Diagnostic Accuracy of the Leishmania OligoC-TesT and NASBA-Oligochromatography for Diagnosis of Leishmaniasis in Sudan

Background

The Leishmania OligoC-TesT and NASBA-Oligochromatography (OC) were recently developed for simplified and standardised molecular detection of Leishmania parasites in clinical specimens. We here present the phase II evaluation of both tests for diagnosis of visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and post kala-azar dermal leishmaniasis (PKDL) in Sudan.

Methodology

The diagnostic accuracy of the tests was evaluated on 90 confirmed and 90 suspected VL cases, 7 confirmed and 8 suspected CL cases, 2 confirmed PKDL cases and 50 healthy endemic controls from Gedarif state and Khartoum state in Sudan.

Principal Findings

The OligoC-TesT as well as the NASBA-OC showed a sensitivity of 96.8% (95% CI: 83.8%–99.4%) on lymph node aspirates and of 96.2% (95% CI: 89.4%–98.7%) on blood from the confirmed VL cases. The sensitivity on bone marrow was 96.9% (95% CI: 89.3%–99.1%) and 95.3% (95% CI: 87.1%–98.4%) for the OligoC-TesT and NASBA-OC, respectively. All confirmed CL and PKDL cases were positive with both tests. On the suspected VL cases, we observed a positive OligoC-TesT and NASBA-OC result in 37.1% (95% CI: 23.2%–53.7%) and 34.3% (95% CI: 20.8%–50.9%) on lymph, in 72.7% (95% CI: 55.8%–84.9%) and 63.6% (95% CI: 46.6%–77.8%) on bone marrow and in 76.9% (95% CI: 49.7%–91.8%) and 69.2% (95% CI: 42.4%–87.3%) on blood. Seven out of 8 CL suspected cases were positive with both tests. The specificity on the healthy endemic controls was 90% (95% CI: 78.6%–95.7%) for the OligoC-TesT and 100% (95% CI: 92.9%–100.0%) for the NASBA-OC test.

Conclusions

Both tests showed high sensitivity on lymph, blood and tissue scrapings for diagnosis of VL, CL and PKDL in Sudan, but the specificity for clinical VL was significantly higher with NASBA-OC.     

High ALDH activity identifies chemotherapy-resistant Ewing's sarcoma stem cells that retain sensitivity to EWS-FLI1 inhibition

Background

Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing''s sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model – remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal.

Methodology/Principal Findings

We have isolated a subpopulation of Ewing''s sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDH^high) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDH^high cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing''s sarcoma cells both in vitro and in vivo.

Conclusions/Significance

Ewing''s sarcoma contains an ALDH^high stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing''s sarcoma stem cells that survive standard chemotherapy.     

Preparation of Intravenous Stealthy Acyclovir Nanoparticles with Increased Mean Residence Time

A major cause of thromboplebitis, during acyclovir (ACV) parenteral administration is the high pH of its reconstituted solution (pH 11). Its plasma half life is 2.5 h, requiring repeated administration which may result in excess of drug solubility leading to possible renal damage and acute renal failure. The present study reports the efficiency of stealthy ACV nanoparticles (NPs) to increase the mean residence time of the drug 29 times. It caused a marked decrease in thrombophlebitis when injected into rabbit’s ear vein. The polymers used were (Poly lactic acid, polylactic-co-glycolic (PLGA) 85/15, PLGA 75/25, PLGA 50/50). Particles were evaluated for their encapsulation efficiency, morphology, particle size and size distribution, zeta potential, and in vitro drug release. Small NPs (280–300 nm) with 60% drug release after 48 h were obtained. Among the block copolymer used, poloxamer 407 was of superior coating properties with a coat thickness in the range of 1.5–8.3 nm and a decreased surface charge.     

