Development and characterization of a bacteriophage cocktail with high lytic efficacy against field-isolated Salmonella enterica.

Salmonella spp. is a prevalent pathogen that causes great public health concern worldwide. Bacteriophage-based cocktails have arisen as an alternative to antibiotics to inhibit the growth of Salmonella. However, the bactericidal effect of bacteriophage cocktails in vivo largely differs from their observed effect in vitro. This is partly because in vitro developments of cocktails do not always consider the bacterial diversity nor the environmental conditions where bacteriophages will have to replicate. Here, we isolated and sequenced 47 bacteriophages that showed variable degrees of lytic activity against 258 Salmonella isolates from a commercial broiler company in Brazil. Three of these bacteriophages were characterized and selected to assemble a cocktail. In vitro quantitative assays determined the cocktail to be highly effective against multiple serovars of Salmonella, including Minnesota and Heidelberg. Remarkably, the in vitro lytic activity of the cocktail was retained or improved in conditions that more closely resembled the chicken gut, such as anaerobiosis, 42°C, and Salmonella mono-strain biofilms. Analysis of bacterial cross-resistance between the 3 bacteriophages composing the cocktail revealed limited or no generation of cross-resistance. Our results highlight the relevance of an optimized flux of work to develop bacteriophage cocktails against Salmonella with high lytic efficacy and strong potential to be applied in vivo in commercial broiler farms.

Interaction analyses of hTAAR1 and mTAAR1 with antagonist EPPTB.

Trace amine-associated receptor 1 (TAAR1) plays a critical role in regulating monoaminergic activity. EPPTB is the only known selective potent antagonist of the mouse (m) TAAR1 presently, while it was shown to be weak at antagonizing human (h) TAAR1. The lack of high-resolution structure of TAAR1 hinders the understanding of the differences in the interaction modes between EPPTB and m/hTARR1. The purpose of this study is to probe these interaction modes using homology modeling, molecular docking, molecular dynamics (MD) simulations, and molecular mechanics-generalized Born surface area (MM-GBSA) binding energy calculations. Eight populated conformers of hTAAR1-EPPTB complex were observed during the MD simulations and could be used in structure-based virtual screening in future. The MM-GBSA binding energy of hTAAR1-EPPTB complex (-96.5 kcal/mol) is larger than that of mTAAR1-EPPTB complex (-106.7 kcal/mol), which is consistent with the experimental finding that EPPTB has weaker binding affinity to hTAAR1. The several residues in binding site of hTAAR1 (F1544.56, T1945.42 and I2907.39) are different from these of mTAAR1 (Y1534.56, A1935.42 and Y2877.39), which might contribute to the binding affinity difference. Our docking analysis on another hTAAR1 antagonist Compound 3 has found that: 1). this compound binds in different pockets of our mTAAR1 and hTAAR1 homology models with a slightly stronger binding affinity to hTAAR1; 2). both antagonists bind to a very similar pocket of hTAAR1.

Probing biased activation of mu-opioid receptor by the biased agonist PZM21 using all atom molecular dynamics simulation.

Morphine is a commonly used opioid drug to treat acute pain by binding to the mu-opioid receptor (MOR), but its effective analgesic efficacy via triggering of the heterotrimeric Gi protein pathway is accompanied by a series of adverse side effects via triggering of the ß-arrestin pathway. Recently, PZM21, a recently developed MOR biased agonist, shows preferentially activating the G protein pathway over ß-arrestin pathway. However, there is no high-resolution receptor structure in complex with PZM21 and its action mechanism remains elusive. In this study, PZM21 and Morphine were docked to the active human MOR-1 homology structure and then subjected to the molecular dynamics (MD) simulations in two different situations (i.e., one situation includes the crystal waters but another does not). Detailed comparisons between the two systems were made to characterize the differences in protein-ligand interactions, protein secondary and tertiary structures and dynamics networks. PZM21 could strongly interact with Y3287.43 of TM7, besides the residues (Asp1493.32 and Tyr1503.33) of TM3. The two systems' network paths to the intracellular end of TM6 were roughly similar but the paths to the end of TM7 were different. The PZM21-bound MOR's intracellular ends of TM5-7 bent outward more along with the distance changes of the three key molecular switches (ionic lock, transmission and Tyr toggle) and the distance increase of some conserved inter-helical residue pairs. The larger intracellular opening of the receptor could potentially facilitate G protein binding.

