Intra-tumoral administration of CHST15 siRNA remodels tumor microenvironment and augments tumor-infiltrating T cells in pancreatic cancer.

The dense stroma is one cause of poor efficacy of T cell-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC). Carbohydrate sulfotransferase 15 (CHST15) is a proteoglycan-synthetic enzyme responsible for remodeling tumor stroma. Intra-tumoral injection of CHST15 small interfering RNA (siRNA) has been shown to increase the tumor-infiltrating T cells (TILs) in patients with unresectable PDAC. However, the mechanism underlying the enhanced accumulation of TILs is not fully explored. Here, we demonstrate that intra-tumoral injection of CHST15 siRNA locally and remotely diminishes myeloid-derived suppressor cells (MDSCs) and enhances TILs in mice. CHST15 was expressed by tumor cells and MDSCs in both tumor and tumor-draining lymph nodes (TDLNs), and CHST15 siRNA repressed stromal density, neutrophil extracellular traps, and Ly6C/G+ MDSCs in vivo. Remarkably, tumor growth inhibition was only observed in the immunocompetent KPC model, which is associated with enhanced TILs. In vitro, CHST15 siRNA significantly downregulated the levels of CHST15 and indoleamine 2,3-dioxygenase mRNA in CD33+ MDSCs derived from human peripheral blood mononuclear cells. These results suggest a dual role for intra-tumorally injected CHST15 siRNA on modulating the tumor immune microenvironment for T cell entry and remotely diminishing CHST15+ MDSCs, decreasing T cell suppression and expanding T cells in the TDLN, ultimately leading to an enhanced accumulation of TILs.

Chloride Ligands on DNA-Stabilized Silver Nanoclusters.

DNA-stabilized silver nanoclusters (AgN-DNAs) are known to have one or two DNA oligomer ligands per nanocluster. Here, we present the first evidence that AgN-DNA species can possess additional chloride ligands that lead to increased stability in biologically relevant concentrations of chloride. Mass spectrometry of five chromatographically isolated near-infrared (NIR)-emissive AgN-DNA species with previously reported X-ray crystal structures determines their molecular formulas to be (DNA)2[Ag16Cl2]8+. Chloride ligands can be exchanged for bromides, which red-shift the optical spectra of these emitters. Density functional theory (DFT) calculations of the 6-electron nanocluster show that the two newly identified chloride ligands were previously assigned as low-occupancy silvers by X-ray crystallography. DFT also confirms the stability of chloride in the crystallographic structure, yields qualitative agreement between computed and measured UV-vis absorption spectra, and provides interpretation of the 35Cl-nuclear magnetic resonance spectrum of (DNA)2[Ag16Cl2]8+. A reanalysis of the X-ray crystal structure confirms that the two previously assigned low-occupancy silvers are, in fact, chlorides, yielding (DNA)2[Ag16Cl2]8+. Using the unusual stability of (DNA)2[Ag16Cl2]8+ in biologically relevant saline solutions as a possible indicator of other chloride-containing AgN-DNAs, we identified an additional AgN-DNA with a chloride ligand by high-throughput screening. Inclusion of chlorides on AgN-DNAs presents a promising new route to expand the diversity of AgN-DNA structure-property relationships and to imbue these emitters with favorable stability for biophotonics applications.

Specific binding of Hg2+ to mismatched base pairs involving 5-hydroxyuracil in duplex DNA.

