Prevalence and Risk Factors for Intestinal Protozoan Infections with Cryptosporidium,Giardia, Blastocystis and Dientamoeba among Schoolchildren in Tripoli,Lebanon

BackgroundIntestinal protozoan infections are confirmed as major causes of diarrhea, particularly in children, and represent a significant, but often neglected, threat to public health. No recent data were available in Lebanon concerning the molecular epidemiology of protozoan infections in children, a vulnerable population at high risk of infection.ConclusionsThis is the first study performed in Lebanon reporting the prevalence and the clinical and molecular epidemiological data associated with intestinal protozoan infections among schoolchildren in Tripoli. A high prevalence of protozoan parasites was found, with Blastocystis spp. being the most predominant protozoans. Although only 50% of children reported digestive symptoms, asymptomatic infection was observed, and these children may act as unidentified carriers. This survey provides necessary information for designing prevention and control strategies to reduce the burden of these protozoan infections, especially in children.     

Notes: β(1-3)Glucanosyltransferase Gel4p Is Essential for Aspergillus fumigatus

The β(1-3)glucanosyltransferase GEL family of Aspergillus fumigatus contains 7 genes, among which only 3 are expressed during mycelial growth. The role of the GEL4 gene was investigated in this study. Like the other Gelps, it encodes a glycosylphosphatidylinositol (GPI)-anchored protein. In contrast to the other β(1-3)glucanosyltransferases analyzed to date, it is essential for this fungal species.β(1-3)Glucan is the main component of the fungal cell wall (11). In fungi, β(1-3)glucans are synthesized by a plasma membrane-bound glucan synthase complex. Neosynthesized glucans are then extruded into the periplasmic space (2, 3, 9), where they become branched and covalently linked to other cell wall components, resulting in the formation of three-dimensional rigid structures. In the search of transglycosidase in the filamentous fungus Aspergillus fumigatus, β(1-3)glucanosyltransferases were identified and classified as a unique family (GH72) in the Carbohydrate-Active enZYmes database (http://www.cazy.org/). These enzymes cleave the β(1-3) bond of a β(1-3)glucan oligosaccharide with at least 10 glucose units and transfer the newly formed reducing end (>5 glucose units) to the nonreducing end of another β(1-3)glucan oligosaccharide, resulting in the elongation of the β(1-3)glucans. This reaction can proceed in vitro until the neosynthetized β(1-3)glucan becomes insoluble. Initially demonstrated biochemically, the requirement for long-chain β(1-3)glucan oligosaccharide has now been confirmed by the analysis of the first crystal structure obtained in this transglycosidase family (7, 8). First discovered in Aspergillus fumigatus and named Gelp for glucan elongase, this activity has been found in all fungal species investigated to date and could be assigned to orthologous proteins, such as Gasp or Phrp, that were known to be involved in cell wall integrity but were endowed with an unknown biochemical function (12, 13, 14).     

Brassicaceae INDEHISCENT genes specify valve margin cell fate and repress replum formation

Members of the Brassicaceae family, including Arabidopsis thaliana and oilseed rape (Brassica napus), produce dry fruits that open upon maturity along a specialised tissue called the valve margin. Proper development of the valve margin in Arabidopsis is dependent on the INDEHISCENT (IND) gene, the role of which in genetic and hormonal regulation has been thoroughly characterised. Here we perform phylogenetic comparison of IND genes in Arabidopsis and Brassica to identify conserved regulatory sequences that are responsible for specific expression at the valve margin. In addition we have taken a comparative development approach to demonstrate that the BraA.IND.a and BolC.IND.a genes from B. rapa and B. oleracea share identical function with Arabidopsis IND since ethyl methanesulphonate (EMS) mutant alleles and silenced transgenic lines have valve margin defects. Furthermore we show that the degree of these defects can be fine‐tuned for crop improvement. Wild‐type Arabidopsis produces an outer replum composed of about six cell files at the medial region of the fruits, whereas Brassica fruits lack this tissue. A strong loss‐of‐function braA.ind.a mutant gained outer replum tissue in addition to its defect in valve margin development. An enlargement of replum size was also observed in the Arabidopsis ind mutant suggesting a general role of Brassicaceae IND genes in preventing valve margin cells from adopting replum identity.     

Towards robust cell culture processes — Unraveling the impact of media preparation by spectroscopic online monitoring

Biopharmaceutical manufacturing processes can be affected by variability in cell culture media, e.g. caused by raw material impurities. Although efforts have been made in industry and academia to characterize cell culture media and raw materials with advanced analytics, the process of industrial cell culture media preparation itself has not been reported so far. Within this publication, we first compare mid‐infrared and two‐dimensional fluorescence spectroscopy with respect to their suitability as online monitoring tools during cell culture media preparation, followed by a thorough assessment of the impact of preparation parameters on media quality. Through the application of spectroscopic methods, we can show that media variability and its corresponding root cause can be detected online during the preparation process. This methodology is a powerful tool to avoid batch failure and is a valuable technology for media troubleshooting activities. Moreover, in a design of experiments approach, including additional liquid chromatography–mass spectrometry analytics, it is shown that variable preparation parameters such as temperature, power input and preparation time can have a strong impact on the physico‐chemical composition of the media. The effect on cell culture process performance and product quality in subsequent fed‐batch processes was also investigated. The presented results reveal the need for online spectroscopic methods during the preparation process and show that media variability can already be introduced by variation in media preparation parameters, with a potential impact on scale‐up to a commercial manufacturing process.     

Serial cultivation of chicken keratinocytes, a composite cell type that accumulates lipids and synthesizes a novel beta-keratin

The epidermis of birds differs from that of mammals by the presence of intracellular lipid droplets and the absence of sebaceous glands. We describe here the cultivation of chicken epidermal keratinocytes; these cells cannot be grown in medium supplemented with the usual fetal bovine serum even in the presence of supporting 3T3 cells, but they can grow from single cells in the presence of supporting 3T3 cells and 10% chicken serum. As revealed by their cell structure, their protein composition, and their gene expression, chicken keratinocytes possess the general properties of mammalian keratinocytes. They ultimately undergo in culture a process of terminal differentiation in which their nucleus is destroyed and a cornified envelope is formed. Chicken keratinocytes also show important properties that mammalian keratinocytes do not possess: they accumulate neutral lipids, usually in the form of a single perinuclear droplet; they accumulate carotenoids; they synthesize beta-keratins; and their multiplication requires a non-lipid factor, present in chicken serum but not in fetal calf serum. The lipid-synthesizing function of sebocytes in mammals is carried out by the keratinocytes themselves in birds. The availability of cultured chicken keratinocytes should allow studies that were hitherto impossible such as the tracing of the keratinocyte lineage during development of the chicken embryo and the investigation of the complete life cycle of viruses that require specific chicken keratinocyte products (such as Marek's disease virus).     

BACE-1 inhibitory activities of new substituted phenyl-piperazine coupled to various heterocycles: chromene, coumarin and quinoline

The protease beta-secretase plays a central role in the synthesis of pathogenic amyloid-beta in Alzheimer's disease. Here, we report a new series of analogues based on the phenyl-piperazine scaffold coupled to various heterocyclic moieties, which demonstrate improved inhibitory activities on BACE-1 (FRET assay) compared to already known naphthyl counterparts. The obtained results suggest further structural modifications to access to more potent BACE-1 inhibitors.     

Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis

During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.     

Mutating RBF Can Enhance Its Pro-Apoptotic Activity and Uncovers a New Role in Tissue Homeostasis

The tumor suppressor retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb (RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, we generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBF^D253A, conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. We show that this overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo.