Metabolite Study and Structural Authentication for the First-in-Human Use Sphingosine-1-phosphate Receptor 1 Radiotracer.

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [11C]CS1P1 has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [11C]CS1P1 detected a radiometabolite peak that is more lipophilic than [11C]CS1P1. Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [11C]CS1P1 in rats, nonhuman primates, and humans. CS1P1 is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1. To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N-oxide 1, also named TZ82121. The MS fragmentation pattern of N-oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [18F]TZ82121 by the oxidation of [18F]FS1P1. Radio-HPLC analysis confirmed that [18F]TZ82121 matched the radiometabolite observed in rat plasma post injection of [18F]FS1P1. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [18F]TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N-oxide [11C]TZ82121, detected in human plasma post injection of [11C]CS1P1, does not enter the brain to confound quantitative PET data analysis. [11C]CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.

Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies.

BACKGROUND: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. METHODS: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines' representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. RESULTS: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. CONCLUSION: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression.

RNA Polymerase II "Pause" Prepares Promoters for Upcoming Transcription during Drosophila Development.

According to previous studies, during Drosophila embryogenesis, the recruitment of RNA polymerase II precedes active gene transcription. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. In addition, the composition of the RNA polymerase II "paused" complexes associated with promoters at different developmental stages are described in detail. For this purpose, we performed ChIP-Seq analysis using antibodies for various modifications of RNA polymerase II (total, Pol II CTD Ser5P, and Pol II CTD Ser2P) as well as for subunits of the NELF, DSIF, and PAF complexes and Brd4/Fs(1)h that control transcription elongation. We found that during metamorphosis, similar to mid-embryogenesis, the promoters were bound by RNA polymerase II in the "paused" state, preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in a "pause" state was phosphorylated at Ser5 and Ser2 of Pol II CTD and bound the NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the "paused" RNA polymerase II complex included Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex was phosphorylated at Ser5 of Pol II CTD, but not at Ser2. These results indicate that, during mid-embryogenesis, RNA polymerase II stalls in the "post-pause" state, being phosphorylated at Ser2 of Pol II CTD (after the stage of p-TEFb action). During metamorphosis, the "pause" mechanism is closer to classical promoter-proximal pausing and is characterized by a low level of Pol II CTD Ser2P.

The Effect of Iodine-Containing Nano-Micelles, FS-1, on Antibiotic Resistance, Gene Expression and Epigenetic Modifications in the Genome of Multidrug Resistant MRSA Strain Staphylococcus aureus ATCC BAA-39.

Application of supplementary drugs which increase susceptibility of pathogenic bacteria to antibiotics is a promising yet unexplored approach to overcome the global problem of multidrug-resistant infections. The discovery of a new drug, an iodine-containing nano-molecular complex FS-1, which has proven to improve susceptibility to antibiotics in various pathogens, including MRSA strain Staphylococcus aureus ATCC BAA-39TM, allowed studying this phenomenon. Chromosomal DNA and total RNA samples extracted from the FS-1 treated strain (FS) and from the negative control (NC) cultures were sequenced by PacBio SMRT and Ion Torrent technologies, respectively. PacBio DNA reads were used to assemble chromosomal DNA of the NC and FS variants of S. aureus BAA-39 and to perform profiling of epigenetically modified nucleotides. Results of transcriptional profiling, variant calling and detection of epigenetic modifications in the FS variant were compared to the NC variant. Additionally, the genetic alterations caused by the treatment of S. aureus BAA-39 with FS-1 were compared to the results of a similar experiment conducted with another model organism, E. coli ATCC BAA-196. Several commonalities in responses of these phylogenetically distant microorganisms to the treatment with FS-1 were discovered, which included metabolic transition toward anaerobiosis and oxidative/osmotic stress response. S. aureus culture appeared to be more sensitive to FS-1 due to a higher penetrability of cells by iodine bound compounds, which caused carbonyl stress associated with nucleotide damaging by FS-1, abnormal epigenetic modifications and an increased rate of mutations. It was hypothesized that the disrupted pattern of adenine methylated loci within methicillin-resistance chromosome cassettes (SCCmec) may promote excision of this antibiotic resistance determinant from chromosomes while the altered pattern of cytosine methylation was behind the adaptive gene regulation in the culture FS. The selection against the antibiotic resistance in bacterial populations caused by abnormal epigenetic modifications exemplifies possible mechanisms of antibiotic resistance reversion induced by iodine-containing compounds. These finding will facilitate development of therapeutic agents against multidrug-resistant infections.

A Genetic Screen for Genes That Impact Peroxisomes in Drosophila Identifies Candidate Genes for Human Disease.

