Description: The utility of 18 F-FDG PET/CT in patients with nasal-type natural killer (NK)/T-cell lymphoma has not been established. Therefore, we evaluated the role of 18 F-FDG PET/CT for determining cancer staging by comparing its results to those of conventional staging methods (CSMs) (physical examination, CT with intravenous contrast, biopsies from primary sites, and bone marrow examinations) in patients with nasal-type NK/T-cell lymphoma. Methods: In this study, 52 consecutive patients (34 men, 18 women; mean age, 49.4 y) with newly diagnosed nasal-type NK/T-cell lymphoma were studied. Anatomic regions ( n = 1,300; 16 nodal and 9 extranodal regions per patient) were assessed with an 18 F-FDG PET/CT scan and with CSMs, and each anatomic region was classified as positive or negative for malignancy. Biopsy and clinical follow-up, including additional imaging studies, were used as the gold standard for diagnosis. Results: Of the 59 nodal and 71 extranodal anatomic regions that were truly positive for malignancy, 18 F-FDG PET/CT detected 58 nodal and 69 extranodal. CSMs, however, detected only 44 of the nodal and 61 of the extranodal anatomic regions that were positive for malignancy (nodal comparison of PET/CT vs. CSMs, P 〈 0.001; extranodal comparison of PET/CT vs. CSMs, P = 0.008). PET/CT scans exhibited a significantly better sensitivity (97.7% vs. 80.7%, P 〈 0.001) than CSMs for the detection of malignant lesions. PET/CT findings altered the original staging category for 12 patients (21.2%) and affected treatment planning in 23 cases (44.2%). Conclusion: Our study demonstrated that 18 F-FDG PET/CT scanning is a valuable modality for staging and treatment planning in patients with nasal-type NK/T-cell lymphoma.

Description: Although Mycobacterium abscessus ( M. abscessus ) is becoming more prevalent in patients without overt immunodeficiency, little is known about the factors that contribute to disease susceptibility. This study was undertaken to investigate how Toll-like receptor 2 (TLR2) functionally contributes to the generation of protective immunity against M. abscessus in a morphotype-specific manner. We found that Tlr2 –/– mice were extremely susceptible to an intravenous (i.v.) model of infection by M. abscessus rough variants, displaying uncontrolled infection in the lungs and a significantly lower survival rate than with wild-type (WT) mice. This uncontrolled infection resulted from failures in the following processes: (i) production of the crucial cytokines gamma interferon (IFN-), tumor necrosis factor alpha (TNF-α), and interleukin 12p70 (IL-12p70); (ii) early infiltration of neutrophils, monocytes, and dendritic cells (DCs) in the lungs of Tlr2 –/– mice; (iii) rapid influx of CD4 + and CD8 + T cells; and (iv) the expansion of memory/effector T cells. Notably, systemic administration of M. abscessus culture filtrate-treated syngeneic DCs from WT mice greatly strengthened immune priming in vivo , resulting in a dramatic reduction in bacterial growth and improved long-term survival in Tlr2 –/– mice, with a recovery of protective immunity. Our findings demonstrate that TLR2 is an essential contributor to instructive and effector immunity during M. abscessus infection in a morphotype-specific manner.

Description: Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo . Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the 1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its 1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. IMPORTANCE While the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the 1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.

Description: Extracellular vesicles (EVs) produced by a sulfur-reducing, hyperthermophilic archaeon, " Thermococcus onnurineus " NA1 T , were purified and characterized. A maximum of four EV bands, showing buoyant densities between 1.1899 and 1.2828 g cm –3 , were observed after CsCl ultracentrifugation. The two major EV bands, B (buoyant density at 25°C [ 25 ] = 1.2434 g cm –3 ) and C ( 25 = 1.2648 g cm –3 ), were separately purified and counted using a qNano particle analyzer. These EVs, showing different buoyant densities, were identically spherical in shape, and their sizes varied from 80 to 210 nm in diameter, with 120- and 190-nm sizes predominant. The average size of DNA packaged into EVs was about 14 kb. The DNA of the EVs in band C was sequenced and assembled. Mapping of the T. onnurineus NA1 T EV (ToEV) DNA sequences onto the reference genome of the parent archaeon revealed that most genes of T. onnurineus NA1 T were packaged into EVs, except for an ~9.4-kb region from TON_0536 to TON_0544. The absence of this specific region of the genome in the EVs was confirmed from band B of the same culture and from bands B and C purified from a different batch culture. The presence of the 3'-terminal sequence and the absence of the 5'-terminal sequence of TON_0536 were repeatedly confirmed. On the basis of these results, we hypothesize that the unpackaged part of the T. onnurineus NA1 T genome might be related to the process that delivers DNA into ToEVs and/or the mechanism generating the ToEVs themselves.

