in silico database analysis, that a specific sequence deletion (indel) occurs in the yeaC gene and exploited it for the specific identification of B. anthracis (27). Nevertheless, a few B. anthracis strains (e.g. B. anthracis A1055) do not have this specific deletion and so may lead to false-negative results (27).In the last few years, protein profiling by MS, essentially based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS), has emerged as an alternative (or a complement) to genotypic or phenotypic methods for the fast and efficient identification of microorganisms (28, 29). Such an approach is based on the reproducible acquisition of global bacterial protein fingerprints/patterns. The combination of MS-based protein patterns and chemometric/bioinformatic tools has been demonstrated to efficiently differentiate members of the B. cereus group from other Bacillus species (30). However, the specific discrimination of B. anthracis from the closely related B. cereus and B. thuringiensis remains difficult (30). This study of Lasch and coworkers, performed on vegetative cells, identified a few ribosomal and spore proteins as being responsible for this clustering (30). Closer inspection of the data revealed that B. anthracis identification was essentially based on one particular isoform of the small acid-soluble spore protein B (SASP-B)¹ (30–34), which is exclusively expressed in spores, as the samples were shown to contain residual spores. However, the specificity of SASP-B has recently been questioned as the published genomes of B. cereus biovar anthracis CI and B. thuringiensis BGSC 4CC1 strains have been shown to share the same SASP-B isoform as B. anthracis (35). Altogether these results underline that the quest for specific markers of B. anthracis needs to be pursued.Mass spectrometry also represents a powerful tool for the discovery and identification of protein markers (36, 37). In the case of B. anthracis, this approach has more commonly been used for the comprehensive characterization of given bacterial proteomes. For example, the proteome of vegetative cells with variable plasmid contents has been extensively studied (38–40), as the proteomes of mature spores (41, 42) and of germinating spores (43, 44). Only one recent study, based on a proteo-genomic approach, was initiated to identify protein markers of B. anthracis (45). In this work, potential markers were characterized but using a very limited number of B. cereus group strains (three B. cereus and two B. thuringiensis). Moreover, this study was done on vegetative cells, whereas the spore proteome is drastically different. To our knowledge, no study has characterized and validated relevant protein markers specific to B. anthracis spores, which constitute the dissemination form of B. anthracis and are often targeted by first-line immunodetection methods (46).Here we report comparative proteomics analyses of Bacillus anthracis/cereus/thuringiensis spores, undertaken to identify proteoforms unique to B. anthracis. Preliminary identification was performed on a limited set of Bacillus species both at the peptide (after enzymatic digestion) and protein levels by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a high resolution/high mass accuracy LTQ-Orbitrap instrument. The pertinence of 11 markers was further demonstrated using proteomics and genomics approaches on a representative larger set of up to 55 different strains, including the closely related B. cereus biovar anthracis CI, CA, and B. thuringiensis 9727. Lastly, as a proof-of-concept study, we also report for four B. anthracis markers the implementation of a targeted LC-MS/MS method using selected reaction monitoring (SRM), based on the extension of a previous one focused on SASP-B (35). Preliminary results regarding method usefulness for the high throughput and accurate detection of B. anthracis spores in complex samples were also obtained and will be reported herein.     

Root Formation in Ethylene-Insensitive Plants

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.     

Genetic mapping of ripening and ethylene-related loci in tomato

 The regulation of tomato fruit development and ripening is influenced by a large number of loci as demonstrated by the number of existing non-allelic fruit development mutations and a multitude of genes showing ripening-related expression patterns. Furthermore, analysis of transgenic and naturally occurring tomato mutants confirms the pivotal role of the gaseous hormone ethylene in the regulation of climacteric ripening. Here we report RFLP mapping of 32 independent tomato loci corresponding to genes known or hypothesized to influence fruit ripening and/or ethylene response. Mapped ethylene-response sequences fall into the categories of genes involved in either hormone biosynthesis or perception, while additional ripening-related genes include those involved in cell-wall metabolism and pigment biosynthesis. The placement of ripening and ethylene-response loci on the tomato RFLP map will facilitate both the identification and exclusion of candidate gene sequences corresponding to identified single gene and quantitative trait loci contributing to fruit development and ethylene response. Received: 26 October 1998 / Accepted: 13 November 1998     

High-throughput screening of Hansenula polymorpha clones in the batch compared with the controlled-release fed-batch mode on a small scale

Most large-scale production processes in biotechnology are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode, which results in the microorganisms being subjected to different physiological conditions. This significantly affects strain selection. To demonstrate differences in ranking during strain selection depending on the operational mode, screenings were performed in batch and fed-batch modes. Two model populations of the methylotrophic yeast Hansenula polymorpha RB11 with vector pC10-FMD (P_FMD-GFP) (220 clones) and vector pC10-MOX (P_MOX-GFP) (224 clones) were applied. For fed-batch cultivations in deep-well microtiter plates, a controlled-release system made of silicone elastomer discs containing glucose was used. Three experimental set-ups were investigated: batch cultivation with (1) glucose as a substrate, which catabolite represses product formation, and (2) glycerol as a carbon source, which is partially repressing, respectively, and (3) fed-batch cultivation with glucose as a limiting substrate using the controlled-release system. These three experimental set-ups showed significant variations in green fluorescent protein (GFP) yield. Interestingly, screenings in fed-batch mode with glucose as a substrate resulted in the selection of yeast strains different from those cultivated in batch mode with glycerol or glucose. Ultimately, fed-batch screening is considerably better than screening in batch mode for fed-batch production processes with glucose as a carbon source.     

Development of a plant transformation selection system based on expression of genes encoding gentamicin acetyltransferases

The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3′ nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium-mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture.     

Calcium-induced sensitization of the central helix of calmodulin to proteolysis.

The rate of proteolysis of trypsin-sensitive bonds was used to examine the nature of the structural changes accompanying Ca2+ and Mg2+ binding to calmodulin. In the Ca(2+)-free form, the rates of proteolysis at Arg-106 and Arg-37 are rapid (greater than 300 and 28 nmol min-1 mL-1, respectively), the bonds at Arg-74, Lys-75, and Lys-77, in the central helix, are cleaved more slowly (10 nmol min-1 mL-1), and a lag in the cleavage at the remaining bonds (Lys-13, Lys-30, Arg-86, Arg-90, and Arg-126) suggests that they are not cleaved in the native protein. High concentrations of Ca2+, but not Mg2+, almost completely abolish proteolysis at Arg-106 and drastically reduce the rate of cleavage at Arg-37. Both Ca2+ and Mg2+ exert a moderate protective effect on the proteolysis of the central helix. These results suggest that the F-helix of domains III and, to a lesser extent, the F-helix of domain I are somewhat flexible in the Ca(2+)-free form and are stabilized by Ca2+. Whereas full occupancy of the four Ca(2+)-binding sites produces little change in the susceptibility of the central helix to proteolytic attack, binding of two Ca2+ produces a 10-fold enhancement of the rate of proteolysis in this part of the molecule. We propose that at intermediate Ca2+ levels the flexibility of the central helix of calmodulin is greatly increased, resulting in the transient formation of intermediates which have not been detected by spectroscopic techniques but are trapped by the irreversible action of trypsin.