Metal ion-nucleic acid interactions contribute significantly to nucleic acid structure and biological activity and have potential applications in nanotechnology. Hg2+ specifically binds to the natural T-T mismatched base pair in duplex DNA to form a T-Hg-T base pair. Metal ions may enhance DNA damage induced by DNA-damaging agents, such as oxidative agents. The interactions between metal ions and damaged DNAs, such as mismatched oxidized bases, have not been well characterized. Here, we examined the possibility of Hg2+ binding to an asymmetric mismatched base pair involving thymine and 5-hydroxyuracil (OHdU), an oxidized base produced by the oxidative deamination of cytosine. UV melting analyses showed that only the melting temperature of the single T-OHdU mismatched duplex DNA increased upon Hg2+ addition. CD spectra indicated no significant change in the higher-order structure of the single T-OHdU mismatched duplex DNA upon Hg2+ addition. X-ray crystallographic structure with two consecutive T-OHdU mismatched base pairs and isothermal titration calorimetric analyses with the single T-OHdU mismatched base pair showed that Hg2+ specifically binds to the N3 positions of both T and OHdU in T-OHdU at 1:1 molar ratio, with a 5×105 M-1 binding constant of to form the T-Hg-OHdU base pair. The Hg2+-bound structure and the Hg2+-binding affinity for T-OHdU was similar to those for T-T. This study on T-Hg-OHdU metal-mediated base pair could aid in studying the molecular mechanism of metal ion-mediated DNA damage and their potential applications in nanotechnology.

Synthesis and properties of fully-modified 4'-selenoRNA, an endonuclease-resistant RNA analog.

A large number of chemically modified oligonucleotides (ONs) have been developed for RNA-based technologies. In most modified RNAs, the characteristic 2'-hydroxyl (2'-OH) groups are removed to enhance both nuclease resistance and hybridization ability. However, the importance of the 2'-OH group in RNA structure and function is well known. Here, we report the synthesis and properties of 4'-selenoRNA in which all four nucleoside units retain the 2'-OH groups but contain a selenium atom instead of an oxygen atom at the 4'-position of the furanose ring. 4'-SelenoRNA has enhanced ability to form duplexes with RNA, and high endonuclease resistance despite the presence of the 2'-OH groups. X-ray crystallography analysis showed that the 4'-selenoRNA duplex adopts an A-conformation, similar to natural RNA, although one 4'-selenocytidine residue has unusual South-type sugar puckering. Furthermore, preliminary studies using 4'-seleno-modified siRNAs suggest that 4'-selenoRNA may be applicable to RNA interference technology. Collectively, our results raise the possibility of a new class of modified RNA in which 2'-OH groups do not need to be masked.

The effect of inosine on the spectroscopic properties and crystal structure of a NIR-emitting DNA-stabilized silver nanocluster.

The effect of replacing guanosines with inosines in the two stabilizing strands (5'-CACCTAGCGA-3') of the NIR emissive DNA-Ag16NC was investigated. The spectroscopic behavior of the inosine mutants is position-dependent: when the guanosine in position 7 was exchanged, the nanosecond fluorescence decay time shortened, while having the inosine in position 9 made the decay time longer. Thanks to structural information gained from single crystal X-ray diffraction measurements, it was possible to propose a mechanistic origin for the observed changes.

A Novel AgI -DNA Rod Comprising a One-Dimensional Array of 11 Silver Ions within a Double Helical Structure.

DNA/RNA duplexes containing metal-ion-mediated base pairs (metallo-base pairs) have potential applications in developing nucleic acid-based nanodevices and genetic code expansion. Many metallo-base pairs are formed within duplexes stabilized by Watson-Crick base pairs. Recently, the crystal structure of an AgI -DNA nanowire with an uninterrupted one-dimensional silver array was determined. Here, we present a new DNA helical wire, the "AgI -DNA rod", containing an uninterrupted array of 11 AgI ions. The AgI -DNA rod consisted of only C-AgI -C, G-AgI -G, G-AgI -5-bromouracil (Br U), and Br U-AgI -Br U metallo base pairs, with no Watson-Crick pairs. The AgI -DNA rods were connected by non-canonical G-G pairs in crystals. Notably, data from our absorbance, circular dichroism, nuclear magnetic resonance, and mass spectrometry analyses suggested that the AgI -DNA rods formed in solution, as well as within crystals.

