The use of phylogeny to interpret cross-cultural patterns in plant use and guide medicinal plant discovery: an example from Pterocarpus (Leguminosae)

Background

The study of traditional knowledge of medicinal plants has led to discoveries that have helped combat diseases and improve healthcare. However, the development of quantitative measures that can assist our quest for new medicinal plants has not greatly advanced in recent years. Phylogenetic tools have entered many scientific fields in the last two decades to provide explanatory power, but have been overlooked in ethnomedicinal studies. Several studies show that medicinal properties are not randomly distributed in plant phylogenies, suggesting that phylogeny shapes ethnobotanical use. Nevertheless, empirical studies that explicitly combine ethnobotanical and phylogenetic information are scarce.

Methodology/Principal Findings

In this study, we borrowed tools from community ecology phylogenetics to quantify significance of phylogenetic signal in medicinal properties in plants and identify nodes on phylogenies with high bioscreening potential. To do this, we produced an ethnomedicinal review from extensive literature research and a multi-locus phylogenetic hypothesis for the pantropical genus Pterocarpus (Leguminosae: Papilionoideae). We demonstrate that species used to treat a certain conditions, such as malaria, are significantly phylogenetically clumped and we highlight nodes in the phylogeny that are significantly overabundant in species used to treat certain conditions. These cross-cultural patterns in ethnomedicinal usage in Pterocarpus are interpreted in the light of phylogenetic relationships.

Conclusions/Significance

This study provides techniques that enable the application of phylogenies in bioscreening, but also sheds light on the processes that shape cross-cultural ethnomedicinal patterns. This community phylogenetic approach demonstrates that similar ethnobotanical uses can arise in parallel in different areas where related plants are available. With a vast amount of ethnomedicinal and phylogenetic information available, we predict that this field, after further refinement of the techniques, will expand into similar research areas, such as pest management or the search for bioactive plant-based compounds.     

Clonality and α-a recombination in the Australian Cryptococcus gattii VGII population--an emerging outbreak in Australia

Background

Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia, Canada), with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure.

Methodology/Principal Findings

A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MAT a VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MAT a populations. Our results also support the hypothesis of a “smouldering” outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion.

Conclusions/Significance

The detection of sexual recombination in MATα-MAT a population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active surveillance procedures.     

Fine mapping of the NRG1 Hirschsprung's disease locus

The primary pathology of Hirschsprung's disease (HSCR, colon aganglionosis) is the absence of ganglia in variable lengths of the hindgut, resulting in functional obstruction. HSCR is attributed to a failure of migration of the enteric ganglion precursors along the developing gut. RET is a key regulator of the development of the enteric nervous system (ENS) and the major HSCR-causing gene. Yet the reduced penetrance of RET DNA HSCR-associated variants together with the phenotypic variability suggest the involvement of additional genes in the disease. Through a genome-wide association study, we uncovered a ～350 kb HSCR-associated region encompassing part of the neuregulin-1 gene (NRG1). To identify the causal NRG1 variants contributing to HSCR, we genotyped 243 SNPs variants on 343 ethnic Chinese HSCR patients and 359 controls. Genotype analysis coupled with imputation narrowed down the HSCR-associated region to 21 kb, with four of the most associated SNPs (rs10088313, rs10094655, rs4624987, and rs3884552) mapping to the NRG1 promoter. We investigated whether there was correlation between the genotype at the rs10088313 locus and the amount of NRG1 expressed in human gut tissues (40 patients and 21 controls) and found differences in expression as a function of genotype. We also found significant differences in NRG1 expression levels between diseased and control individuals bearing the same rs10088313 risk genotype. This indicates that the effects of NRG1 common variants are likely to depend on other alleles or epigenetic factors present in the patients and would account for the variability in the genetic predisposition to HSCR.     

Sustained fixation induced changes in phoria and convergence peak velocity

Purpose

This study sought to investigate the influence of phoria adaptation on convergence peak velocity from responses located at different initial vergence positions.

Methods

Symmetrical 4° convergence step responses and near dissociated phoria (measured at 40 cm from the subject''s midline) were recorded from six subjects with normal binocular vision using an infrared limbus tracking system with a haploscope. Two different sustained fixations (1° and 16° convergent rotation along the subject''s midline) were used to study whether phoria had an influence on the peak velocity of convergence responses located at two initial vergence positions (1° or ‘far’ steps and 12° or ‘near’ steps).

Results

Phoria was significantly adapted after a sustained fixation task at near (16°) and far (1°) (p<0.002). A repeated measures ANOVA showed that convergence far steps were significantly faster than the near steps (p<0.03). When comparing convergence steps with the same initial vergence position, steps measured after near phoria adaptation were faster than responses after far adaptation (p<0.02). A regression analysis demonstrated that the change in phoria and the change in convergence peak velocity were significantly correlated for the far convergence steps (r = 0.97, p = 0.001). A weaker correlation was observed for the near convergence steps (r = 0.59, p = 0.20).

