Identifying the molecular mechanisms responsible for persistent effects of developmental exposure to chlorpyrifos on behavior

Chlorpyrifos (CPF) is one of the most widely used organophosphorus insecticides (OPs). The developmental exposure to low levels of CPF results in the inhibition of the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) and in altered emotional behavior (increased social play) without affecting the acetylcholinesterase, the canonical target of OPs. However, the molecular mechanisms responsible for this increased social play are not known. In this study, male rat pups were exposed orally to either corn oil, 0.75 mg/kg CPF, or 0.02 mg/kg PF-04457845 (PF; a specific inhibitor of FAAH) daily from postnatal day 10 (PND10) - PND16. This dosage of CPF does not alter brain cholinergic activity but inhibits FAAH. Once these rats reached adolescence (PND38), they were divided into two cohorts and each cohort contained all treatments. One cohort underwent social behavior testing and the other cohort remained naïve to behavioral testing. Following testing, the amygdala was collected from each cohort and protein expression was determined using a labelree shotgun proteomic approach. The obtained differentially expressed proteins from the different cohorts were analyzed by DAVID and Ingenuity Pathway Analysis software. Comparison of control non-behavior and control behavior rats suggests that social play altered the systems involved in the regulation of reward such as the opioid, dopaminergic, and serotonergic systems. These data also suggest that synaptic levels of GABA and glutamate increased during play. Comparison of non-behavior control and treated rats suggests that FAAH inhibition resulting from developmental exposure to CPF and PF persistently affects glutamatergic and GABAergic signaling. These data also suggest that there is a similar pattern of protein expression between CPF and PF. Comparison of the data from the behavioral groups of rats suggests that alterations in glutamatergic and GABAergic signaling and improper activation of opioid signaling could be responsible for the increased social play behavior. These alterations in the neurotransmitter signaling were observed in both CPF and PF treated rats. Overall, the results suggest that FAAH inhibition by either CPF or PF leads to alterations in opioid, glutamatergic, and GABAergic signaling that could be responsible for increased levels of social play

Evaluation of Dikamali as a Tablet Binder in Zidovudine Tablets

The aim of the present study is to evaluate the gum, Dikamali, as a tablet binder employing zidovudine as a model drug. Zidovudine tablets were prepared by wet granulation technique using Dikamali as a tablet binder. The Dikamali was used in wet form and dry form. Granules were evaluated for pre-compression parameters:  tapped density, bulk density, compressibility index, hausner ratio, and angle of repose. All the parameters were found to be within the acceptable limits. The tablets were evaluated for hardness, friability, weight variation, disintegration, content uniformity, and dissolution. For the formulations F1-F3D; F1-F3W; F4-F7 (see Table 1) the parameters of friability, disintegration time, and hardness were measured and their values range from 0.57-0.73% (w/w), 0.83-0.97% (w/w), 0.69-0.99% (w/w); 12-13 min, 10-12 min, 10-12 min; and 5-6.9 kg/cm2, 4.5-5.1 kg/cm2, 4.1-5.2 kg/cm2; respectively. The binding efficacy of Dikamali was compared with the standard binders, starch mucilage and polyvinyl pyrrolidone, using dissolution studies. The binders, Dikamali and starch, were compared at similar concentrations [2.5% (w/v), 5% (w/v), and 7.5% (w/v)], and the finalized formulation (F1D) was compared with a 10% (w/v) concentration of starch mucilage and a 10% (w/v) concentration of polyvinyl pyrrolidone (PVP). Dikamali [2.5% (w/v)] in dry form (i.e. F1D) showed the same percent drug release as that of the 10% (w/v) of starch mucilage and of polyvinyl pyrrolidone. In conclusion, Dikamali could well be used as a binding agent in the formulation of tablet dosage forms, and Dikamali is more effective in dry form than the wet form

Multiplex TaqMan® Quantitative PCR Assays for Host-Tick-Pathogen Studies Using the Guinea Pig-Tick-Rickettsia System

Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever Rickettsiosis (SFR); its immune system is more like the human immune system than that of the murine model, and guinea pigs develop characteristic clinical signs. Thus, we have a compelling interest in developing, expanding, and optimizing tools for use in our guinea pig-Amblyomma-Rickettsia system for understanding host-tick-pathogen interactions. With the design and optimization of the three multiplex TaqMan® qPCR assays described here, we can detect the two SFGR, their respective primary Amblyomma sp. vectors, and the guinea pig model as part of controlled experimental studies using tick-transmission of SFGR to guinea pigs. We developed qPCR assays that reliably detect each specific target down to 10 copies by producing plasmid standards for each assay target, optimizing the individual primer-probe sets, and optimizing the final multiplex reactions in a methodical, stepwise fashion. We anticipate that these assays, currently designed for in vivo studies, will serve as a foundation for optimal SFGR detection in other systems, including fieldwork

UCH-L5 regulates inflammasome activity in chicken macrophages.

