Detection of lymph node metastasis in patients with nodal prostate cancer relapse using (18)F/(11)C-choline positron emission tomography/computerized tomography.

PURPOSE: We evaluated the diagnostic accuracy of choline positron emission tomography/computerized tomography for nodal relapse of prostate cancer according to topographical site and tumor infiltration size in lymph nodes. MATERIALS AND METHODS: A total of 72 patients with nodal prostate cancer relapse after primary therapy underwent pelvic and/or retroperitoneal salvage lymph node dissection. Salvage was done after whole body positron emission tomography/computerized tomography with (11)C-choline or (18)F-fluoroethylcholine showed positron emission tomography positive lymph nodes but no other detectable metastasis. Diagnostic accuracy was evaluated in 160 dissected lymph node regions (pelvic left/right and retroperitoneal), 498 subregions (common, external and internal iliac, obturator, presacral, aortic bifurcation, aortal, vena caval and interaortocaval) and 2,122 lymph nodes. RESULTS: Lymph node metastasis was present in 32% of resected lymph nodes (681 of 2,122), resulting in 238 positive subregions and 111 positive regions. Positron emission tomography/computerized tomography was positive for 110 regions and 209 subregions. Sensitivity, specificity, positive and negative predictive values, and accuracy were 91.9%, 83.7%, 92.7%, 82.0% and 89.4% (region based), 80.7%, 93.5%, 91.9%, 84.1% and 87.3% (subregion based), and 57.0%, 98.4%, 94.5%, 82.6% and 84.9% (lesion based), respectively. Of 393 positive lymph node metastases detected by this method 278 (70.7%) were in lymph nodes with a less than 10 mm short axis diameter. Imaging sensitivity was 13.3%, 57.4% and 82.8% for a tumor infiltration depth of 2 or greater to less than 3 mm, 5 or greater to less than 6 mm and 10 or greater to less than 11 mm, respectively. Lymph node metastasis site and the radiotracer ((11)C-choline/(18)F-fluoroethylcholine) had no substantial impact on diagnostic accuracy. CONCLUSIONS: Choline positron emission tomography/computerized tomography detects affected lymph node regions (pelvic left/right and retroperitoneal) in patients with prostate cancer relapse with high accuracy and it seems helpful for guiding salvage lymph node dissection. Sensitivity decreases with the size of metastatic infiltration in lymph nodes. This technique detects metastasis in a significant fraction of lymph nodes that are not pathologically enlarged on computerized tomography.

The molecular basis of FimT-mediated DNA uptake during bacterial natural transformation.

Naturally competent bacteria encode sophisticated protein machinery for the uptake and translocation of exogenous DNA into the cell. If this DNA is integrated into the bacterial genome, the bacterium is said to be naturally transformed. Most competent bacterial species utilise type IV pili for the initial DNA uptake step. These proteinaceous cell-surface structures are composed of thousands of pilus subunits (pilins), designated as major or minor according to their relative abundance in the pilus. Here, we show that the minor pilin FimT plays an important role in the natural transformation of Legionella pneumophila. We use NMR spectroscopy, in vitro DNA binding assays and in vivo transformation assays to understand the molecular basis of FimT's role in this process. FimT binds to DNA via an electropositive patch, rich in arginines, several of which are well-conserved and located in a conformationally flexible C-terminal tail. FimT orthologues from other Gammaproteobacteria share the ability to bind to DNA. Our results suggest that FimT plays an important role in DNA uptake in a wide range of competent species.

Proteins at the polypeptide tunnel exit of the yeast mitochondrial ribosome.

Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.

Single-Domain Antibodies for Targeting, Detection, and In Vivo Imaging of Human CD4+ Cells.

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+ cells in regulating immunological processes, we generated novel single-domain antibodies [nanobodies (Nbs)] that specifically recognize human CD4. After in-depth analysis of their binding properties, recognized epitopes, and effects on T-cell proliferation, activation, and cytokine release, we selected CD4-specific Nbs that did not interfere with crucial T-cell processes in vitro and converted them into immune tracers for noninvasive molecular imaging. By optical imaging, we demonstrated the ability of a high-affinity CD4-Nb to specifically visualize CD4+ cells in vivo using a xenograft model. Furthermore, quantitative high-resolution immune positron emission tomography (immunoPET)/MR of a human CD4 knock-in mouse model showed rapid accumulation of 64Cu-radiolabeled CD4-Nb1 in CD4+ T cell-rich tissues. We propose that the CD4-Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

Lysine-specific demethylase 1 regulates differentiation onset and migration of trophoblast stem cells.

Propagation and differentiation of stem cell populations are tightly regulated to provide sufficient cell numbers for tissue formation while maintaining the stem cell pool. Embryonic parts of the mammalian placenta are generated from differentiating trophoblast stem cells (TSCs) invading the maternal decidua. Here we demonstrate that lysine-specific demethylase 1 (Lsd1) regulates differentiation onset of TSCs. Deletion of Lsd1 in mice results in the reduction of TSC number, diminished formation of trophectoderm tissues and early embryonic lethality. Lsd1-deficient TSCs display features of differentiation initiation, including alterations of cell morphology, and increased migration and invasion. We show that increased TSC motility is mediated by the premature expression of the transcription factor Ovol2 that is directly repressed by Lsd1 in undifferentiated cells. In summary, our data demonstrate that the epigenetic modifier Lsd1 functions as a gatekeeper for the differentiation onset of TSCs, whereby differentiation-associated cell migration is controlled by the transcription factor Ovol2.

PRK1/PKN1 controls migration and metastasis of androgen-independent prostate cancer cells.

The major threat in prostate cancer is the occurrence of metastases in androgen-independent tumor stage, for which no causative cure is available. Here we show that metastatic behavior of androgen-independent prostate tumor cells requires the protein-kinase-C-related kinase (PRK1/PKN1) in vitro and in vivo. PRK1 regulates cell migration and gene expression through its kinase activity, but does not affect cell proliferation. Transcriptome and interactome analyses uncover that PRK1 regulates expression of migration-relevant genes by interacting with the scaffold protein sperm-associated antigen 9 (SPAG9/JIP4). SPAG9 and PRK1 colocalize in human cancer tissue and are required for p38-phosphorylation and cell migration. Accordingly, depletion of either ETS domain-containing protein Elk-1 (ELK1), an effector of p38-signalling or p38 depletion hinders cell migration and changes expression of migration-relevant genes as observed upon PRK1-depletion. Importantly, a PRK1 inhibitor prevents metastases in mice, showing that the PRK1-pathway is a promising target to hamper prostate cancer metastases in vivo. Here we describe a novel mechanism controlling the metastatic behavior of PCa cells and identify PRK1 as a promising therapeutic target to treat androgen-independent metastatic prostate cancer.