Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.     

BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies

The vasculature develops primarily through two processes, vasculogenesis and angiogenesis. Although much work has been published on angiogenesis, less is known of the mechanisms regulating the de novo formation of the vasculature commonly called vasculogenesis. Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health-registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium-2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P<0.05). Analysis of the expression of 45 genes by quantitative real time-polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (alpha-smooth muscle actin [alpha SMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.     

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

Background

Cytokinin is a plant hormone that plays a crucial role in several processes of plant growth and development. In recent years, major breakthroughs have been achieved in the elucidation of the metabolism, the signal perception and transduction, as well as the biological functions of cytokinin. An important activity of cytokinin is the involvement in chloroplast development and function. Although this biological function has already been known for 50 years, the exact mechanisms remain elusive.

Results

To elucidate the effects of altered endogenous cytokinin content on the structure and function of the chloroplasts, chloroplast subfractions (stroma and thylakoids) from transgenic Pssu-ipt and 35S:CKX1 tobacco (Nicotiana tabacum) plants with, respectively, elevated and reduced endogenous cytokinin content were analysed using two different 2-DE approaches. Firstly, thykaloids were analysed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (BN/SDS-PAGE). Image analysis of the gel spot pattern thus obtained from thylakoids showed no substantial differences between wild-type and transgenic tobacco plants. Secondly, a quantitative DIGE analysis of CHAPS soluble proteins derived from chloroplast subfractions indicated significant gel spot abundance differences in the stroma fraction. Upon identification by MALDI-TOF/TOF mass spectrometry, these proteins could be assigned to the Calvin-Benson cycle and photoprotective mechanisms.

Conclusion

Taken together, presented proteomic data reveal that the constitutively altered cytokinin status of transgenic plants does not result in any qualitative changes in either stroma proteins or protein complexes of thylakoid membranes of fully developed chloroplasts, while few but significant quantitative differences are observed in stroma proteins.     

Membrane proteomic signatures of karyotypically normal and abnormal human embryonic stem cell lines and derivatives

Cultured human embryonic stem cells (hESCs) and derived derivatives contain heterogeneous cell populations with varying degrees of differentiation and karyotypic stability. The inability to isolate homogenous population presents a challenge toward cell-based applications and therapies. A proteomics approach was utilized to discover novel membrane proteins able to distinguish between the hESC lines BG01, WA09, and abBG02 (trisomy 12, 14, 17 and an extra copy of the X chromosome), along with WA09-derived human neural progenitor (hNP) cells. Membrane protein signatures were developed using sucrose-gradient isolation, 1-D gel electrophoresis followed by in-gel digestion and analysis by reverse phase chromatography coupled to ion trap-FT-ICR. At a ≤1.0% false discovery rate, 1918 proteins were identified; 775 were annotated as membrane proteins and 720 predicted to contain transmembrane spanning regions. Flow cytometry was used to validate cell surface expression of selected proteins. Junctional adhesion molecule 1 expression was shared by BG01, BG02 and abBG02 hESC lines. Dysferlin expression was specific to the WA09 hESC line and not the derived neural or mesenchymal progenitors. Ciliary neurotrophic factor receptor distinguished WA09-derived human neural progenitor cells from the parent hESC population, and WA09-derived mesenchymal progenitor cells. This study expands the current membrane protein data set for hESCs.     

Quantifying transmission of Campylobacter spp. among broilers

Campylobacter species are frequently identified as a cause of human gastroenteritis, often from eating or mishandling contaminated poultry products. Quantitative knowledge of transmission of Campylobacter in broiler flocks is necessary, as this may help to determine the moment of introduction of Campylobacter in broiler flocks more precisely. The aim of this study was to determine the transmission rate parameter in broiler flocks. Four experiments were performed, each with four Campylobacter-inoculated chicks housed with 396 contact chicks per group. Colonization was monitored by regularly testing fecal samples for Campylobacter. A mathematical model was used to quantify the transmission rate, which was determined to be 1.04 new cases per colonized chick per day. This would imply that, for example, in a flock of 20,000 broilers, the prevalence of Campylobacter would increase from 5% to 95% within 6 days after Campylobacter introduction. The model and the estimated transmission rate parameter can be used to develop a suitable sampling scheme to determine transmission in commercial broiler flocks, to estimate whether control measures can reduce the transmission rate, or to estimate when Campylobacter was introduced into a colonized broiler flock on the basis of the time course of transmission in the flock.     

Cytogenetic analyses of eight species in the genus <Emphasis Type="Italic">Leptodactylus</Emphasis> Fitzinger, 1843 (Amphibia,Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations

Background

The karyotypes of Leptodactylus species usually consist of 22 bi-armed chromosomes, but morphological variations in some chromosomes and even differences in the 2n have been reported. To better understand the mechanisms responsible for these differences, eight species were analysed using classical and molecular cytogenetic techniques, including replication banding with BrdU incorporation.

