The catalytic domain of human hepatitis delta virus RNA A proton nuclear magnetic resonance study

AbstractWe have obtained and analyzed the 600 MHz proton NMR spectra of a 74-mer RNA derived from the catalytic domain of hepatitis delta virus genomic RNA (HDV RNA) to determine its secondary structure. Deconvolution of the NMR spectrum obtained at 32°C indicates that part of the 74-mer RNA molecule may exist in multiple conformations in equilibrium. The major conformer contains two A-U base pairs and 14 ± 2 G-C base pairs. It appears to contain no standard G-U base pairs. Our NMR melting study suggests that this conformer has at least two stem-loop regions. One of the regions has been identified to be a tetra-loop. We have assigned five imino proton resonances of the tetraloop stem. Our data is consistent with the pseudoknot model of Perrotta and Been

Gold Ion–Angiotensin Peptide Interaction by Mass Spectrometry

Stimulated by the interest in developing gold compounds for treating cancer, gold ion– angiotensin peptide interactions are investigated by mass spectrometry. Under the experimental conditions used, the majority of gold ion–angiotensin peptide complexes contain gold in the oxidation states I and III. Both ESI-MS and MALDI-TOF MS detect singly/multiply charged ions for mononuclear/multinuclear gold-attached peptides, which are represented as [peptide+a Au (I)+b Au(III)+(e - a -3b) H]e+, where a,b≥0 and e is charge. ESI-MS data shows singly/multiply charged ions of Au(I)-peptide and Au(III)-peptide complexes. This study reveals that MALDITOF MS mainly detects singly charged Au(I)-peptide complexes, presumably due to the ionization process. The electrons in the MALDI plume seem to efficiently reduce Au(III) to Au(I). MALDI also tends to enhance the higher polymeric forms of gold-peptide complexes regardless of the laser power used. Collision-induced dissociation experiments of the mononuclear and dinuclear gold-attached peptide ions for angiotensin peptides show that the gold ion (a soft acid) binding sites are in the vicinity of Cys (a soft ligand), His (a major anchor of peptide for metal ion chelation), and the basic residue Arg. Data also suggests that the abundance of gold-attached peptides increases with higher gold concentration until saturation, after which an increase in gold ion concentration leads to the aggregation and/or precipitation of gold-bound peptides

The Helicobacter pylori Genome Project : insights into H. pylori population structure from analysis of a worldwide collection of complete genomes

Helicobacter pylori, a dominant member of the gastric microbiota, shares co-evolutionary history with humans. This has led to the development of genetically distinct H. pylori subpopulations associated with the geographic origin of the host and with differential gastric disease risk. Here, we provide insights into H. pylori population structure as a part of the Helicobacter pylori Genome Project (HpGP), a multi-disciplinary initiative aimed at elucidating H. pylori pathogenesis and identifying new therapeutic targets. We collected 1011 well-characterized clinical strains from 50 countries and generated high-quality genome sequences. We analysed core genome diversity and population structure of the HpGP dataset and 255 worldwide reference genomes to outline the ancestral contribution to Eurasian, African, and American populations. We found evidence of substantial contribution of population hpNorthAsia and subpopulation hspUral in Northern European H. pylori. The genomes of H. pylori isolated from northern and southern Indigenous Americans differed in that bacteria isolated in northern Indigenous communities were more similar to North Asian H. pylori while the southern had higher relatedness to hpEastAsia. Notably, we also found a highly clonal yet geographically dispersed North American subpopulation, which is negative for the cag pathogenicity island, and present in 7% of sequenced US genomes. We expect the HpGP dataset and the corresponding strains to become a major asset for H. pylori genomics

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA

The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDIα showed two potential protein kinase A (PKA) phosphorylation motifs, Ser174 and Thr182. Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDIα-GST fusion protein and PKA motif-containing GDIα peptide at Ser174, but not Thr182. Transfection of COS-7 cells with mutated full-length GDIα at Ser174 to Ala174 (GDIα-Ser174A) abrogated the ability of cAMP to phosphorylate GDIα. However, mutation of Thr182 to Ala182 (GDIα-Thr182A) did not abrogate, and cAMP increased phosphorylation of GDIα to a similar extent as wild-type GDIα transfectants. The mutant GDIα-Ser174A, but not GDIα-Thr182A, was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDIα-Ser174A was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIα binding with RhoA, suggesting that phosphorylation of Ser174 regulated preformed GDIα-RhoA complexes. The findings provide strong support that the selective phosphorylation at Ser174 by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury

HPLC-MTT assay: anti-cancer activity of aqueous garlic extract is from allicin

A strategy using RP-HPLC, TLC, MS, NMR, chemical synthesis, and MTT cell viability assay to identify allicin as the active anti-cancer compound in the aqueous extract of garlic (AGE) is described. Change the pH of the AGE from 7 to 5 eliminated interfering molecules and enabled a clean HPLC separation of the constituents in AGE. MTT assay of the HPLC fractions identify an active fraction. Further analysis by TLC, MS, and NMR verify the active HPLC fraction as allicin. Chemically synthesized allicin is used to provide further confirmation. The results clearly identify the active compound in AGE as allicin