Description: Although ingenol 3,20-dibenzoate (IDB) is known as a selective novel protein kinase C (PKC) agonist, its biologic actions and underlying mechanisms remain incompletely understood. In this study, we identified IDB as a proliferative agent for an erythropoietin (EPO)-dependent cell line, UT-7/EPO, through the screening of a natural compound library. To clarify the underlying mechanism of IDB’s EPO-like activities, we thoroughly analyzed the mutual relation between EPO and IDB in terms of in vitro and in vivo activities, signaling molecules, and a cellular receptor. IDB substantially induced the proliferation of UT-7/EPO cells, but not as much as EPO. IDB also lessened the anemia induced by 5-fluorouracil in an in vivo mouse model. Interestingly, IDB showed a synergistic effect on EPO at low concentration, but an antagonistic effect at higher concentration. Physical interaction and activation of PKCs by IDB- and EPO-competitive binding of IDB to EPO receptor (EPOR) explain these synergistic and antagonistic activities, respectively. Importantly, we addressed IDB’s mechanism of action by demonstrating the direct binding of IDB to PKCs, and by identifying EPOR as a novel molecular target of IDB. Based on these dual targeting properties, IDB holds promise as a new small molecule modulator of EPO-related pathologic conditions.

Description: The usefulness of 18 F-FDG PET in gastric cancer recurrence is limited by low sensitivity. Given that detectability by PET is dependent on the tumor’s metabolic characteristics, we tested whether the performance of PET for gastric cancer recurrence is enhanced in patients with 18 F-FDG–avid primary tumors. Methods: Three hundred sixty-eight patients with advanced gastric cancer underwent 18 F-FDG PET/CT for initial staging and for recurrence surveillance after curative surgery. On initial PET/CT, primary tumors were 18 F-FDG–avid if they displayed focal uptake with an SUV max 4 or more. Follow-up 18 F-FDG PET/CT was evaluated for recurrent disease. Results: On initial PET/CT, the primary tumor was 18 F-FDG–avid in 236 of 368 (64.1%) and nonavid in 132 patients (35.9%). During follow-up for 18.9 ± 13.3 mo, 72 patients (19.6%) had recurrence. Of the 63 PET scans with recurrence, 42 (66.7%) and 21 (33.3%) were scans of patients with 18 F-FDG–avid and nonavid primary tumors, respectively. PET sensitivity was higher in scans of patients with 18 F-FDG–avid than nonavid tumors for all recurrences (81.0% vs. 52.4%; P = 0.018) and nonanastomosis site recurrences (82.1% vs. 47.4%; P = 0.006). The sensitivity for detecting peritoneal recurrence was also higher for the avid tumor group. PET specificity was similarly high (97.1% and 97.5%) for both groups. Adding cell type and Lauren classification to tumor 18 F-FDG avidity further enhanced PET sensitivity. Conclusion: Surveillance 18 F-FDG PET/CT after resection of gastric cancer has significantly higher sensitivity in patients with 18 F-FDG–avid primary tumors and may have greater value in this group.

