SYBR Green Real-Time PCR-RFLP Assay Targeting the Plasmodium Cytochrome B Gene – A Highly Sensitive Molecular Tool for Malaria Parasite Detection and Species Determination

A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5–10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as ‘final positive’ if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5–100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7–100%) when compared against ‘final positive’ samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings.     

Noninvasive Assessment of Pulse-Wave Velocity and Flow-Mediated Vasodilation in Anesthetized G?ttingen Minipigs

Few methods for noninvasive assessment of arterial stiffness and endothelial dysfunction in porcine models are available. The aim of this study was to evaluate methods for assessment of arterial stiffness and endothelial dysfunction in anesthetized Göttingen minipigs. Pulse-wave velocity (PWV) was assessed in male Göttingen minipigs (n = 8; age approximately 60 wk) by using applanation tonometry of the carotid and femoral arteries. In addition, flow-mediated vasodilation (FMD) was assessed by using vascular ultrasonography of the brachial artery to evaluate endothelial dysfunction. To evaluate the reproducibility of the methods, minipigs were anesthetized by intravenous infusion of ketamine and midazolam and examined every other day for a total of 3 trials. Neither examination day nor systolic, diastolic, or mean arterial blood pressure statistically influenced PWV or FMD. The median interexamination coefficient of variation was 17% for PWV and 59% for FMD. Measured values of PWV corresponded largely to those in clinically healthy humans, but FMD values were lower than expected for lean, young animals. Although the ketamine–midazolam anesthesia we used has been associated with minor hemodynamic effects in vivo, in vitro studies suggest that both drugs are vasodilatory. Therefore anesthesia might have influenced the endothelial response, contributing to the modest FMD response and the concurrent high coefficients of variation that we noted. We conclude that PWV—but not FMD—showed acceptable interexamination variation for its potential application in porcine models.Abbreviations: FMD, flow-mediated vasodilation; FVI, integrated flow velocity; GTN, glyceryl trinitrate; PWV, pulse-wave velocity; T, transit timeCardiovascular disease has become a global challenge in public health,⁴² and the development and characterization of comparative animal models are of increasing importance. Several animal models of atherosclerosis, including porcine, have been described.^11,12,34 Due to similarities to humans in the anatomy of the cardiovascular system and metabolic physiology, pigs represent a generally useful model in regard to preclinical evaluation and pharmacology.³⁶ Assessment of changes related to atherosclerosis in vivo would be valuable in for example longitudinal assessment of drug effect, but few noninvasive methods for evaluating structural and functional changes in the arteries of pigs are available. In humans, increased arterial stiffness, which occurs with advanced age, also is caused by the pathophysiologic changes associated with atherosclerosis,²⁶ and noninvasive methods for assessing arterial stiffness have been established. The evaluation of pulse-wave velocity (PWV) by using pressure transducers, such as applanation tonometry, is a method recognized as an independent predictor for cardiovascular events in epidemiologic studies.²¹ The method evaluates the velocity with which the pulse wave is propagated through the arterial tree, with arterial stiffness causing increased velocity.^{1,22,29,37,39} Flow-mediated vasodilation (FMD), assessed by vascular ultrasonography, represents a noninvasive evaluation of endothelial-dependent vasodilation. A decrease in vasodilation as a response to increased shear stress has been recognized as a marker of endothelial dysfunction, which precedes the development of atherosclerosis.^5,7 Recent studies have shown that the FMD method is applicable in large animals (that is, dogs and horses) and that a decreased FMD response occurs in dogs with valvular heart disease.^10,15,23,28The aim of this study was to evaluate the reproducibility of methods for assessing arterial stiffness (PWV) and endothelial function (FMD response) in anesthetized Göttingen minipigs, including the influences of arterial blood pressure, heart rate, and room and body temperatures on these methods.     

