Metal-dependent activity control of a compact-sized 8-17 DNAzyme based on metal-mediated unnatural base pairing.

A compact 8-17 DNAzyme was modified with a CuII-meditated artificial base pair to develop a metal-responsive allosteric DNAzyme. The base sequence was rationally designed based on the reported three-dimensional structure. The activity of the modified DNAzyme was enhanced 5.1-fold by the addition of one equivalent of CuII ions, showing good metal responsiveness. Since it has been challenging to modify compactly folded DNAzymes without losing their activity, this study demonstrates the utility of the metal-mediated artificial base pairing to create stimuli-responsive functional DNAs.

Metal-mediated DNA strand displacement and molecular device operations based on base-pair switching of 5-hydroxyuracil nucleobases.

Rational design of self-assembled DNA nanostructures has become one of the fastest-growing research areas in molecular science. Particular attention is focused on the development of dynamic DNA nanodevices whose configuration and function are regulated by specific chemical inputs. Herein, we demonstrate the concept of metal-mediated base-pair switching to induce inter- and intramolecular DNA strand displacement in a metal-responsive manner. The 5-hydroxyuracil (UOH) nucleobase is employed as a metal-responsive unit, forming both a hydrogen-bonded UOH-A base pair and a metal-mediated UOH-GdIII-UOH base pair. Metal-mediated strand displacement reactions are demonstrated under isothermal conditions based on the base-pair switching between UOH-A and UOH-GdIII-UOH. Furthermore, metal-responsive DNA tweezers and allosteric DNAzymes are developed as typical models for DNA nanodevices simply by incorporating UOH bases into the sequence. The metal-mediated base-pair switching will become a versatile strategy for constructing stimuli-responsive DNA nanostructures, expanding the scope of dynamic DNA nanotechnology.

Superior immunogenicity of high-dose quadrivalent inactivated influenza vaccine versus Standard-Dose vaccine in Japanese Adults ≥ 60 years of age: Results from a phase III, randomized clinical trial.

BACKGROUND: A high-dose, split-virion inactivated quadrivalent influenza vaccine (IIV4-HD; Sanofi) is being used for the prevention of influenza in multiple countries. This study examined the immunogenicity and safety of the IIV4-HD vaccine administered intramuscularly (IM) compared with a locally licensed standard-dose influenza vaccine (IIV4-SD) administered subcutaneously (SC) in Japan. METHODS: This was a phase III, randomized, modified double-blind, active-controlled, multi-center study in older adults ≥ 60 years of age conducted during the Northern Hemisphere (NH) influenza season of 2020-21 in Japan. Participants were randomized in a 1:1 ratio to receive a single IM injection of IIV4-HD or SC injection of IIV4-SD. Hemagglutination inhibition antibody and seroconversion rates were measured at baseline and day 28. Solicited reactions were collected for up to 7 days after vaccination, unsolicited adverse events up to 28 days after vaccination, and serious adverse events throughout the study. RESULTS: The study included 2100 adults ≥ 60 years of age. IIV4-HD given IM induced superior immune responses versus IIV4-SD given SC as assessed by geometric mean titers for all four influenza strains. Superior seroconversion rates were also observed for IIV4-HD compared to IIV4-SD for all influenza strains. The safety profiles of IIV4-HD and IIV4-SD were similar. IIV4-HD was well tolerated in participants, with no safety concerns identified. CONCLUSIONS: IIV4-HD provided superior immunogenicity versus IIV4-SD and was well tolerated in participants ≥ 60 years of age in Japan. With superior immunogenicity based on the multiple randomized controlled trials and real-world evidence of trivalent high-dose formulation, IIV4-HD is expected to be the first differentiated influenza vaccine in Japan that offer a greater protection against influenza and its complications in adults 60 years of age and older. STUDY REGISTRATION: NCT04498832 (clinicaltrials.gov); U1111-1225-1085 (who.int).

Hysteresis behavior in the unfolding/refolding processes of a protein trapped in metallo-cages.

Confinement of molecules in a synthetic host can physically isolate even their unstable temporary structures, which has potential for application to protein transient structure analysis. Here we report the NMR snapshot observation of protein unfolding and refolding processes by confining a target protein in a self-assembled coordination cage. With increasing acetonitrile content in CD3CN/H2O media (50 to 90 vol%), the folding structure of a protein sharply denatured at 83 vol%, clearly revealing the regions of initial unfolding. Unfavorable aggregation of the protein leading to irreversible precipitation is completely prevented because of the spatial isolation of the single protein molecule in the cage. When the acetonitrile content reversed (84 to 70 vol%), the once-denatured protein started to regain its original folded structure at 80 vol%, showing that the protein folding/unfolding process can be referred to as a phase transition with hysteresis behavior.

Immunogenicity and Safety of a Quadrivalent Meningococcal Tetanus Toxoid-Conjugate Vaccine (MenACYW-TT) in Meningococcal Vaccine-Naïve Participants across a Broad Age Range (2-55 Years) in Japan: a Phase III Randomized Study.

