Whole-brain mapping of direct inputs to midbrain dopamine neurons

Recent studies indicate that dopamine neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) convey distinct signals. To explore this difference, we comprehensively identified each area's monosynaptic inputs using the rabies virus. We show that dopamine neurons in both areas integrate inputs from a more diverse collection of areas than previously thought, including autonomic, motor, and somatosensory areas. SNc and VTA dopamine neurons receive contrasting excitatory inputs: the former from the somatosensory/motor cortex and subthalamic nucleus, which may explain their short-latency responses to salient events; and the latter from the lateral hypothalamus, which may explain their involvement in value coding. We demonstrate that neurons in the striatum that project directly to dopamine neurons form patches in both the dorsal and ventral striatum, whereas those projecting to GABAergic neurons are distributed in the matrix compartment. Neuron-type-specific connectivity lays a foundation for studying how dopamine neurons compute outputs.     

Structural and functional analysis of a conjugated bile salt hydrolase from Bifidobacterium longum reveals an evolutionary relationship with penicillin V acylase

Bile salt hydrolase (BSH) is an enzyme produced by the intestinal microflora that catalyzes the deconjugation of glycine- or taurine-linked bile salts. The crystal structure of BSH reported here from Bifidobacterium longum reveals that it is a member of N-terminal nucleophil hydrolase structural superfamily possessing the characteristic alphabetabetaalpha tetra-lamellar tertiary structure arrangement. Site-directed mutagenesis of the catalytic nucleophil residue, however, shows that it has no role in zymogen processing into its corresponding active form. Substrate specificity was studied using Michaelis-Menten and inhibition kinetics and fluorescence spectroscopy. These data were compared with the specificity profile of BSH from Clostridium perfrigens and pencillin V acylase from Bacillus sphaericus, for both of which the three-dimensional structures are available. Comparative analysis shows a gradation in activity toward common substrates, throwing light on a possible common route toward the evolution of pencillin V acylase and BSH.     

Antimicrobial Use and Resistance in Swine Waste Treatment Systems

Chlortetracycline and the macrolide tylosin were identified as commonly used antimicrobials for growth promotion and prophylaxis in swine production. Resistance to these antimicrobials was measured throughout the waste treatment processes at five swine farms by culture-based and molecular methods. Conventional farm samples had the highest levels of resistance with both culture-based and molecular methods and had similar levels of resistance despite differences in antimicrobial usage. The levels of resistance in organic farm samples, where no antimicrobials were used, were very low by a culture-based method targeting fecal streptococci. However, when the same samples were analyzed with a molecular method detecting methylation of a specific nucleotide in the 23S rRNA that results in resistance to macrolides, lincosamides, and streptogramin B (MLS_B), an unexpectedly high level of resistant rRNA (approximately 50%) was observed, suggesting that the fecal streptococci were not an appropriate target group to evaluate resistance in the overall microbial community and that background levels of MLS_B resistance may be substantial. All of the feed samples tested, including those from the organic farm, contained tetracycline resistance genes. Generally, the same tetracycline resistance genes and frequency of detection were found in the manure and lagoon samples for each commercial farm. The levels of tetracycline and MLS_B resistance remained high throughout the waste treatment systems, suggesting that the potential impact of land application of treated wastes and waste treatment by-products on environmental levels of resistance should be investigated further.     

Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts

Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic antitumor effects and the highest frequency of E7-specific CD8⁺ T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8⁺ T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic antitumor effects. The clinical implications of the study are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Serum antibody responses to Wolbachia surface protein in patients with human lymphatic filariasis

Wolbachia surface protein (WSP), which is the most abundantly expressed protein of Wolbachia from the human filarial parasite Brugia malayi, was chosen for the present study. B‐cell epitope prediction of the WSP protein sequence indicates a high antigenicity, surface probability and hydrophilicity by DNA STAR software analysis. ProPred analysis suggests the presence of HLA class II binding regions in the WSP protein that contribute to T‐cell responses and isotype reactivity. In order to validate these findings, the gene coding for endosymbiont WSP was PCR‐amplified from the genomic DNA of the human filarial parasite Brugia malayi and cloned in T‐7 expression vector pRSET‐A. Western blot and ELISA at the total IgG level with recombiant WSP indicated a significantly elevated reactivity in CP compared to MF, EN and NEN individuals. Isotype ELISA also suggested an elevated reactivity in CP patients at the IgG1 level. In contrast, WSP‐specific IgG4 levels were found to be elevated in MF patients compared to CP and EN. Besides this, WSP‐specific IgE levels indicated an elevated reactivity in CP and MF patients compared to normals. Observations from ELISA supported the in silico predictions that indicate the presence of B‐ and T‐cell epitopes. Hence, a combinatorial approach of in silico predictions and wet‐lab studies provides interesting insights into the role of Wolbachia proteins in filarial pathogenesis.     

Combination of apigenin treatment with therapeutic HPV DNA vaccination generates enhanced therapeutic antitumor effects

Background  

It is important to develop innovative therapies for advanced stage cancers in addition to the conventional therapies including chemotherapy, radiation and surgery. Antigen-specific immunotherapy has emerged as a novel alternate therapy for advanced stage cancers, which may be employed in conjunction with conventional therapies.     

Proteomic changes in the hypothalamus and retroperitoneal fat from male F344 rats subjected to repeated light–dark shifts

Chronic circadian desynchronization induced by repeated 12 h light–dark cycle shifts conducted twice weekly resulted in elevated food intake, body weight gain, and retroperitoneal fat mass in male F344 rats. Using a proteomic approach, we found that repeated light–dark shifts caused changes in expression levels of five hypothalamic (four upregulated) and 22 retroperitoneal fat (13 upregulated) 2‐DE protein spots. Proteins involved in carbohydrate metabolism and in the citric acid cycle were upregulated, indicating a positive energy balance status. In addition, the hypothalamic gamma‐amino butyric acid (GABA) aminotransferase was upregulated, thus suggesting a connection between the brain GABAeric system and the modulation of food intake. Furthermore, the upregulation of fatty acid‐binding protein 4 and the downregulation of 78 kDa glucose‐regulated protein in the fat implicated the development of insulin resistance. We observed the upregulation of two antioxidant enzymes that might serve as protection against insulin dysfunction associated with oxidative stress. Finally, the downregulation of hypothalamic voltage‐dependent anion‐selective channel protein 1 and fat ATP synthase suggested a reduction in synthesis of mitochondrial ATP. These findings are in partial agreement with those of studies of obesity induced by genotype and a high‐fat diet.