Development of a microarray assay that measures hybridization stoichiometry in moles

Microarray data is most useful when it can be compared with other genetic detection technologies. In this report, we designed a microarray assay format that transforms raw data into a defined scientific unit (i.e., moles) by measuring the amount of array feature present and the cDNA sequence hybridized. This study profiles a mouse reference universal RNA sample on a microarray consisting of PCR products. In measuring array features, a labeled DNA sequence was designed that hybridizes to a conserved sequence that is present in every array feature. To measure the amount of cDNA sample hybridized, the RNA sample was processed to ensure consistent dye to DNA ratio for every labeled target cDNA molecule, using labeled branched dendrimers rather than by incorporation. A dye printing assay was then performed in order to correlate molecules of cyanine dye to signal intensity. We demonstrate that by using this microarray assay design, raw data can be transformed into defined scientific units, which will facilitate interpretation of other experiments, such as data deposited at the Gene Expression Omnibus and ArrayExpress.     

Saving the endangered Mary River turtle: Enhancing conservation outcomes through community engagement

Australian biodiversity is facing an extinction crisis; yet, government spending on conservation is wholly inadequate. The involvement of local communities in fundraising, direct actions, and habitat restoration is becoming vital in the fate of threatened species. Here, we review the research outputs and impact generated from 22 years of conservation-driven collaboration between researchers and a local community focused on saving the endangered Mary River turtle (Elusor macrurus). The study found that this collaboration generated a significant body of research that advanced the ecological knowledge of the species and ensured the findings were being applied towards the conservation of the turtle, locally and nationally. While the national listing status of E. macrurus as endangered has not changed over the past 22 years, the knowledge gained about the turtle's biology and its use to better advise development and water resources in the catchment suggests that the species' future is brighter than when it was first discovered in 1994. This review demonstrates the potential of local communities in driving and supporting conservation initiatives and provides a blueprint for scientific endeavours that inform adaptive community conservation programmes for threatened species.     

Comparison of three methods for DNA extraction from bone remains

Extraction of amplifiable DNA is a frequent problem when working with degraded specimens like bone samples. The possibility of obtaining as much information as possible from these samples has a particular significance in many forensic investigations. The present investigation was aimed to assess the efficiency of three organic extraction methods for purifying amplifiable DNA from bone samples. The amount of nucleic acids obtained, the success rate in the amplification of DNA microsatellite (STR) markers and amelogenin by PCR, the influence of PCR inhibitors and environmental conditions, and where the samples were found before their processing in the laboratory, were all evaluated in this investigation for the three methods. Results showed that method A (a modification of FBI method for DNA extraction) performed better in producing not a higher amount but a better quality amplifiable DNA, in comparison with the other two methods evaluated. It was also demonstrated that the quality of the DNA to be amplified by PCR was influenced by the presence of inhibitors and/or contaminants and the environmental conditions where the bone sample was taken from. The worst conditions were observed from aquatic environments. The results suggest that the implementation of some specific modifications in the method A (use of purification columns, reliable quantification methods and different dilutions) would help to obtain better DNA extracts intended to be used in different molecular identification tests.     

When motion is not perceived: evidence from adaptation and dynamical stability

Adaptation was used to probe the perceiver's activation state when either motion or nonmotion percepts are formed for bistable, single-element apparent motion stimuli. Although adaptation was not observed in every instance, when it was observed its effect was to increase the probability of both motion-to-nonmotion and nonmotion-to-motion switches, the time scale of adaptation corresponding to neurophysiological observations for directionally selective cortical cells (Giaschi et al. 1993). This susceptibility to de-stabilizing adaptation effects indicated that the nonmotion percept was not the result of inadequate stimulation producing subthreshold levels of motion detector activation; if that were the case, activation-dependent adaptation would have decreased the nonmotion-to-motion switching rate by reducing activation further below threshold. Above-threshold activation levels are therefore associated with both nonmotion and motion perceptual states, and the failure to perceive motion despite the presence of adequate motion detector stimulation can be attributed to inhibitory competition between detectors activated by motion-specifying stimulus information and detectors activated to similar levels by motion-independent stimulus information, consistent with the dynamical quality of single-element apparent motion.     

A statistical view of FMRFamide neuropeptide diversity

FMRFamide-like peptide (FLP) amino acid sequences have been collected and statistically analyzed. FLP amino acid composition as a function of position in the peptide is graphically presented for several major phyla. Results of total amino acid composition and frequencies of pairs of FLP amino acids have been computed and compared with corresponding values from the entire GenBank protein sequence database. The data for pairwise distributions of amino acids should help in future structure-function studies of FLPs. To aid in future peptide discovery, a computer program and search protocol was developed to identify FLPs from the GenBank protein database without the use of keywords.     

