Toxoplasma gondii grown in human cells uses GalNAc-containing glycosylphosphatidylinositol precursors to anchor surface antigens while the immunogenic Glc-GalNAc-containing precursors remain free at the parasite cell surface

Toxoplasma gondii is a ubiquitous parasite that infects nearly all warm-blooded animals. Developmental switching in T. gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for the disease propagation after alteration of the immune status of the carrier. The redifferentiation event is characterized by an over expression of a tachyzoite specific set of glycosylphosphatidylinositol anchored surface antigens and free GPIs. T. gondii grown in animal cells uses two glycosylphosphatidylinositol precursors to anchor the parasite surface proteins. The first form has an N-acetylgalactosamine residue bound to a conserved three-mannosyl core glycan, while the second structure contains an additional terminal glucose linked to the N-acetylgalactosamine side branch. Sera from persons infected with T. gondii reacted only with the glucose-N-acetylgalactosamine-containing structure. Here we report that T. gondii cultured in human cells uses predominantly the N-acetylgalactosamine-containing structure to anchor the parasite surface antigens. On the other hand, glycosylphosphatidylinositol structures having an additional terminal glucose are found exclusively on the parasite cell surface as free glycolipids participating in the production of cytokines that are implicated in the pathogenesis of T. gondii. We also provide evidence that such free glycosylphosphatidylinositols are restricted mainly to the lipid microdomains in the parasite cell surface membrane and mostly associated with proteins involved in the parasite motility as well as invasion of the host cell.     

Inhibitors of glycosyl-phosphatidylinositol anchor biosynthesis

Glycosyl-phosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa such as Trypanosoma brucei, Leishmania major, Plasmodium falciparum and Toxoplasma gondii, and represent the major carbohydrate modification of many cell-surface parasite proteins. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. Therefore, the characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. In vitro and in vivo studies using sugars and substrate analogues as well as natural compounds have shown that it is possible to interfere with GPI biosynthesis at different steps in a species-specific manner. Here we review the recent and promising progress in the field of GPI inhibition.     

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.     

Assessment of losses in honey yield due to the chalkbrood disease, with reference to the determination of its economic injury levels in Egypt

In Egypt, the chalk brood (CHB) disease caused by the fungus, Ascosphaera apis Maassen started again infesting the honeybee, Apis melifera L (Hymenoptera: Apidae) colonies after a cessation pause of seven to nine years. For the first time, an attempt has been made to look into the problem of the CHB disease from the view point of assessing losses in both clover (Trifolium alexandrinum) and chinus (Chinus molus) honey yields. In this regard, two techniques were adopted. Under the natural conditions of the experimental apiary (first technique), loss in clover honey was 18.412 +/- 0.663%, on average, whereas the average loss in chinus honey was 18.332 +/- 1.536%. In the second technique of loss appraisal (artificial infection). The tested honeybee colonies were artificially infected with four sources of A. apis infection, I.e. black mummies, white mummies, pollen grains and water, in addition to controls to create the so-called "different levels of infection". The mean percentages of losses in clover honey were 30.06 +/- 1.807, 27.95 +/- 1.062; 21.13 +/- 0.987; 16.96 +/- 0.672 and 0.00 +/- 0.00 for black mummies, white mummies, pollen grains, water and control, respectively. Taking into account the relationship between number of resulted mummies in each source of infection and % loss in clover honey, it could be concluded that as the resulted mummies increased, the corresponding clover honey yield decreased in each source of infection. It was noticed that the % loss in clover honey differed as the used technique differed. For example, the percentage loss in clover honey produced from colonies exposed to the natural conditions was relatively less than that of the artificially infected ones. This has been discussed in the text. However, the causative pathogen of CHB disease resulted in serious decrease in honey production. Loss appraisal is a perquisite step for the determination of the economic injury levels (EILs). By regressing % loss in clover honey yield against the total number of fallen mummies, the linear equation was worked out to be: Y = 0.242 + 0.040 x (r2 = 0.99, F = 62408.865). Depending on the total costs of controlling CHB disease, the EIL values were 31.875 and 11.250 mummies/colony for cedar oil and thymol granules, subsequently. As to the chinus honey the corresponding EILs were 18.940 and 6.683 mummies/colony for cedar oil and thymol granules, in respect, as a result of adopting the regression formula: Y = -6.762 + 0.101 X. As for the clover honey (artificial infection) the EIL values were 39.844 and 14.063 mummies/ colony in case of using cedar oil and thymol grains, subsequently. These values were worked out using the formula Y = 5.871 + 0.032 X. From the practical point of view, apiarists should not use thymol or cedar oil as fallen mummies don't reach the suggested EILs values to minimize control costs as much as possible.     