Poplar vulnerability to xylem cavitation acclimates to drier soil conditions

Xylem vulnerability to cavitation differs between tree species according to their drought resistance, more xerophilous species being more resistant to xylem cavitation. Variability in xylem vulnerability to cavitation is also found within species, especially between in situ populations. The origin of this variability has not been clearly identified. Here we analyzed the response of xylem hydraulic traits of Populus tremula×Populus alba trees to three different soil water regimes. Stem xylem vulnerability was scored as the xylem water potential causing 12, 50 and 88% loss of conductivity (P₁₂, P₅₀ and P₈₈). Vulnerability to cavitation was found to acclimate to growing conditions under different levels of soil water content, with P₅₀ values of ?1.82, ?2.03 and ?2.45 MPa in well‐watered, moderately water‐stressed and severely water‐stressed poplars, respectively. The value of P₁₂, the xylem tension at which cavitation begins, was correlated with the lowest value of midday leaf water potential (ψm) experienced by each plant, the difference between the two parameters being approximately 0.5 MPa, consistent with the absence of any difference in embolism level between the different water treatments. These results support the hypothesis that vulnerability to cavitation is a critical trait for resistance to drought. The decrease in vulnerability to cavitation under growing conditions of soil drought was correlated with decreased vessel diameter, increased vessel wall thickness and a stronger bordered pit field (t/b)². The links between these parameters are discussed.     

Natural Killer Cells Induce Eosinophil Activation and Apoptosis

Eosinophils are potent inflammatory cells with numerous immune functions, including antigen presentation and exacerbation of inflammatory responses through their capacity to release a range of largely preformed cytokines and lipid mediators. Thus, timely regulation of eosinophil activation and apoptosis is crucial to develop beneficial immune response and to avoid tissue damage and induce resolution of inflammation. Natural Killer (NK) cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and eosinophils. Co-culture experiments revealed that human NK cells could trigger autologous eosinophil activation, as shown by up-regulation of CD69 and down-regulation of CD62L, as well as degranulation, evidenced by increased CD63 surface expression, secretion of eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). Moreover, NK cells significantly and dose dependently increased eosinophil apoptosis as shown by annexin V and propidium iodide (PI) staining. Direct contact was necessary for eosinophil degranulation and apoptosis. Increased expression of phosphorylated extracellular signal-regulated kinase (ERK) in cocultured eosinophils and inhibition of eosinophil CD63 expression by pharmacologic inhibitors suggest that MAPK and PI3K pathways are involved in NK cell-induced eosinophil degranulation. Finally, we showed that NK cells increased reactive oxygen species (ROS) expression by eosinophils in co-culture and that mitochondrial inhibitors (rotenone and antimycin) partially diminished NK cell-induced eosinophil apoptosis, suggesting the implication of mitochondrial ROS in NK cell-induced eosinophil apoptosis. Pan-caspase inhibitor (ZVAD-FMK) only slightly decreased eosinophil apoptosis in coculture. Altogether, our results suggest that NK cells regulate eosinophil functions by inducing their activation and their apoptosis.     

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

Activated ezrin-radixin-moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate apical structures, including microvilli. Among many kinases implicated in ERM activation are the homologues LOK and SLK. CRISPR/Cas9 was used to knock out all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM-activating phosphorylation. The apical domains of cells lacking LOK/SLK or ERMs are strikingly similar and selectively altered, with loss of microvilli and with junctional actin replaced by ectopic myosin-II–containing apical contractile structures. Constitutively active ezrin can reverse the phenotypes of either ERM or LOK/SLK knockouts, indicating that a central function of LOK/SLK is to activate ERMs. Both knockout lines have elevated active RhoA with concomitant enhanced myosin light chain phosphorylation, revealing that active ERMs are negative regulators of RhoA. As RhoA-GTP activates LOK/SLK to activate ERM proteins, the ability of active ERMs to negatively regulate RhoA-GTP represents a novel local feedback loop necessary for the proper apical morphology of epithelial cells.     

A Novel Approach for the Synthesis of seco C-Nucleoside Analogues

Abstract

The synthesis of 4-amino-3-(D-gluco- or D-galacto-pentitol-1.yl)-5-mercapto-1,2,4-triazoles and their conversion to the respective 6-methyl-3-(1,2,3,4,5-penta-O-acetyl-pentitol-1-yl)1,2,4-triazolo[3,4-b]1,3,4-thiadiazoles have been achieved. The vicinal coupling constants were used to deduce the favored conformations.     

