Zebrafish whole-adult-organism chemogenomics for large-scale predictive and discovery chemical biology

The ability to perform large-scale, expression-based chemogenomics on whole adult organisms, as in invertebrate models (worm and fly), is highly desirable for a vertebrate model but its feasibility and potential has not been demonstrated. We performed expression-based chemogenomics on the whole adult organism of a vertebrate model, the zebrafish, and demonstrated its potential for large-scale predictive and discovery chemical biology. Focusing on two classes of compounds with wide implications to human health, polycyclic (halogenated) aromatic hydrocarbons [P(H)AHs] and estrogenic compounds (ECs), we generated robust prediction models that can discriminate compounds of the same class from those of different classes in two large independent experiments. The robust expression signatures led to the identification of biomarkers for potent aryl hydrocarbon receptor (AHR) and estrogen receptor (ER) agonists, respectively, and were validated in multiple targeted tissues. Knowledge-based data mining of human homologs of zebrafish genes revealed highly conserved chemical-induced biological responses/effects, health risks, and novel biological insights associated with AHR and ER that could be inferred to humans. Thus, our study presents an effective, high-throughput strategy of capturing molecular snapshots of chemical-induced biological states of a whole adult vertebrate that provides information on biomarkers of effects, deregulated signaling pathways, and possible affected biological functions, perturbed physiological systems, and increased health risks. These findings place zebrafish in a strategic position to bridge the wide gap between cell-based and rodent models in chemogenomics research and applications, especially in preclinical drug discovery and toxicology.     

Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice

The family Acetobacteraceae currently includes three known nitrogen-fixing species, Gluconacetobacter diazotrophicus, G. johannae and G. azotocaptans. In the present study, acetic acid-producing nitrogen-fixing bacteria were isolated from four different wetland rice varieties cultivated in the state of Tamilnadu, India. Most of these isolates were identified as G. diazotrophicus on the basis of their phenotypic characteristics and PCR assays using specific primers for that species. Based on 16S rDNA partial sequence analysis and DNA: DNA reassociation experiments the remaining isolates were identified as Acetobacter peroxydans, another species of the Acetobacteraceae family, thus far never reported as diazotrophic. The presence of nifH genes in A. peroxydans was confirmed by PCR amplification with nifH specific primers. Scope for the findings: This is the first report of the occurrence and association of N2-fixing Gluconacetobacter diazotrophicus and Acetobacter peroxydans with wetland rice varieties. This is the first report of diazotrophic nature of A. peroxydans.     

Regulation of digestive enzyme activity in the larvae of Catopsilia crocale (Lepidoptera)

Amylase and invertase activity in the midgut tissue of the lepidopterous larvae Catopsilia crocale was studied in relation to the developmental stage and food availability. The enzyme activity increased significantly with advancing age of the larva. A direct positive correlation between food intake and enzyme activity was observed. A possible relationship between feeding and the regulation of enzyme activity is discussed.     

Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology

Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its’ reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c) or intramammary (i/mam) routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ) produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05) higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05). There was significant reduction (p<0.05) in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its’ native state was apparently not a suitable candidate for inclusion in a cell-free vaccine formulation against mastitis.     

Aggregation‐induced emission enhancement of anthracene‐derived Schiff base compounds and their application as a sensor for bovine serum albumin and optical cell imaging

Three anthracene‐based Schiff base complexes, R1–R3 ( R1 = (E)‐N´‐((anthracen‐10‐yl)methylene)benzohydrazide; R2 = (E)‐1‐((anthracen‐10‐yl)methylene)‐4‐phenylsemicarbazide; and R3 = (E)‐1‐((anthracen‐10‐yl)methylene)‐4‐phenylthiosemicarbazide) were synthesized from 9‐anthracenecarboxaldehyde, benzohydrazide, 4‐phenylsemicarbazide and 4‐phenylthiosemi‐carbazide respectively, and characterized by various spectral techniques. The absorption spectral characteristics of R1–R3 were bathochromically tuned to the visible region by extending the π conjugation. These target compounds were weakly fluorescent in tetrahydrofuran (THF) solution because of rapid isomerization of the C=N double bond in the excited state. However, the aqueous dispersion of R1–R3 in the THF/water mixture by the gradual addition of water up to 90% resulted in an increase in the fluorescence intensity mainly due to aggregation‐induced emission enhancement (AIEE) properties. The formation of nanoaggregates of R1–R3 were confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. The compounds R1–R3 are ideal probes for the fluorescence sensing of bovine serum albumin (BSA) and breast cancer cells by optical cell imaging.     

Sperm-mediated gene transfer in the silkworm Bombyx mori

Plasmid DNA containing the CAT reporter gene was injected into the testis of V instar silkworm larvae. The persistence, expression, and transmission of the injected DNA were monitored in the injected individuals till eclosion as well as in the progeny. The DNA injected into the testis persisted extrachromosomally during the entire period of metamorphosis and was also transferred into the egg via sperm during fertilization. Injected plasmids were rescued from the moths that emerged from the injected larvae and also from the eggs laid by the moths that copulated with injected males. Positive signals for CAT assay in the experimental samples suggested that the injected DNA was internalized in the testicular cells and sperm. The persistence, expression, and transmission of the DNA injected into the testes indicate that sperm-mediated gene transfer is possible in the silkworm, Bombyx mori. Arch. Insect Biochem. Physiol. 37:168–177, 1998. © 1998 Wiley-Liss, Inc.