Bacterial Endosymbioses of Gutless Tube-Dwelling Worms in Nonhydrothermal Vent Habitats

Gutless tube-dwelling worms of pogonophorans (also known as frenulates) and vestimentiferans depend on primary production of endosymbiotic bacteria. The endosymbionts include thiotrophs that oxidize sulfur for autotrophic production and methanotrophs that oxidize and assimilate methane. Although most of the pogonophoran and vestimentiferan tube worms possess single thiotrophic 16S rRNA genes (16S rDNA) related to γ-proteobacteria, some pogonohorans are known to bear single methanotroph species or even dual symbionts of thiotrophs and methanotrophs. The vestimentiferan Lamellibrachia sp. L1 shows symbiotic 16S rDNA sequences of α-, β-, γ-, and ε-proteobacteria, varying among specimens, with RuBisCO form II gene (cbbM) sequences related to β-proteobacteria. An unidentified pogonophoran from the world’s deepest cold seep, 7326-m deep in the Japan Trench, hosts a symbiotic thiotroph based on 16S rDNA with the RuBisCO form I gene (cbbL). In contrast, a shallow-water pogonophoran (Oligobrachia mashikoi) in coastal Japan Sea has a methanotrophic 16S rDNA and thiotrophic cbbL, which may suggest the feature of type X methanotrophs. These observations demonstrate that pogonophoran and vestimentiferan worms have higher plasticity in bacterial symbioses than previously suspected.     

Comparative analysis of cytokines and growth factors in the conditioned media of stem cells from the pulp of deciduous,young, and old permanent tooth

ObjectivesTo compare and analyze the secretome profile of stem cells obtained from the deciduous tooth (SHEDs), young (yDPSCs), and old permanent tooth (oDPSCs).MethodsAll the stem cells were assessed for mesenchymal stem cell markers. The stem cells were differentiated into osteoblasts and chondrocytes using lineage-specific differentiation media. Conditioned media was collected from growing stem cells, and a cytometric bead array was performed to estimate secreted cytokines and growth factor levels by flow cytometry. Gene expresseion levels were assessed by real-time quantitative polymerase chain reaction.ResultsAge did not affect the mesenchymal characteristics of dental stem cells from various age groups. The secretomes of SHEDs and young yDPSCs exhibit more growth factors and lesser pro-inflammatory cytokines than oDPSCs. Osteo and chondrogenic differentiation potential were higher in SHEDs and young yDPSCs than in the oDPSCs. TLR1, TLR2, TLR3 show decreased expression levels with age and TLR5, TLR6 show increased expression with age.ConclusionThe superior regenerative potential of SHEDs and yDPSCs may be due to the higher growth factors and lower pro-inflammatory cytokine profile.     