The scramble for Africa: pan-temperate elements on the African high mountains

The composition of isolated floras has long been thought to be the result of relatively rare long-distance dispersal events. However, it has recently become apparent that the recruitment of lineages may be relatively easy and that many dispersal events from distant but suitable habitats have occurred, even at an infraspecific level. The evolution of the flora on the high mountains of Africa has been attributed to the recruitment of taxa not only from the African lowland flora or the Cape Floristic Region, but also to a large extent from other areas with temperate climates. We used the species rich, pan-temperate genera Carex, Ranunculus and Alchemilla to explore patterns in the number of recruitment events and region of origin. Molecular phylogenetic analyses, parametric bootstrapping and ancestral area optimizations under parsimony indicate that there has been a high number of colonization events of Carex and Ranunculus into Africa, but only two introductions of Alchemilla. Most of the colonization events have been derived from Holarctic ancestors. Backward dispersal out of Africa seems to be extremely rare. Thus, repeated colonization from the Northern Hemisphere in combination with in situ radiation has played an important role in the composition of the flora of African high mountains.     

Beta-D-(1→4)-galactan-containing side chains in RG-I regions of pectic polysaccharides from Biophytum petersianum Klotzsch. contribute to expression of immunomodulating activity against intestinal Peyer's patch cells and macrophages

The aerial parts of the medicinal plant Biophytum petersianum have a long tradition for being used in Mali and other West-African countries against various ailments such as wound healing and malaria. Previous studies on polysaccharides from water extracts of the aerial parts showed the presence of pectic like polymers with an effect on the human complement system as well as the ability to activate macrophages and dendritic cells.The present study shows that pectic polysaccharide fragments (BPII.1 and BPII.2) as well as the original pectic polysaccharide (BPII) expressed immunomodulating activity against Peyer’s patch immunocompetent cells. Exo-β-d-(1 → 3)-galactanase digestion succeeded to decrease IL-6 production enhancing activity against Peyer’s patch cells of BPII.2, but the activity of BPII.1 did not decrease. Endo-β-d-(1 → 4)-galactanase digestion reduced the activities of both BPII.1 and BPII.2. BPII.1 and BPII.2 also stimulated IL-6 production enhancing activity against macrophages, and the activities of both pectic fragments were significantly decreased by either enzymic digestion with exo-β-d-(1 → 3)-galactanase or endo-β-d-(1 → 4)-galactanase. Trimming of terminal GlcA by exo-β-d-glucuronidase digestion did not affect IL-6 production enhancing activity against macrophages of both pectic fragments. Methylation analyses of endo-β-d-(1 → 4)-galactanase digestion products showed the characteristic decrement of 4-linked Gal residues in the pectic fragments. These results suggest that β-d-(1 → 4)-galactan-containing side chains in BPII.1 and BPII.2 play an important role for expression of immunomodulating activity against both Peyer’s patch immunocompetent cells and macrophages in addition to β-d-(1 → 3,6)-galactan chains.     

An immunomodulating pectic polymer from Glinus oppositifolius

An immunomodulating pectic polymer, GOA1, obtained from the aerial parts of the Malian medicinal plant Glinus oppositifolius (L.) Aug. DC. (Aizoaceae) has previously been reported to consist of arabinogalactans type I and II, probably linked to a rhamnogalacturonan backbone. To further elucidate the structure of the polymer GOA1, enzymatic degradation studies and weak acid hydrolysis were performed. Five different glycosidases were used, endo-alpha-D-(1-->4)-polygalacturonase, exo-alpha-L-arabinofuranosidase, endo-alpha-L-(1-->5)-arabinanase, endo-beta-D-(1-->4)-galactanase and exo-beta-D-galactosidase. It appears that GOA1 may contain a structural moiety consisting of a 1,3-linked galactopyranosyl (Galp) main chain with 1,6-linked Galp side chains attached to position 6 of the main chain. The 1,6-linked Galp side chain may be branched in position 3 with arabinofuranosyl (Araf) side chains. A 1,4-linked Galp backbone which might carry side chains or glycosyl units attached to position 3 is also a structural element in the polymer. We further show that GOA1 induce proliferation of B cells and the secretion of IL-1beta by macrophages, in addition to a marked increase of mRNA for IFN-gamma in NK-cells. To elucidate structure-activity relations the native polymer and the digested fractions were tested for complement fixing activity and intestinal immune stimulating activity. The partial removal of Araf residues after enzymatic degradations did not affect the bioactivities, while the acid hydrolysed fraction showed reduced complement fixing activity. A decrease in Araf units, 1,3,6-linked Galp units and a partial hydrolysed rhamnogalacturonan backbone, in addition to a reduction in molecular weight are factors that might have contributed to reduced bioactivity.     