Epistasis,inbreeding depression,and the evolution of self-fertilization

Inbreeding depression resulting from partially recessive deleterious alleles is thought to be the main genetic factor preventing self-fertilizing mutants from spreading in outcrossing hermaphroditic populations. However, deleterious alleles may also generate an advantage to selfers in terms of more efficient purging, while the effects of epistasis among those alleles on inbreeding depression and mating system evolution remain little explored. In this article, we use a general model of selection to disentangle the effects of different forms of epistasis (additive-by-additive, additive-by-dominance, and dominance-by-dominance) on inbreeding depression and on the strength of selection for selfing. Models with fixed epistasis across loci, and models of stabilizing selection acting on quantitative traits (generating distributions of epistasis) are considered as special cases. Besides its effects on inbreeding depression, epistasis may increase the purging advantage associated with selfing (when it is negative on average), while the variance in epistasis favors selfing through the generation of linkage disequilibria that increase mean fitness. Approximations for the strengths of these effects are derived, and compared with individual-based simulation results.     

Histochemical and ultrastructural investigations on the renal parenchyma of the Egyptian Nile Monitor Lizard (Varanus niloticus)

Varanus niloticus (Linnaeus, 1766), the Nile monitor lizard, is considered the largest lizard in Africa and one of the most widespread. The Egyptian Nile monitor lizard exists in variable habitats, from grasslands to rainforests. This study pointed to investigate the light and ultrastructural features of the renal tissue of this lizard. Microscopically, the lizard nephron deprived from loop of henle and no demarcation could be detected between the cortical and medullary tissues. The renal corpuscles were small, but complex structures. The proximal convoluted tubules were lined by acidophilic cuboidal cells with hemosiderin pigment in their apical cytoplasm. The cytoplasm of the distal convoluted tubular cells reacted strongly with alcian blue stain. The sexual segment of the lizard kidney was lined with high columnar cells with massive periodic acid–Schiff-positive/alcian blue-negative supra-nuclear granules. Ultrastructurally, the basal infoldings of the proximal convoluted cells were evident. Supra-nuclear electron-dense vesicles were detected in the cytoplasm of the sexual segment cells. In summary, the kidney of the Egyptian Nile monitor lizard shares many histochemical features with other reptiles. However, they own several structural specializations in order to adapt to their harsh environments. Future studies focusing on the histochemical components of the sexual segment secretion would be required.     

4,4′-Bis(tert-butyl)-2,2′-bipyridinedichlorometal(II) - Synthesis, structure and EPR spectroscopy

Due to the better solubility of the 4,4′-substituted bipyridine ligand a series of 4,4′-bis(tert-butyl)-2,2′-bipyridinedichlorometal(II) complexes, [M(tbbpy)Cl₂], with M = Cu, Ni, Zn, Pd, Pt was synthesised and characterised. The blue copper complex 4,4′-bis(tert-butyl)-2,2′-bipyridinedichlorocopper(II) was isolated in two different polymorphic forms, as prisms 1 with a solvent inclusion and solvent-free as needles 2. Both structures were determined by X-ray structure analysis. They crystallise in the monoclinic space group P2₁/c with four molecules in the unit cell, but with different unit cells and packing motifs. Whereas in the prisms 1, with the unit cell parameters a = 12.1613(12), b = 10.6363(7), c = 16.3074(15) Å, β = 94.446(8)°, the packing is dominated by intra- and intermolecular hydrogen bonds, in the needles 2, with a = 7.738(1), b = 18. 333(2), c = 13.291(3) Å, β = 97.512(15)°, only intramolecular hydrogen bonds appear and the complex molecules are arranged in columns which are stabilised by π-π-stacking interactions. In both complexes the copper has a tetrahedrally distorted coordination sphere. These copper complexes were also studied by EPR spectroscopy in solution, as frozen glass and diamagnetically diluted powder with the analogue [Pd(tbbpy)Cl₂] as host lattice.