Constipation Is a Frequent Problem Associated with Vascular Complications in Patients with Type 2 Diabetes: A Cross-sectional Study.

Objective Diabetes is recognized as an underlying disease of constipation. However, the prevalence of constipation varies according to the diagnostic criteria applied. We investigated the prevalence of constipation based on the new guideline for constipation in Japanese patients with type 2 diabetes and examined the relationship with the clinical background, including diabetic vascular complications. Methods Questionnaire surveys including items concerning the diagnosis and treatment status of constipation were administered to 410 patients with type 2 diabetes. Results Although 29% of the patients considered that they had experienced constipation (self-judged), only 14% had consulted a physician about constipation. The prevalence of chronic constipation based on the guideline was 26%. After including laxative users, constipation was finally found in 36%. Despite the use of laxatives (n=81), 51% of the patients were still diagnosed with chronic constipation. Patients with constipation (chronic constipation or laxative use) were significantly older and had a longer duration of diabetes than those without constipation. The body mass index (BMI) of patients with constipation (24.9±3.8 kg/m2) was significantly lower than that of those without constipation (26.3±4.6 kg/m2). Diabetic neuropathy (49% vs. 32%) and coronary heart disease (CHD) (27% vs. 13%) were significantly more frequent in the patients with constipation than in those without constipation. A multivariate logistic regression analysis revealed that gender, BMI, diabetic neuropathy, insulin use, and CHD were significantly associated with constipation. Conclusion An accurate diagnosis of constipation is desirable in patients with type 2 diabetes because constipation is independently associated with CHD.

Efficacy of dulaglutide after switching from incretin-related drugs in patients with type 2 diabetes and inadequate glycemic control.

AIMS: The efficacy of dulaglutide was assessed according to the pretreatments administered before the initiation of dulaglutide in patients with type 2 diabetes. METHODS: In total, 89 patients treated using dulaglutide (0.75 mg, once a weekly) were investigated. The subjects were divided into the three groups based on the form in which therapy was started: additional therapy (n = 35), switched from dipeptidyl peptidase-4 (DPP-4) inhibitors (n = 32) and switched from daily glucagon-like peptide-1 receptor agonists (GLP-1 RAs, n = 30). The changes in medication adherence were determined in the daily GLP-1 RAs group using questionnaire surveys. RESULTS: The HbA1c values significantly decreased after the initiation of dulaglutide in all groups (additional therapy group, - 1.4 ± 1.6%; DPP-4 inhibitors group, - 1.2 ± 1.3%; and daily GLP-1 RAs group, - 0.5 ± 0.7%). Forty-six percent of the subjects in the daily GLP-1 RAs group reported that the incidence of forgetting injections of GLP-1 RA was decreased. The reduction of HbA1c was significantly greater in the subjects who reported a decrease in the incidence of forgetting injections (0.9 ± 0.9%) in comparison to those in which there was no change (0.1 ± 0.4%). CONCLUSIONS: Dulaglutide is considered effective in patients with type 2 diabetes and inadequate glycemic control, regardless of whether their pretreatment includes daily GLP-1 RA treatment.

Effect of cytosine-Ag+-cytosine base pairing on the redox potential of the Ag+/Ag couple and the chemical reduction of Ag+ to Ag by tetrathiafulvalene.

The redox properties of metallo-base pairs remain to be elucidated. Herein, we report the detailed 1H/13C/109Ag NMR spectroscopic and cyclic voltammetric characterisation of the [Ag(cytidine)2]+ complex as isolated cytosine-Ag+-cytosine (C-Ag+-C) base pairs. We also performed comparative studies between cytidine/Ag+ and other nucleoside/Ag+ systems by using cyclic voltammetry measurements. In addition, to evaluate the effect of [Ag(cytidine)2]+ formation on the chemical reduction of Ag+ to Ag, we utilised the redox reaction between Ag+ and tetrathiafulvalene (TTF). We found that Ag+-mediated base pairing lowers the redox potential of the Ag+/Ag couple. In addition, C-Ag+-C base pairing makes it more difficult to reduce captured Ag+ ions than in other nucleoside/Ag+ systems. Remarkably, the cytidine/Ag+ system can be utilised to control the redox potential of the Ag+/Ag couple in DMSO. This feature of the cytidine/Ag+ system may be exploited for Ag nanoparticle synthesis by using the redox reaction between Ag+ and TTF.