Triphala extract negates arecoline-induced senescence in oral mucosal epithelial cells in vitro

BackgroundArecoline found in areca nut causes oral submucous fibrosis. Triphala is an Ayurvedic medicinal preparation used to improve overall physical wellness that has also been shown to improve oral health.ObjectivesTo assess the activity of Triphala extract on arecoline-induced senescence in oral mucosal epithelial cells in vitro.Materials and methodsOral mucosal epithelial cells were isolated and cultured in vitro. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to assess the viability of treated cells, while senescence was assessed by senescence-associated-β-galactosidase staining. Cell surface marker expression was analyzed by flow cytometry. Finally, real-time quantitative polymerase chain reaction was performed to examine gene expression levels.ResultsTriphala extract (5 µg/mL) reversed the cell senescence activity of arecoline, as evidenced by reduced β-galactosidase activity, increased Ki-67 marker expression, and reduced expression of senescence-related genes p16 and p21.ConclusionTriphala extract helped to reduce the pathological effects of arecoline-induced pathogenesis.Clinical relevance.Arecoline found in the areca nut causes oral pathological conditions including oral submucous fibrosis. Our results showed that Triphala counteracted the adverse effects of arecoline, in particular, negating senescence in oral mucosal epithelial cells. As a translational effect, Triphala treatment could restore normal epithelial thickness in oral submucous fibrosis, thus reducing the clinical severity of the disease. This reestablishment of oral homeostasis would help to improve oral health-related quality of life in patients with oral submucous fibrosis.     

Oxidative stress and DNA damage in relation to transition metals overload in Abu-Qir Bay,Egypt

The aim of the present study is to evaluate the transition metals overload in Abu-Qir Bay in Egypt, as compared to a less polluted area (reference area) through some biomarkers of oxidative stress. Catalase enzyme activity, malondialdehyde (MDA) concentration and DNA damage (number of apurinic/apyrimidinic sites) were the tested biomarkers. The levels of iron and copper in Mugil cephalus liver tissues were significantly higher in samples from the polluted area as compared to the reference area: Fe: 407 ± 38 vs. 216 ± 21 μg/g wet wt; p = 0.008, Cu: 54 ± 6 vs. 17.7 ± 4 μg/g wet wt; p = 0.0001. This could account for the observed increase in MDA concentration (15.7 ± 5.7 vs. 2.5 ± 0.5 U/g; p = 0.035), and the elevated number of AP sites (13.9 ± 2.6 vs. 0.37 ± 0.2 AP site/1 × 10⁵ bp; p = 0.0001). Similarly, the activity of catalase enzyme responsible for the cellular defense was significantly high (58.3 ± 12.2 vs. 28.4 ± 4.0 U/mg; p = 0.032). The present data indicated a clear relationship between the pollution degree of the above marine environment and both biochemical and molecular responses of the piscine system.     

Optimizing anti-gene oligonucleotide ‘Zorro-LNA’ for improved strand invasion into duplex DNA

Zorro-LNA (Zorro) is a newly developed, oligonucleotide (ON)-based, Z-shaped construct with the potential of specific binding to each strand of duplex DNA. The first-generation Zorros are formed by two hybridized LNA/DNA mixmers (2-ON Zorros) and was hypothesized to strand invade. We have now established a method, which conclusively demonstrates that an LNA ON can strand invade into duplex DNA. To make Zorros smaller in size and easier to design, we synthesized 3′–5′–5′–3′ single-stranded Zorro-LNA (ssZorro) by using both 3′- and 5′-phosphoramidites. With ssZorro, a significantly greater extent and rate of double-strand invasion (DSI) was obtained than with conventional 2-ON Zorros. Introducing hydrophilic PEG-linkers connecting the two strands did not significantly change the rate or extent of DSI as compared to ssZorro with a nucleotide-based linker, while the longest alkyl-chain linker tested (36 carbons) resulted in a very slow DSI. The shortest alkyl-chain linker (3 carbons) did not reduce the extent of DSI of ssZorro, but significantly decreased the DSI rate. Collectively, ssZorro is smaller in size, easier to design and more efficient than conventional 2-ON Zorro in inducing DSI. Analysis of the chemical composition of the linker suggests that it could be of importance for future therapeutic considerations.