<p><b>(A-B). UCH-L5 overexpression leads to reduced cell viability in infected as well as uninfected HD11 macrophages, which depends on its catalytic activity.</b> UCH-L5 was overexpressed in HD11 macrophages and empty vector was used as a control (Ctrl). 24-hours past overexpression, the cells were treated with b-AP15 (UCH-L5 inhibitor) used at indicated concentrations for 18 hours. The cell viability was measured by Presto Blue assay (A). Alternatively, UCH-L5 was overexpressed in HD11 macrophages for 2 days prior to infection with <i>Salmonella</i> Typhimurium for 1 hour. The cell viability was measured by Presto Blue assay. The p-values were calculated by using Student T test. <b>(C) UCH-L5 overexpression leads to an increase in caspase-1 activity in HD11 macrophages.</b> UCH-L5 was overexpressed in HD11 macrophages (UCH-L5) and empty vector was used as a control (ctrl). 24-hours past overexpression, the cells were subjected to <i>Salmonella</i> infection at MOI of 50:1 for 18-hours (inf) or left uninfected. The caspase-1 activity was measured by using specific inhibitor. The p-values were calculated by using Student T test. <b>(D). Overexpressed UCH-L5 is increased in infected cells.</b> The protein cell lysates from (C) were analyzed by SDS-PAGE, followed by anti-FLAG western blotting to demonstrate UCH-L5 expression; anti-GAPDH western blotting was used as a loading control. <b>(E). Caspase-1 activity in HD11 macrophages is dampened upon b-AP15 inhibitor treatment.</b> UCH-L5 was overexpressed in HD11 macrophages (UCH-L5-FLAG) and empty vector was used as a control. 24-hours past overexpression, cells were primed with 1μg/ml LPS for 4 hrs followed by treatment with 1uM b-AP15 or vehicle control (DMSO) for 15 minutes to inhibit the activity of UCH-L5. Cells were then treated with 10μM nigericin for 1 hour to induce inflammasome. The cell pellets were collected and caspase-1 activity was measured by using Z-YVAD-AFC substrate. The p-values were calculated by using Student T test. <b>(F). Exposure of cells to b-AP15 inhibitor leads to decrease in IL-1β secretion in HD11 macrophages upon inflammasome activation.</b> The HD11 cells were seeded on 6-well plates (1x10⁶ cells, 3 replicates each) and primed with LPS (1μg/ml) for 4 hours followed by treatment with 1uM b-AP15 (or vehicle control, DMSO) for 15 minutes. Cells were then treated with 10uM nigericin (or not) for 1 hour to induce inflammasome. Media were collected and used for detection of chicken IL-1β by western blot. <b>(G). Exposure of chicken HD11 macrophages to b-AP15 inhibitor leads to decrease in IL-1β secretion during <i>Salmonella</i> infection.</b> HD11 cells were treated with 1μM b-AP15 or DMSO (vehicle control) for 60 min. They were then infected (or not) with <i>Salmonella</i> Typhimurium wild-type at MOI 50:1 for 60 minutes. Media were collected for ELISA-based quantitation of chicken IL-1β (MyBioSource, Inc., USA).</p

Activity profiling of DUBs in <i>Salmonella</i>-infected cells by using ubiquitin-specific active-site probe.