Different renoprotective effects of luseogliflozin depend on the renal function at the baseline in patients with type 2 diabetes: A retrospective study during 12 months before and after initiation.

AIMS: The safety and efficacy, particularly, the factors associated with the renal prognosis, were assessed over 12 months after the initiation of luseogliflozin therapy in Japanese patients with type 2 diabetes and renal impairment. METHODS: In total, 238 patients treated with luseogliflozin (2.5 mg, once daily) were studied as the safety analysis set. Two hundred and two subjects whose medication was continued over 12 months were investigated as the full analysis set. The subjects were divided into 3 groups based on the estimated glomerular filtration rate (eGFR): high eGFR (n = 49), normal eGFR (n = 116) and low eGFR (n = 37) groups. RESULTS: The body weight, systolic blood pressure, HbA1c and urinary protein excretion gradually decreased from baseline in all eGFR groups. While the eGFR was significantly reduced from baseline in the high and normal eGFR groups, the eGFR did not significantly differ over time in the low eGFR group. There was no marked difference in the frequency of adverse events that were specific for SGLT2 inhibitors among the 3 groups in the safety analysis set. CONCLUSIONS: Luseogliflozin can preserve the renal function in the medium term in patients with type 2 diabetes and renal impairment without an increase in specific adverse events.

Chemical reduction of Ag+ to Ag employing organic electron donors: evaluation of the effect of Ag+-mediated cytosine-cytosine base pairing on the aggregation of Ag nanoparticles.

Ag+-mediated base pairing is valuable for synthesising DNA-based silver nanoparticles (AgNPs) and nanoclusters (AgNCs). Recently, we reported the formation of a [Ag(cytidine)2]+ complex in dimethyl sulfoxide (DMSO), which facilitated the evaluation of the effect of cytosine-Ag+-cytosine (C-Ag+-C) base pairing on the degree of AgNP aggregation in solution. As an aprotic solvent, DMSO was expected to dissolve the [Ag(cytidine)2]+ complex, and powerful reducing agents, such as organic electron donors. In this study, the chemical reduction of a cytidine/Ag+ system using a powerful reducing agent tetrakis(dimethylamino)ethylene (TDAE) was investigated. 1H/13C/15N NMR spectroscopic evidence was obtained to identify the iminium dication (TDAE2+), which is an oxidised form of TDAE. The results were compared with those obtained using another organic electron donor, tetrathiafulvalene (TTF), which exhibits a relatively lower reduction activity than TDAE. AgNPs prepared via redox reaction between [Ag(cytidine)2]+ and organic electron donors (TDAE and TTF) were characterised using UV-Vis spectroscopy and nanoparticle tracking analysis. It was found that the formation of C-Ag+-C base pairing inhibited the aggregation of AgNPs in solution. In addition, in the presence of cytidine, the total concentration of the AgNP solution was affected by the reduction activity of the reducing agent.

Removal of the A10 adenosine in a DNA-stabilized Ag16 nanocluster.

The role of the terminal adenosine (A10) on the spectroscopic and structural properties of a previously described DNA-stabilized Ag16 nanocluster (DNA:Ag16NC) is presented. In the original DNA:Ag16NCs (5'-CACCTAGCGA-3'), the A10 nucleobase was involved in an Ag+-mediated interaction with an A10 in a neighboring asymmetric unit, and did not interact with the Ag16NC. Therefore, we synthesized AgNCs embedded in the corresponding 9-base sequence in order to investigate the crystal structure of these new DNA-A10:Ag16NCs and analyze the photophysical properties of the solution and crystalline state. The X-ray crystallography and spectroscopic measurements revealed that the 3'-end adenosine has little importance with respect to the photophysics and structure of the Ag16NCs. Additionally, the new crystallographic data was recorded with higher spatial resolution leading to a more detailed insight in the interactions between the nucleotides and Ag atoms.