Description: The role of AMP-activated protein kinase (AMPK) in promoting fatty acid (FA) oxidation in various tissues, such as liver and muscle, has been well understood. However, the role of AMPK in lipolysis and FA metabolism in adipose tissue has been controversial. To investigate the role of AMPK in the regulation of adipose lipolysis in vivo , we generated mice with adipose-tissue-specific knockout of both the α1 and α2 catalytic subunits of AMPK (AMPK-ASKO mice) by using aP2-Cre and adiponectin-Cre. Both models of AMPK-ASKO ablation show no changes in desnutrin/ATGL levels but have defective phosphorylation of desnutrin/ATGL at S406 to decrease its triacylglycerol (TAG) hydrolase activity, lowering basal lipolysis in adipose tissue. These mice also show defective phosphorylation of hormone-sensitive lipase (HSL) at S565, with higher phosphorylation at protein kinase A sites S563 and S660, increasing its hydrolase activity and isoproterenol-stimulated lipolysis. With higher overall adipose lipolysis, both models of AMPK-ASKO mice are lean, having smaller adipocytes with lower TAG and higher intracellular free-FA levels. Moreover, FAs from higher lipolysis activate peroxisome proliferator-activated receptor delta to induce FA oxidative genes and increase FA oxidation and energy expenditure. Overall, for the first time, we provide in vivo evidence of the role of AMPK in the phosphorylation and regulation of desnutrin/ATGL and HSL and thus adipose lipolysis.

Description: The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans . The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans , EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.

Description: Stable isotope probing (SIP) is a cultivation-free methodology that provides information about the identity of microorganisms participating in assimilatory processes in complex communities. In this study, a Herminiimonas -related bacterium was identified as the dominant member of a denitrifying microcosm fed [ 13 C]toluene. The genome of the uncultivated toluene-degrading bacterium was obtained by applying pyrosequencing to the heavy DNA fraction. The draft genome comprised ~3.8 Mb, in 131 assembled contigs. Metabolic reconstruction of aromatic hydrocarbon (toluene, benzoate, p -cresol, 4-hydroxybenzoate, phenylacetate, and cyclohexane carboxylate) degradation indicated that the bacterium might specialize in anaerobic hydrocarbon degradation. This characteristic is novel for the order Burkholderiales within the class Betaproteobacteria . Under aerobic conditions, the benzoate oxidation gene cluster (BOX) system is likely involved in the degradation of benzoate via benzoyl coenzyme A. Many putative genes for aromatic hydrocarbon degradation were closely related to those in the Rhodocyclaceae (particularly Aromatoleum aromaticum EbN1) with respect to organization and sequence similarity. Putative mobile genetic elements associated with these catabolic genes were highly abundant, suggesting gene acquisition by Herminiimonas via horizontal gene transfer.

Description: 11 C-LY2795050 is a novel -selective antagonist PET tracer. The in vitro binding affinities ( K i ) of LY2795050 at the -opioid (KOR) and μ-opioid (MOR) receptors are 0.72 and 25.8 nM, respectively. Thus, the in vitro KOR/MOR binding selectivity is about 36:1. Our goal in this study was to determine the in vivo selectivity of this new KOR antagonist tracer in the monkey. Methods: To estimate the ED 50 value (dose of a compound [or drug] that gives 50% occupancy of the target receptor) of LY2795050 at the MOR and KOR sites, 2 series of blocking experiments were performed in 3 rhesus monkeys using 11 C-LY2795050 and 11 C-carfentanil with coinjections of various doses of unlabeled LY2795050. Kinetic modeling was applied to calculate regional binding potential ( BP ND ), and 1- and 2-site binding curves were fitted to these data to measure 11 C-LY2795050 binding selectivity. Results: The LY2795050 ED 50 at MOR was 119 μg/kg based on a 1-site model for 11 C-carfentanil. The 1-site binding model was also deemed sufficient to describe the specific binding of 11 C-LY2795050 at KOR. The ED 50 at KOR estimated from the 1-site model was 15.6 μg/kg. Thus, the ED 50 ratio for MOR:KOR was 7.6. Conclusion: The in vivo selectivity of 11 C-LY2795050 for KOR over MOR is 7.6. 11 C-LY2795050 has 4.7-fold-lower selectivity at KOR over MOR in vivo as compared with in vitro. Nevertheless, on the basis of our finding in vivo, 88% of the PET-observed specific binding of 11 C-LY2795050 under baseline conditions will be due to binding of the tracer at the KOR site in a region with similar prevalence of KOR and MOR. 11 C-LY2795050 is sufficiently selective for KOR over MOR in vivo to be considered an appropriate probe for studying the KOR with PET.