Peroxisomes are subcellular organelles that are essential for proper function of eukaryotic cells. In addition to being the sites of a variety of oxidative reactions, they are crucial regulators of lipid metabolism. Peroxisome loss or dysfunction leads to multi-system diseases in humans that strongly affect the nervous system. In order to identify previously unidentified genes and mechanisms that impact peroxisomes, we conducted a genetic screen on a collection of lethal mutations on the X chromosome in Drosophila Using the number, size and morphology of GFP tagged peroxisomes as a readout, we screened for mutations that altered peroxisomes based on clonal analysis and confocal microscopy. From this screen, we identified eighteen genes that cause increases in peroxisome number or altered morphology when mutated. We examined the human homologs of these genes and found that they are involved in a diverse array of cellular processes. Interestingly, the human homologs from the X-chromosome collection are under selective constraint in human populations and are good candidate genes particularly for dominant genetic disease. This in vivo screening approach for peroxisome defects allows identification of novel genes that impact peroxisomes in vivo in a multicellular organism and is a valuable platform to discover genes potentially involved in dominant disease that could affect peroxisomes.

Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila.

Regulatory decisions in Drosophila require Polycomb group (PcG) proteins to maintain the silent state and Trithorax group (TrxG) proteins to oppose silencing. Since PcG and TrxG are ubiquitous and lack apparent sequence specificity, a long-standing model is that targeting occurs via protein interactions; for instance, between repressors and PcG proteins. Instead, we found that Pc-repressive complex 1 (PRC1) purifies with coactivators Fs(1)h [female sterile (1) homeotic] and Enok/Br140 during embryogenesis. Fs(1)h is a TrxG member and the ortholog of BRD4, a bromodomain protein that binds to acetylated histones and is a key transcriptional coactivator in mammals. Enok and Br140, another bromodomain protein, are orthologous to subunits of a mammalian MOZ/MORF acetyltransferase complex. Here we confirm PRC1-Br140 and PRC1-Fs(1)h interactions and identify their genomic binding sites. PRC1-Br140 bind developmental genes in fly embryos, with analogous co-occupancy of PRC1 and a Br140 ortholog, BRD1, at bivalent loci in human embryonic stem (ES) cells. We propose that identification of PRC1-Br140 "bivalent complexes" in fly embryos supports and extends the bivalency model posited in mammalian cells, in which the coexistence of H3K4me3 and H3K27me3 at developmental promoters represents a poised transcriptional state. We further speculate that local competition between acetylation and deacetylation may play a critical role in the resolution of bivalent protein complexes during development.

Perception of vowels and prosody by cochlear implant recipients in noise.

UNLABELLED: The aim of the present study was to compare the ability of cochlear implant (CI) recipients to recognise speech prosody in the presence of speech-weighted noise to their ability to recognise vowels in the same test paradigm and listening condition. All test materials were recorded from four different speakers (two male, two female). Two prosody recognition tasks were developed, both using single words as stimuli. The first task involved a question/statement distinction, while the second task required listeners to make a judgement about the speaker's attitude. Vowel recognition tests were conducted using vowel pairs selected on the basis of specific acoustic cues (frequencies of the first two formants and duration). Ten CI users and ten normal-hearing controls were tested in both quiet and an adaptive noise condition, using a two-alternative forced-choice test paradigm for all the tests. Results indicated that vowel recognition was significantly better than prosody recognition in both listener groups in both quiet and noise, and that question/statement discrimination was the most difficult task for CI listeners in noise. Data from acoustic analyses were used to interpret differences in performance on different tasks and with different speakers. LEARNING OUTCOMES: As a result of this activity, readers will be able to (1) describe suitable methods for comparing vowel and prosody perception in noise, (2) compare performance on vowel and prosody perception tasks in quiet in normal-hearing listeners and cochlear implant recipients, (3) compare performance on vowel and prosody perception tasks in noise in normal-hearing listeners and cochlear implant recipients and (4) relate performance on prosody tasks in quiet to performance on these tasks in noise.

Characterization of Vibrio cholerae O139 of an Aquatic Isolate in Northern Vietnam.

During the cholera survey in Namdinh province (northern Vietnam) in July, 2010, one strain of Vibrio cholerae O139 was isolated from 7 environmental water samples positive for ctxA, toxR,VCO139 genes and named as V. cholerae O139, ND1 strain. This strain was lysogenic harbouring a genome similar to the filamentous phage fs1. The replicative form DNA of this phage (named as ND1-fs1, 6856 bp) was sequenced and compared with the other filamentous phages. The filamentous phage ND1-fs1 integrates into the region between ctxB and rtxA genes. The genetic organization of the CTXϕ of V. cholerae O139, strain ND1 was determined and the schematic representation of the genetic organization was shown together with the ND1-fs1 prophage.