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-ß superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica.

STP position paper: interpreting the significance of increased alveolar macrophages in rodents following inhalation of pharmaceutical materials.

The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) appointed a working group to address risk assessment for increases in alveolar macrophages following inhalation of pharmaceutical materials. This position paper provides recommendations for inhalation study-specific terminology and interpretation based on literature and information from marketed inhaled drugs. Based on a weight-of-the-evidence approach, and with appropriate consideration of the physical and pharmacological characteristics of the compound, uncomplicated increases in the size or number of alveolar macrophages in nonclinical species are interpreted as nonadverse.

Chloroquine causes similar electroretinogram modifications, neuronal phospholipidosis and marked impairment of synaptic vesicle transport in albino and pigmented rats.

Retinal toxicity of chloroquine has been known for several years, but the mechanism(s) of toxicity remain controversial; some author support the idea that the binding of chloroquine to melanin pigments in the retinal pigmented epithelium (RPE) play a major toxic role by concentrating the drug in the eye. In our study, 12 albinos Sprague-Dawley (SD) and 12 pigmented Brown Norway (BN) rats were treated orally for 3 months with chloroquine to compare functional and pathological findings. On Flash electroretinograms (ERG) performed in scotopic conditions, similar and progressive (time-dependent) delayed onset and decreased amplitudes of oscillatory potentials (from Day 71) and b-waves (on Day 92) were identified in both BN and SD rats. In both strains, identical morphological changes consisted of neuronal phospholipidosis associated with UV auto-fluorescence without evidence of retinal degeneration and gliosis; the RPE did not show any morphological lesions or autofluorescence. IHC analyses demonstrated a decrease in GABA expression in the inner nuclear layer. In addition, a marked accumulation of synaptic vesicles coupled with a marked disruption of neurofilaments in the optic nerve fibers was identified. In conclusion, ERG observations were very similar to those described in humans. Comparable ERG modifications, histopathology and immunohistochemistry findings were observed in the retina of both rat strains suggesting that melanin pigment is unlikely involved. chloroquine-induced impairment of synaptic vesicle transport, likely related to disruption of neurofilaments was identified and non-previously reported. This new mechanism of toxicity may also be responsible for the burry vision described in humans chronically treated with chloroquine.

Proliferative and nonproliferative lesions of the rat and mouse respiratory tract.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the respiratory tract of laboratory rats and mice, with color photomicrographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for respiratory tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

1st international ESTP expert workshop: "Larynx squamous metaplasia". A re-consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment.

Invited international experts participated in a 2-day workshop organized by the European Society of Toxicologic Pathology (ESTP) to evaluate and discuss spontaneous and induced laryngeal lesions in rodents. The main purpose of the workshop was to agree upon the terminology and relevance of a range of laryngeal changes that varied from very subtle epithelial alterations up to severe metaplastic or neoplastic lesions. The workshop experts concluded that minimal, focal epithelial changes of the laryngeal epithelium, predominantly occurring at the base of the epiglottis, should be given the descriptive term of "epithelial alteration" and assessed as "non-adverse". Although observed as induced effects they may also occur in non-treated animals and were not considered to have a potential for a laryngeal dysfunction. Also, cases of minimal to slight laryngeal squamous metaplasia that are not observed diffusely could occur spontaneously or as treatment-induced lesions and should be assessed as "non-adverse". Cases of moderate to severe laryngeal squamous metaplasia observed diffusely in multiple levels should be regarded as "adverse", as there is a potential for dysfunction of the larynx. The occurrence of dysplasia or cellular atypia linked to laryngeal squamous metaplasia should always be reported separately and described in detail. In the evaluation of treatment-related effects of the larynx in studies utilizing aged animals, it has to be considered that moderate or even severe cases of focal laryngeal squamous metaplasia may occasionally be found as age-related, spontaneous lesions. Although inhalation exposure of rodents to non-genotoxic compounds may cause laryngeal squamous metaplasia, none of the workshop experts were aware of any reported cases of tumor induction in the larynx with a non-genotoxic compound. Therefore, for non-genotoxic compounds, the workshop experts did not regard laryngeal squamous metaplasia by itself as a precancerous lesion.