<p><b>(A). DUB probe anatomy.</b> Ubiquitin-specific active-site probes consist of a retrieval element (tag), such as HA, which enables visualization and purification of a probe-bound DUB, as well as a ubiquitin molecule used for site recognition, and warhead (such as hereby used vinyl sulfone), which is a reactive group that interacts with DUB’s cysteine by formation of a thioester. <b>(B). Activity profiling by ubiquitin-specific active-site probe in <i>Salmonella</i>-infected macrophages.</b> HD11 macrophages were infected in duplicate for 0, 1, 2 and 18 hours with <i>Salmonella</i> Typhimurium at multiplicity of infection (MOI) of 50:1. After infection, the protein content was obtained and subjected to the enzymatic reaction with Ub-VS-HA probe. The proteins were separated by SDS-PAGE and subjected to anti-HA western blotting to visualize the active DUBs. One representative blot is shown. <b>(C, D)</b>. <b>Identification of DUBs regulated in HD11 macrophages during <i>Salmonella</i> infection by chemical proteomics.</b> HD11 macrophages were infected for 0 and 18 hours with <i>Salmonella</i> Typhimurium at MOI of 50:1. After infection, the protein content was obtained and subjected to the enzymatic reaction with the Ub-VS-HA probe. Probe-bound DUBs were immunoprecipitated by using anti-HA agarose and subjected to tryptic digestion. The peptide mixtures were then analyzed by quantitative proteomics (HPLC-MS/MS). The table shows names of the identified proteins, their accession numbers (NCBI), molecular weight, Fisher’s exact test p-values, fold change (calculated from the weighted spectral count in infected versus uninfected samples), protein identification probabilities as well as percent of protein sequence coverage [%] in individual replicas. Only the DUBs identified with high confidence are shown (C). A graph displays fold change (calculated using weighted spectral count) of DUBs and other relevant ubiquitin-binding proteins in infected versus uninfected samples (D). The abbreviations refer to the names of proteins from the table (C). <b>(E-F). Identification of UCH-L5 upregulated in infected HD11 macrophages upon infection.</b> Immunoprecipitated DUBs obtained from uninfected and infected HD11 macrophages as described in (C) were resolved on SDS-PAGE and subjected to silver staining prior to band excision and tryptic digestion of the indicated band. The identified DUB corresponded to chicken UCH-L5 (F). Accession, protein name, protein score, protein sequence coverage [%], number of identified peptides and Peptide spectrum matches (PSMs) as well as expected molecular weight are shown for the identified protein. For each one of three UCH-L5 peptides, sequence, modification, XCorr value, number of missed cleavages, delta Cn value, peptide rank, search engine rank, peptide charge, molecular weight of a precursor ion and molecular weight of the calculated singly charged peptide are shown, as well as delta mass [ppm], retention time [minute] and number of ions matched.</p

Skin in the Game: An Assay to Monitor Leukocyte Infiltration in Dermal Lesions of a Guinea Pig Model for Tick-Borne Rickettsiosis

Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host–pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens, Staphylococcus aureus infection, and Buruli ulcer caused by Mycobacterium ulcerans

UCH-L5 regulates inflammasome activity in human macrophages.

<p><b>(A). Exposure of THP-1 macrophages to b-AP15 inhibitor leads to decrease in IL-1β upon inflammasome activation.</b> The THP-1 cells were seeded on 6-well plates, treated with 100nM PMA for 24 hours and exposed to LPS treatment for hours, followed by treatment with 1μM b-AP15 or DMSO (vehicle control) for 60 min and treatment with nigericin for 60 minutes. Media were collected for western blotting of human IL-1β. <b>(B). Exposure of THP-1 macrophages to b-AP15 leads to decrease in IL-1β secretion upon <i>Salmonella</i> infection at different time points of infection.</b> The THP-1 cells were seeded on 24-well plates, treated with 100nM PMA for 24 hours and exposed to 1μM b-AP15 or DMSO (vehicle control) for 60 min. They were then infected with <i>Salmonella</i> Typhimurium wild-type at MOI 50:1 for indicated time points. Human IL-1β ELISA was used to quantify the amount of IL-1β I into the cell culture medium, which is shown in pg/ml. <b>(C). IL-1β secretion from <i>Salmonella</i>-infected control and NRLP3-deficient cells exposed and not exposed to b-AP15 inhibitor.</b> The THP-1 cells control cells as well as NLRP3-deficient cells (Invivogen Inc, USA) were seeded on 24-well plates, treated with 100nM PMA for 24 hours and exposed to b-AP15 or DMSO (vehicle control) for 60 min. They were then infected with <i>Salmonella</i> Typhimurium wild-type at MOI 50:1 for 1 hour. Human IL-1β ELISA was used to quantify the amount of IL-1β I into the cell culture medium, which is shown in pg/ml. <b>(D). The b-AP15 inhibitor does not affect IL-1β secretion from uninfected cells.</b> The THP-1 were treated with 100nM PMA for 24 hours and exposed to b-AP15 or DMSO (vehicle control) for 60 min. They were then infected or not with <i>Salmonella</i> Typhimurium wild-type for 30 minutes. Human IL-1β ELISA was used to quantify the amount of IL-1β I into the cell culture medium, which is shown in pg/ml. <b>(E-F). Partial knock-down of UCH-L5 in THP-1 macrophages leads to attenuation in IL-1β secretion in <i>Salmonella</i>-infected cells.</b> UCH-L5 was knocked-down in THP-1 macrophages by UCH-L5 siRNA (negative control siRNA was used as a control). After nucleofection was complete, new medium was added onto cells and cells were incubated for 24 hours prior to infection with <i>Salmonella</i> Typhimurium, MOI 50:1 for 1 hour. The cells were lysed and obtained proteins were resolved on SDS-PAGE and subjected to western blotting (Fig 4E; anti-UCH-L5, anti-GAPDH for loading control). IL-1β secretion to medium was quantified by ELISA (F). <b>(E). Model of UCH-L5’s effect on inflammasome activation in macrophages infected with <i>Salmonella</i> Typhimurium.</b></p