Results

Distinct chromosome numbers were found: 2n = 22 in Leptodactylus chaquensis, L. labyrinthicus, L. pentadactylus, L. petersii, L. podicipinus, and L. rhodomystax; 2n = 20 in Leptodactylus sp. (aff. podicipinus); and 2n = 24 in L. marmoratus. Among the species with 2n = 22, only three had the same basic karyotype. Leptodactylus pentadactylus presented multiple translocations, L. petersii displayed chromosome morphological discrepancy, and L. podicipinus had four pairs of telocentric chromosomes. Replication banding was crucial for characterising this variability and for explaining the reduced 2n in Leptodactylus sp. (aff. podicipinus). Leptodactylus marmoratus had few chromosomes with a similar banding patterns to the 2n = 22 karyotypes. The majority of the species presented a single NOR-bearing pair, which was confirmed using Ag-impregnation and FISH with an rDNA probe. In general, the NOR-bearing chromosomes corresponded to chromosome 8, but NORs were found on chromosome 3 or 4 in some species. Leptodactylus marmoratus had NORs on chromosome pairs 6 and 8. The data from C-banding, fluorochrome staining, and FISH using the telomeric probe helped in characterising the repetitive sequences. Even though hybridisation did occur on the chromosome ends, telomere-like repetitive sequences outside of the telomere region were identified. Metaphase I cells from L. pentadactylus confirmed its complex karyotype constitution because 12 chromosomes appeared as ring-shaped chain in addition to five bivalents.

Conclusions

Species of Leptodactylus exhibited both major and minor karyotypic differences which were identified by classical and molecular cytogenetic techniques. Replication banding, which is a unique procedure that has been used to obtain longitudinal multiple band patterns in amphibian chromosomes, allowed us to outline the general mechanisms responsible for these karyotype differences. The findings also suggested that L. marmoratus, which was formerly included in the genus Adenomera, may have undergone great chromosomal repatterning.

     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Tolerability and effects of OROS® MPH (Concerta®) on functioning, severity of disease and quality of life in children and adolescents with ADHD: results from a prospective, non-interventional trial

Attention-deficit/hyperactivity disorder may have substantial impact on family life, peer interactions, and quality of life. Stimulants are recommended as first-line pharmacotherapy for ADHD. OROS(?) MPH (Concerta(?)) is a long-acting preparation with duration of effect for up to 12 h. In this 8-week, prospective, open-label, non-interventional trial the impact of therapy with OROS(?) MPH on functioning in four different areas of life (school, recreation, family life, and peer interaction), severity of disease, and quality of life (QoL) as well as tolerability were investigated under daily routine care. 306 patients, aged 10.2±2.3 years, were either transitioned to OROS(?) MPH from short-acting, immediate-release MPH (-IR) preparations (n=231; 75%), or treatment was initiated with OROS(?) MPH in MPH-na?ve patients (n =75; 25%). In both groups, therapy with OROS(?) MPH was associated with significant improvements in daily functioning, severity of disease, and QoL. Adverse events (AE) were documented in 160 patients (52.3%). In 95 patients (31.0%) a causal relationship was assessed as at least possible. Four serious AEs were reported in 2 patients and rated as doubtfully related to study medication. Most frequent AEs (≥5% of patients) were insomnia, anorexia, ineffectiveness of medication, and headache. In 12.1% of patients AE led to discontinuation of study participation. Considering the limitations of this non-interventional study, the results refer to the importance of a therapy that covers not only school-time, but also takes other areas of life into account. Initiating treatment with long-acting preparations, such as OROS(?) MPH in MPH-na?ve patients might be a feasible option.     

Vergleich eines "glatten" und eines "rauhen" Stammes von Pseudomonas syringae pv. phaseolicola

Comparison of a “smooth” and a “rough” isolate of Pseudomonas syringae pv. phaseolicola The “smooth” (S) wild strain of Pseudomonas syringae pv. phaseolicola was compared with a “rough” (R) variant of low virulence. Both strains grew nearly equally well on a sucrose containing medium with yeast extract and casamino acids, and the strains did not differ markedly in the quantity of produced EPS (= extracellular polysaccharides). Principally the same results were obtained for high and medium concentrations of sucrose, or when sucrose was replaced by glucose or fructose. However, on glucose and fructose considerably lower quantities of EPS were produced. The biological activity of S-EPS was higher than that of R-EPS. This difference between the EPS preparations was not as marked as leaf inoculation with both bacterial isolates. After prolonged bacterial culture the EPS-production increased further, so that the differences between both strains decreased. A different EPS type was produced on the glycerol containing medium of KING B. Variations in the composition of this medium resulted in different morphology of the agar grown cultures, and the relative differences between S and R bacteria changed. When 62 different physiological tests for both bacterial strains were compared, the “rough” bacteria revealed a lowered range of positive reactions, with a few exceptions. However, it appeared unlikely that the reduced virulence of the “rough” bacteria was due to these differences. Obviously, defects in the extracellular products, but not in levan, were responsible for the reduction of virulence.