A Novel DNA Helical Wire Containing HgII -Mediated T:T and T:G Pairs.

Numerous applications of metal-mediated base pairs (metallo-base-pairs) to nucleic acid based nanodevices and genetic code expansion have been extensively studied. Many of these metallo-base-pairs are formed in DNA and RNA duplexes containing Watson-Crick base pairs. Recently, a crystal structure of a metal-DNA nanowire with an uninterrupted one-dimensional silver array was reported. We now report the crystal structure of a novel DNA helical wire containing HgII -mediated T:T and T:G base pairs and water-mediated C:C base pairs. The Hg-DNA wire does not contain any Watson-Crick base pairs. Crystals of the Hg-DNA wire, which is the first DNA wire structure driven by HgII ions, were obtained by mixing the short oligonucleotide d(TTTGC) and HgII ions. This study demonstrates the potential of metallo-DNA to form various structural components that can be used for functional nanodevices.

Crystal structure of a NIR-Emitting DNA-Stabilized Ag16 Nanocluster.

DNA has been used as a scaffold to stabilize small, atomically monodisperse silver nanoclusters, which have attracted attention due to their intriguing photophysical properties. Herein, we describe the X-ray crystal structure of a DNA-encapsulated, near-infrared emitting Ag16 nanocluster (DNA-Ag16 NC). The asymmetric unit of the crystal contains two DNA-Ag16 NCs and the crystal packing between the DNA-Ag16 NCs is promoted by several interactions, such as two silver-mediated base pairs between 3'-terminal adenines, two phosphate-Ca2+ -phosphate interactions, and π-stacking between two neighboring thymines. Each Ag16 NC is confined by two DNA decamers that take on a horse-shoe-like conformation and is almost fully shielded from the solvent environment. This structural insight will aid in the determination of the structure/photophysical property relationship for this class of emitters and opens up new research opportunities in fluorescence imaging and sensing using noble-metal clusters.

Conformational adaptation of UNCG loops upon crowding.

If the A-form helix is the major structural motif found in RNA, the loops that cap them constitute the second most important family of motifs. Among those, two are overrepresented, GNRA and UNCG tetraloops. Recent surveys of RNA structures deposited in the PDB show that GNRA and UNCG tetraloops can adopt tertiary folds that are very different from their canonical conformations, characterized by the presence of a U-turn of a Z-turn, respectively. Crystallographic data from both a lariat-capping (LC) ribozyme and a group II intron ribozyme reveal that a given UUCG tetraloop can adopt a distinct fold depending on its structural environment. Specifically, when the crystal packing applies relaxed constraints on the loop, the canonical Z-turn conformation is observed. In contrast, a highly packed environment induces "squashing" of the tetraloop by distorting its sugar-phosphate backbone in a specific way that expels the first and fourth nucleobases out of the loop, and falls in van der Waals distance of the last base pair of the helix, taking the place of the pair formed between the first and fourth residues in Z-turn loops. The biological relevance of our observations is supported by the presence of similarly deformed loops in the highly packed environment of the ribosome and in a complex between a dsRNA and a RNase III. The finding that Z-turn loops change conformation under higher molecular packing suggests that, in addition to their demonstrated role in stabilizing RNA folding, they may contribute to the three-dimensional structure of RNA by mediating tertiary interactions with distal residues.

Crystal structure of a DNA duplex cross-linked by 6-thioguanine-6-thioguanine disulfides: reversible formation and cleavage catalyzed by Cu(ii) ions and glutathione.