2n+n Hybridization of Apomictic Paspalum dilatatum with Diploid Paspalum Species

Common dallisgrass (Paspalum dilatatum) is an apomictic pentaploid (2n=5x=50) of hybrid origin with irregular meiosis and with the genome formula IIJJX. The I and J genomes are homologous to those of diploid P. intermedium and P. jurgensii, respectively, but the source of the X genome is unknown. Members of the X genome may have genes of special biological significance, including those controlling apomixis. Common dallisgrass was crossed with several diploid Paspalum species in an attempt to identify the source of the X genome. Since common dallisgrass is apomictic, all hybrids produced will be formed by fertilization of an unreduced egg (2n+n). Any hybrid showing 30 chromosome bivalents at meiosis would indicate that the male diploid parent has a chromosome set that is homologous to the X genome of dallisgrass. Over 36,000 spikelets of dallisgrass were emasculated and dusted with pollen of 15 different diploid species (diploid species bearing I or J genomes were excluded). Only five (P. chaseanum, P. equitans, P. fasciculatum, P. notatum, and P. simplex) produced 2n+n hybrids with P. dilatatum. Meiotic chromosome behavior was similar in all hexaploid hybrids showing ca. 20 bivalents and 20 univalents. Results indicated a very low rate of 2n+n hybridization; none of the five diploid species possessed the X genome. Because several diploid species failed to hybridize with 5x dallisgrass, other methods should be attempted. Molecular markers specific for the X genome may help solve the question.     

Early post-larval development of the endoparasitic platyhelminth Mesocestoides corti: trypsin provokes reversible tegumental damage leading to serum-induced cell proliferation and growth

Mesocestoides corti is a suitable in vitro model for studying the development of human endoparasitic platyhelminthes. Treatment with trypsin, supplemented with fetal bovine serum (FBS), induces M. corti development from larvae (tetrathyridia) to segmented adult worm; however, the role of this protease and of FBS in post-larval development induction remains unknown. To characterize the participation of trypsin enzymatic activity and of FBS in the induction of tetrathyridia growth and development, both stimuli were added to the larvae either together or sequentially. Additionally, specific inhibition of trypsin activity was also monitored. Finally, the effect of the enzyme on the parasite tegument as well as the proliferative activity and location of proliferating cells after induction of tetrathyridia development were also studied. We conclude that trypsin-induced tetrathyridia development to adult worm is FBS-dependent and that the effect of serum factors is dependent upon a previous trypsin-induced reversible damage to the larva tegument. In dividing and non-dividing tetrathyridia, proliferative activity of cells is mainly located within the apical massif in the anterior region and nerve cords of larvae, respectively. In tetrathyridia stimulated to develop to adult worms, an intense proliferative activity is evident along the nerve cords. Our results suggest that in natural infections the tetrathyridia tegument is temporally made permeable to growth factors by proteolytic enzyme activity in the intestine juice of the definitive host, thus leading to development to adult worms.     

Mass Administration of Ivermectin for the Elimination of Onchocerciasis Significantly Reduced and Maintained Low the Prevalence of Strongyloides stercoralis in Esmeraldas,Ecuador

Objectives

To evaluate the effect of ivermectin mass drug administration on strongyloidiasis and other soil transmitted helminthiases.

Methods

We conducted a retrospective analysis of data collected in Esmeraldas (Ecuador) during surveys conducted in areas where ivermectin was annually administered to the entire population for the control of onchocerciasis.Data from 5 surveys, conducted between 1990 (before the start of the distribution of ivermectin) and 2013 (six years after the interruption of the intervention) were analyzed. The surveys also comprised areas where ivermectin was not distributed because onchocerciasis was not endemic.Different laboratory techniques were used in the different surveys (direct fecal smear, formol-ether concentration, IFAT and IVD ELISA for Strongyloides stercoralis).

Results

In the areas where ivermectin was distributed the strongyloidiasis prevalence fell from 6.8% in 1990 to zero in 1996 and 1999. In 2013 prevalence in children was zero with stool examination and 1.3% with serology, in adult 0.7% and 2.7%.In areas not covered by ivermectin distribution the prevalence was 23.5% and 16.1% in 1996 and 1999, respectively. In 2013 the prevalence was 0.6% with fecal exam and 9.3% with serology in children and 2.3% and 17.9% in adults.Regarding other soil transmitted helminthiases: in areas where ivermectin was distributed the prevalence of T. trichiura was significantly reduced, while A. lumbricoides and hookworms were seemingly unaffected.

Conclusions

Periodic mass distribution of ivermectin had a significant impact on the prevalence of strongyloidiasis, less on trichuriasis and apparently no effect on ascariasis and hookworm infections.     

Paraptosis Cell Death Induction by the Thiamine Analog Benfotiamine in Leukemia Cells

Benfotiamine is a synthetic thiamine analogue that stimulates transketolase, a cellular enzyme essential for glucose metabolism. Currently, benfotiamine is used to treat diabetic neuropathy. We recently reported that oral benfotiamine induced a temporary but remarkable recovery from acute myeloid leukemia in an elderly patient who was ineligible for standard chemotherapy due to dementia and renal failure. In the present study we present evidences that benfotiamine possess antitumor activity against leukemia cells. In a panel of nine myeloid leukemia cell lines benfotiamine impaired the viability of HL-60, NB4, K562 and KG1 cells and also inhibited the growing of primary leukemic blasts. The antitumor activity of benfotiamine is not mediated by apoptosis, necrosis or autophagy, but rather occurs though paraptosis cell death induction. Mechanistic studies revealed that benfotiamine inhibited the activity of constitutively active ERK1/2 and concomitantly increased the phosphorylation of JNK1/2 kinase in leukemic cells. In addition, benfotiamine induced the down regulation of the cell cycle regulator CDK3 which resulted in G1 cell cycle arrest in the sensitive leukemic cells. Moreover, combination index studies showed that benfotiamine enhanced the antiproliferative activities of cytarabine against leukemia cells. These findings suggest that benfotiamine has antitumor therapeutic potential.