Conclusion

As a result of sustained fixation, phoria was adapted and the peak velocity of the near and far convergence steps was modified. This study has clinical considerations since prisms, which evoke phoria adaptation, can be prescribed to help alleviate visual discomfort. Future investigations should include a systematic study of how prisms may influence convergence and divergence eye movements for those prescribed with prisms within their spectacles.     

SARS-CoV 9b protein diffuses into nucleus, undergoes active Crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus

BACKGROUND: 9b is an accessory protein of the SARS-CoV. It is a small protein of 98 amino acids and its structure has been solved recently. 9b is known to localize in the extra-nuclear region and has been postulated to possess a nuclear export signal (NES), however the role of NES in 9b functioning is not well understood. PRINCIPAL FINDINGS/METHODOLOGY: In this report, we demonstrate that 9b in the absence of any nuclear localization signal (NLS) enters the nucleus by passive transport. Using various cell cycle inhibitors, we have shown that the nuclear entry of 9b is independent of the cell cycle. Further, we found that 9b interacts with the cellular protein Crm1 and gets exported out of the nucleus using an active NES. We have also revealed that this NES activity influences the half-life of 9b and affects host cell death. We found that an export signal deficient SARS-CoV 9b protein induces apoptosis in transiently transfected cells and showed elevated caspase-3 activity. CONCLUSION/SIGNIFICANCE: Here, we showed that nuclear shuttling of 9b and its interaction with Crm1 are essential for the proper degradation of 9b and blocking the nuclear export of this protein induces apoptosis. This phenomenon may be critical in providing a novel role to the 9b accessory protein of SARS-CoV.     

Ziehl-Neelsen staining technique can diagnose paragonimiasis

Background

We evaluated the Ziehl-Neelsen staining (ZNS) technique for the diagnosis of paragonimiasis in Laos and compared different modifications of the ZNS techniques.

Methodology

We applied the following approach: We (1) examined a paragonimiasis index case''s sputum with wet film direct examination (WF) and ZNS; (2) re-examined stored ZNS slides from two provinces; (3) compared prospectively WF, ZNS, and formalin-ether concentration technique (FECT) for sputum examination of patients with chronic cough; and (4) compared different ZNS procedures. Finally, we assessed excess direct costs associated with the use of different diagnostic techniques.

Principal Findings

Paragonimus eggs were clearly visible in WF and ZNS sputum samples of the index case. They appeared brownish-reddish in ZNS and were detected in 6 of 263 archived ZNS slides corresponding to 5 patients. One hundred sputum samples from 43 patients were examined with three techniques, which revealed that 6 patients had paragonimiasis (13 positive samples). Sensitivity per slide of the FECT, ZNS and the WF technique was 84.6 (p = 0.48), 76.9 (p = 0.25) and 61.5% (p = 0.07), respectively. Percentage of fragmented eggs was below 19% and did not differ between techniques (p = 0.13). Additional operational costs per slide were 0 (ZNS), 0.10 US$ (WF), and 0.79 US$ (FECT). ZNS heated for five minutes contained less eggs than briefly heated slides (29 eggs per slide [eps] vs. 42 eps, p = 0.01). Bloodstained sputum portions contained more eggs than unstained parts (3.3 eps vs. 0.7 eps, p = 0.016).

Conclusions/Significance

Paragonimus eggs can easily be detected in today''s widely used ZNS of sputum slides. The ZNS technique appears superior to the standard WF sputum examination for paragonimiasis and eliminates the risk of tuberculosis transmission. Our findings suggest that ZNS sputum slides should also be examined routinely for Paragonimus eggs. ZNS technique has potential in epidemiological research on paragonimiasis.     

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.     

Nucleosome-remodelling machines and other molecular motors observed at the single-molecule level

Through its capability to transiently pack and unpack our genome, chromatin is a key player in the regulation of gene expression. Single-molecule approaches have recently complemented conventional biochemical and biophysical techniques to decipher the complex mechanisms ruling chromatin dynamics. Micromanipulations with tweezers (magnetic or optical) and imaging with molecular microscopy (electron or atomic force) have indeed provided opportunities to handle and visualize single molecules, and to measure the forces and torques produced by molecular motors, along with their effects on DNA or nucleosomal templates. By giving access to dynamic events that tend to be blurred in traditional biochemical bulk experiments, these techniques provide critical information regarding the mechanisms underlying the regulation of gene activation and deactivation by nucleosome and chromatin structural changes. This minireview describes some single-molecule approaches to the study of ATP-consuming molecular motors acting on DNA, with applications to the case of nucleosome-remodelling machines.     

1H, 13C, 15N resonance assignment of the chitin-binding protein CBP21 from Serratia marcescens

The 18.8 kDa chitin-binding protein CBP21 from Serratia marcescens has been isotopically labeled and recombinantly expressed. In this paper, we report the ¹H, ¹³C, ¹⁵N resonance assignment of CBP21.