Herein, we determined the crystal structure of a DNA duplex containing consecutive 6-thioguanine-6-thioguanine disulfides. The disulfide bonds were reversibly formed and cleaved in the presence of Cu(ii) ions and glutathione. To our knowledge, this is the first reaction in which metal ions efficiently accelerated disulfide bond formation between thio-bases in duplexes.

Structural Bases for the Fitness Cost of the Antibiotic-Resistance and Lethal Mutations at Position 1408 of 16S rRNA.

To understand a structural basis for the fitness cost of the A1408G antibiotic-resistance mutation in the ribosomal A-site RNA, we have determined crystal structures of its A1408C and A1408U lethal mutants, and made comparison with previously solved structures of the wild type and the antibiotic-resistant mutant. The A-site RNA containing an asymmetric internal loop functions as a molecular switch to discriminate a single cognate tRNA from several near-cognate tRNAs by its conformational ON/OFF switching. Overall structures of the "off" states of the A1408C/U lethal mutants are very similar to those of the wild type and the A1408G antibiotic-resistant mutant. However, significant differences are found in local base stacking interactions including the functionally important A1492 and A1493 residues. In the wild type and the A1408G antibiotic-resistant mutant "off" states, both adenines are exposed to the solvent region. On the other hand, one of the corresponding adenines of the lethal A1408C/U mutants stay deeply inside their A-site helices by forming a purine-pyrimidine AoC or A-U base pair and is sandwiched between the upper and lower bases. Therefore, the ON/OFF switching might unfavorably occur in the lethal mutants compared to the wild type and the A1408G antibiotic-resistant mutant. It is probable that bacteria manage to acquire antibiotic resistance without losing the function of the A-site molecular switch by mutating the position 1408 only from A to G, but not to pyrimidine base C or U.

Structure-Based Design of a Eukaryote-Selective Antiprotozoal Fluorinated Aminoglycoside.

Aminoglycosides (AG) are antibiotics that lower the accuracy of protein synthesis by targeting a highly conserved RNA helix of the ribosomal A-site. The discovery of AGs that selectively target the eukaryotic ribosome, but lack activity in prokaryotes, are promising as antiprotozoals for the treatment of neglected tropical diseases, and as therapies to read-through point-mutation genetic diseases. However, a single nucleobase change A1408G in the eukaryotic A-site leads to negligible affinity for most AGs. Herein we report the synthesis of 6'-fluorosisomicin, the first 6'-fluorinated aminoglycoside, which specifically interacts with the protozoal cytoplasmic rRNA A-site, but not the bacterial A-site, as evidenced by X-ray co-crystal structures. The respective dispositions of 6'-fluorosisomicin within the bacterial and protozoal A-sites reveal that the fluorine atom acts only as a hydrogen-bond acceptor to favorably interact with G1408 of the protozoal A-site. Unlike aminoglycosides containing a 6'-ammonium group, 6'-fluorosisomicin cannot participate in the hydrogen-bonding pattern that characterizes stable pseudo-base-pairs with A1408 of the bacterial A-sites. Based on these structural observations it may be possible to shift the biological activity of aminoglycosides to act preferentially as antiprotozoal agents. These findings expand the repertoire of small molecules targeting the eukaryotic ribosome and demonstrate the usefulness of fluorine as a design element.

Crystal structure of a DNA duplex containing four Ag(i) ions in consecutive dinuclear Ag(i)-mediated base pairs: 4-thiothymine-2Ag(i)-4-thiothymine.

Herein, we determined a high-resolution crystal structure of a B-form DNA duplex containing consecutive dinuclear metal ion-mediated base pairs, namely, 4-thiothymine-2Ag(i)-4-thiothymine (S-2Ag(i)-S), and four Ag(i) ions form a rectangular network and the distances between the Ag(i) ions are 2.8-3.2 Å, which may indicate the existence of metallophilic attractions.