A rapid and inexpensive method for the direct PCR amplification of DNA from plants

? Premise of the study: We present a rapid and inexpensive alternative to DNA isolation for polymerase chain reaction (PCR) amplification from plants. ? Methods and Results: The method involves direct PCR amplification from material macerated in one buffer, followed by dilution and incubation in a second buffer. We describe the procedure and demonstrate its application for nuclear and plastid DNA amplification across a broad range of vascular plants. ? Conclusions: The method is fast, easy to perform, cost-effective, and consequently ideal for large sample numbers. It represents a considerable simplification of present approaches requiring DNA isolation prior to PCR amplification and will be useful in plant systematics and biotechnology, including applications such as DNA barcoding.     

Effect of sub-lethal exposure to ultraviolet radiation on the escape performance of Atlantic cod larvae (Gadus morhua)

The amount of ultraviolet (UV) radiation reaching the earth's surface has increased due to depletion of the ozone layer. Several studies have reported that UV radiation reduces survival of fish larvae. However, indirect and sub-lethal impacts of UV radiation on fish behavior have been given little consideration. We observed the escape performance of larval cod (24 dph, SL: 7.6±0.2 mm; 29 dph, SL: 8.2±0.3 mm) that had been exposed to sub-lethal levels of UV radiation vs. unexposed controls. Two predators were used (in separate experiments): two-spotted goby (Gobiusculus flavescens; a suction predator) and lion's mane jellyfish (Cyanea capillata; a "passive" ambush predator). Ten cod larvae were observed in the presence of a predator for 20 minutes using a digital video camera. Trials were replicated 4 times for goby and 5 times for jellyfish. Escape rate (total number of escapes/total number of attacks ×100), escape distance and the number of larvae remaining at the end of the experiment were measured. In the experiment with gobies, in the UV-treated larvae, both escape rate and escape distance (36%, 38±7.5 mm respectively) were significantly lower than those of control larvae (75%, 69±4.7 mm respectively). There was a significant difference in survival as well (UV: 35%, Control: 63%). No apparent escape response was observed, and survival rate was not significantly different, between treatments (UV: 66%, Control: 74%) in the experiment with jellyfish. We conclude that the effect and impact of exposure to sub-lethal levels of UV radiation on the escape performance of cod larvae depends on the type of predator. Our results also suggest that prediction of UV impacts on fish larvae based only on direct effects are underestimations.     

New genetic markers for identifying Cronartium flaccidum and Peridermium pini and examining genetic variation within and between lesions of Scots pine blister rust in Sweden

Microsatellite markers were developed as an identification tool and for analysis of the genetic variation in the pathogens Cronartium flaccidum and Peridermium pini, causing Scots pine blister rust in Pinus spp. Six reference aeciospore samples from Finland were used to examine genetic differences between the two pathogens. Genetic variation within and between 27 lesions on Scots pines from seven locations in Sweden was also investigated. Aeciospores were collected from single aecia within the lesions. Reference samples from P. pini were homozygous for all seven microsatellite loci investigated, while the three C.?flaccidum samples contained heterozygous loci. These results confirm previous studies, where homozygous aeciospores were indicated to be characteristic for P. pini. The majority of aeciospores had two nuclei in both heterozygotic and homozygotic samples. Five of the Swedish lesions contained only homozygotic aecia, while the aecia in the remaining 22 lesions were heterozygotic. All lesions with homozygotic aecia contained only one single multilocus genotype, while many of the lesions with heterozygotic aecia contained several genotypes. The latter indicates the occurrence of multiple matings within a lesion between the resident spermogonia and alien fertilizing spermatia.