The hepatic transcriptome as a window on whole-body physiology and pathophysiology.

Transcriptomics can be a valuable aid to pathologists. The information derived from microarray studies may soon include the entire transcriptomes of most cell types, tissues and organs for the major species used for toxicology and human disease risk assessment. Gene expression changes observed in such studies relate to every aspect of normal physiology and pathophysiology. When interpreting such data, one is forced to look "far from the lamp post:' and in so doing, face one's ignorance of many areas of biology. The central role of the liver in toxicology, as well as in many aspects of whole-body physiology, makes the hepatic transcriptome an excellent place to start your studies. This article provides data that reveals the effects of fasting and circadian rhythm on the rat hepatic transcriptome, both of which need to be kept in mind when interpreting large-scale gene expression in the liver. Once you become comfortable with evaluating mRNA expression profiles and learn to correlate these data with your clinical and morphological observations, you may wonder why you did not start your studies of transcriptomics sooner. Additional study data can be viewed at the journal website at (www.toxpath.org). Two data files are provided in Excel format, which contain the control animal data from each of the studies referred to in the text,including normalized signal intensity data for each animal (n=5) in the 6-hour, 24-hour, and 5-day time points. These files are briefly described in the associated 'Readme' file, and the complete list of GenBank numbers and Affymetrix IDs are provided in a separate txt file. These files are available at http://taylorandfrancis.metapress.comlopenurl.asp?genre=journal&issn=0192-6233. Click on the issue link for 33(1), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through (www.toxpath.org).

Complementary roles for toxicologic pathology and mathematics in toxicogenomics, with special reference to data interpretation and oscillatory dynamics.

Toxicogenomics is an emerging multidisciplinary science that will profoundly impact the practice of toxicology. New generations of biologists, using evolving toxicogenomics tools, will generate massive data sets in need of interpretation. Mathematical tools are necessary to cluster and otherwise find meaningful structure in such data. The linking of this structure to gene functions and disease processes, and finally the generation of useful data interpretation remains a significant challenge. The training and background of pathologists make them ideally suited to contribute to the field of toxicogenomics, from experimental design to data interpretation. Toxicologic pathology, a discipline based on pattern recognition, requires familiarity with the dynamics of disease processes and interactions between organs, tissues, and cell populations. Optimal involvement of toxicologic pathologists in toxicogenomics requires that they communicate effectively with the many other scientists critical for the effective application of this complex discipline to societal problems. As noted by Petricoin III et al (Nature Genetics 32, 474-479, 2002), cooperation among regulators, sponsors and experts will be essential for realizing the potential of microarrays for public health. Following a brief introduction to the role of mathematics in toxicogenomics, "data interpretation" from the perspective of a pathologist is briefly discussed. Based on oscillatory behavior in the liver, the importance of an understanding of mathematics is addressed, and an approach to learning mathematics "later in life" is provided. An understanding of pathology by mathematicians involved in toxicogenomics is equally critical, as both mathematics and pathology are essential for transforming toxicogenomics data sets into useful knowledge.

Promotion of colon tumors in C57BL/6J-APC(min)/+ mice by thiazolidinedione PPARgamma agonists and a structurally unrelated PPARgamma agonist.

Thiazolidinedione PPARgamma agonists (troglitazone and rosiglitazone) were previously shown to promote colon tumor formation in C57BL/6J-APC(min)/+ mice, a model for human familial adenomatous polyposis. This study was conducted to determine if another thiazolidinedione PPARgamma agonist, pioglitazone, and a PPARgamma agonist structurally unrelated to the thiazolidinedione family, NID525, (a tetrazole-substituted phenoxymethylquinolone), would also promote colon tumors in this mouse model. Mice were treated in-feed with the thiazolidinediones troglitazone (150 mg/kg/day), rosiglitazone (20 mg/kg/day), or pioglitazone (150 mg/kg/day), or with NID525 (150 mg/kg/day) for 8 weeks. An increased incidence in colon tumors compared to controls was observed for all of the thiazolidinedione-treated groups as well as the NID525-treated group. These results indicate that the tumor-promoting effect of PPARgamma agonists in the colon of C57BL/6J-APC(min)/+ mice is likely related to the pharmacological activity of this group of drugs and not the thiazolidinedione structure.