Sensitive Detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by Loop-Mediated Isothermal Amplification

Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 10⁴ to 10⁵ CFU ml⁻¹, while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens.     

Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool

Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger. All enzymes were active toward p-nitrophenyl-β-d-galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose β-1,4 linked to either N-acetylglucosamine or fucose, such as N-glycans derived from bovine fibrin and Caenorhabditis elegans. In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae, all enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.     

From frog integument to human skin: dermatological perspectives from frog skin biology

For over a century, frogs have been studied across various scientific fields, including physiology, embryology, neuroscience, (neuro)endocrinology, ecology, genetics, behavioural science, evolution, drug development, and conservation biology. In some cases, frog skin has proven very successful as a research model, for example aiding in the study of ion transport through tight epithelia, where it has served as a model for the vertebrate distal renal tubule and mammalian epithelia. However, it has rarely been considered in comparative studies involving human skin. Yet, despite certain notable adaptations that have enabled frogs to survive in both aquatic and terrestrial environments, frog skin has many features in common with human skin. Here we present a comprehensive overview of frog (and toad) skin ontogeny, anatomy, cytology, neuroendocrinology and immunology, with special attention to its unique adaptations as well as to its similarities with the mammalian integument, including human skin. We hope to provide a valuable reference point and a source of inspiration for both amphibian investigators and mammalian researchers studying the structural and functional properties of the largest organ of the vertebrate body.     

High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei

In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite''s infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo. Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.     

A large genomic island allows Neisseria meningitidis to utilize propionic acid,with implications for colonization of the human nasopharynx

Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid‐generating bacteria. Data from classical microbiological and sequence‐based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp‐containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.     

A promising biological control agent for the invasive alien plant,Pereskia aculeata Miller (Cactaceae), in South Africa

Pereskia aculeata Miller (Cactaceae) is an invasive alien plant from Central and South America that has become a problematic environmental weed in South Africa. A potential biological control agent, the stem-wilter, Catorhintha schaffneri Brailovsky & Garcia (Coreidae), was collected in southern Brazil and imported into quarantine in South Africa. Field host range data suggested that C. schaffneri has a host range restricted to P. aculeata. No-choice nymph survival tests were then conducted on 27 test plant species in 9 families. Survival to the adult stage was only recorded on P. aculeata and the closely related Pereskia grandifolia Haw. (Cactaceae). Mortality was significantly higher on P. grandifolia with only 3% of the nymphs reaching the adult stage compared with 74% on P. aculeata indicating that P. aculeata is the primary host plant. P. grandifolia is native in South America and is of no agricultural importance in South Africa so any feeding on P. grandifolia in South Africa would have no negative environmental or economic consequences. In other tests, adult survival on P. aculeata [25.8 days (SE ± 3.74)] was significantly longer than on other test plant species [4.3 days (SE ± 0.36)] further confirming the host specificity of the species. Impact studies conducted in quarantine indicated that C. schaffneri is damaging to P. aculeata, significantly reducing the number of leaves and the shoot lengths of plants, even at relatively low insect densities. C. schaffneri is safe for release in South Africa and is likely to be a damaging and effective agent.     

Epitope detection chromatography: a method to dissect the structural heterogeneity and inter‐connections of plant cell‐wall matrix glycans

Plant cell walls are complex, multi‐macromolecular assemblies of glycans and other molecules and their compositions and molecular architectures vary extensively. Even though the chemistry of cell‐wall glycans is now well understood, it remains a challenge to understand the diversity of glycan configurations and interactions in muro, and how these relate to changes in the biological and mechanical properties of cell walls. Here we describe in detail a method called epitope detection chromatography analysis of cell‐wall matrix glycan sub‐populations and inter‐connections. The method combines chromatographic separations with use of glycan‐directed monoclonal antibodies as detection tools. The high discrimination capacity and high sensitivity for the detection of glycan structural features (epitopes) provided by use of established monoclonal antibodies allows the study of oligosaccharide motifs on sets of cell‐wall glycans in small amounts of plant materials such as a single organ of Arabidopsis thaliana without the need for extensive purification procedures. We describe the use of epitope detection chromatography to assess the heterogeneity of xyloglucan and pectic rhamnogalacturonan I sub‐populations and their modulation in A. thaliana organs.     

Collective Epithelial Migration Drives Kidney Repair after Acute Injury

Acute kidney injury (AKI) is a common and significant medical problem. Despite the kidney’s remarkable regenerative capacity, the mortality rate for the AKI patients is high. Thus, there remains a need to better understand the cellular mechanisms of nephron repair in order to develop new strategies that would enhance the intrinsic ability of kidney tissue to regenerate. Here, using a novel, laser ablation-based, zebrafish model of AKI, we show that collective migration of kidney epithelial cells is a primary early response to acute injury. We also show that cell proliferation is a late response of regenerating kidney epithelia that follows cell migration during kidney repair. We propose a computational model that predicts this temporal relationship and suggests that cell stretch is a mechanical link between migration and proliferation, and present experimental evidence in support of this hypothesis. Overall, this study advances our understanding of kidney repair mechanisms by highlighting a primary role for collective cell migration, laying a foundation for new approaches to treatment of AKI.