MenACYW-TT is a quadrivalent meningococcal tetanus toxoid-conjugate vaccine designed to prevent invasive meningococcal disease. The primary objective of this study was to demonstrate non-inferiority of the vaccine seroresponse to a single dose of MenACYW-TT compared with MCV4-DT, a licensed meningococcal quadrivalent diphtheria-conjugate vaccine. This Phase III double-blind, multicenter trial was conducted in meningococcal vaccine-naïve individuals aged 2-55 years in Japan (NCT04368429; jRCT2080225192). Participants were randomized 1:1 to receive either MenACYW-TT (n = 180) or MCV4-DT (n = 180). Functional antibodies against meningococcal serogroups A, C, W, and Y were measured using a serum bactericidal antibody assay with human complement (hSBA) at baseline (D0) and 30 days after vaccination (D30). Seroresponse was defined as a post-vaccination titer ≥1:16 in participants with a baseline titer <1:8; or a ≥4-fold increase in titer in participants with a baseline titer ≥1:8. Safety data were collected for 30 days. Non-inferiority of the seroresponse to MenACYW-TT vs. MCV4-DT was demonstrated on D30 for each serogroup tested (A: 85.6% vs. 65.4%; C: 96.6% vs. 62.6%; W: 87.4% vs. 49.2%; Y: 97.7% vs. 63.5%). MenACYW-TT was well tolerated with no safety concerns identified. A single dose of MenACYW-TT was well tolerated, with a non-inferior seroresponse compared with MCV4-DT. MenACYW-TT could thus be used as an alternative vaccine in meningococcal vaccine-naïve individuals.

Site-specific polymerase incorporation of consecutive ligand-containing nucleotides for multiple metal-mediated base pairing.

An enzymatic method has been developed for the synthesis of DNA oligomers containing consecutive artificial ligand-type nucleotides. Three hydroxypyridone ligand-containing nucleotides forming CuII-mediated unnatural base pairs were continuously incorporated at a pre-specified position by a lesion-bypass Dpo4 polymerase. This enzymatic synthesis was applied to the development of a CuII-responsive DNAzyme. Accordingly, this research will open new routes for the construction of metal-responsive DNA architectures that are manipulated by multiple metal-mediated base pairing.

Sharp Switching of DNAzyme Activity through the Formation of a CuII -Mediated Carboxyimidazole Base Pair.

DNAzymes are widely used as functional units for creating DNA-based sensors and devices. Switching of DNAzyme activity by external stimuli is of increasing interest. Herein we report a CuII -responsive DNAzyme rationally designed by incorporating one of the most stabilizing artificial metallo-base pairs, a CuII -mediated carboxyimidazole base pair (ImC -CuII -ImC ), into a known RNA-cleaving DNAzyme. Cleavage of the substrate was suppressed without CuII , but the reaction proceeded efficiently in the presence of CuII ions. This is due to the induction of a catalytically active structure by ImC -CuII -ImC pairing. The on/off ratio was as high as 12-fold, which far exceeds that of the previously reported DNAzyme with a CuII -mediated hydroxypyridone base pair. The DNAzyme activity can be regulated specifically in response to CuII ions during the reaction through the addition, removal, or reduction of CuII . This approach should advance the development of stimuli-responsive DNA systems with a well-defined sharp switching function.

Allosteric Regulation of DNAzyme Activities through Intrastrand Transformation Induced by Cu(II)-Mediated Artificial Base Pairing.

Allosteric regulation is gaining increasing attention as a basis for the production of stimuli-responsive materials in many research areas including DNA nanotechnology. We expected that metal-mediated artificial base pairs, consisting of ligand-type nucleotides and a bridging metal ion, could serve as allosteric units that regulate the function of DNA molecules. In this study, we established a rational design strategy for developing CuII-responsive allosteric DNAzymes by incorporating artificial hydroxypyridone ligand-type nucleotides (H) that form a CuII-mediated base pair (H-CuII-H). We devised a new enzymatic method using a standard DNA polymerase and a ligase to prepare DNA strands containing H nucleotides. Previously reported DNAzymes were modified by introducing a H-H pair into the stem region, and the stem-loop sequences were altered so that the structure becomes catalytically inactive in the absence of CuII ions. The formation of a H-CuII-H base pair triggers intrastrand transformation from the inactive to the active structure, enabling allosteric regulation of the DNAzyme activity in response to CuII ions. The activity of the H-modified DNAzyme was reversibly switched by the addition and removal of CuII ions under isothermal conditions. Similarly, by incorporating a H-CuII-H pair into an in vitro-selected AgI-dependent DNAzyme, we have developed a DNAzyme that exhibits an AND logic-gate response to CuII and AgI ions. The rational design strategy and the easy enzymatic synthetic method presented here provide a versatile way to develop a variety of metal-responsive allosteric DNA materials, including molecular machines and logic circuits, based on metal-mediated artificial base pairing.

Efficacy and safety of insulin glargine/lixisenatide fixed-ratio combination (iGlarLixi 1:1) in Japanese patients with type 2 diabetes mellitus inadequately controlled on oral antidiabetic drugs: A randomized, 26-week, open-label, multicentre study: The LixiLan JP-O2 randomized clinical trial.

AIMS: To assess efficacy and safety of 26-week treatment with insulin glargine/lixisenatide fixed-ratio combination (iGlarLixi) compared with insulin glargine U100 (iGlar) in Japanese patients with type 2 diabetes mellitus (T2DM) inadequately controlled on oral antidiabetic drugs (OADs). MATERIALS AND METHODS: This phase 3, multicentre, open-label, 1:1 randomized, parallel-group study compared efficacy of iGlarLixi and iGlar in patients with T2DM, HbA1c of ≥7.5% to ≤9.5% and fasting plasma glucose ≤10.0 mmol/L (180 mg/dL). The primary endpoint was change in HbA1c from baseline to week 26. RESULTS: Patients were randomized to iGlarLixi (n = 260) or iGlar (n = 261) (mean age 59.7 years, baseline BMI 26.04 kg/m2 , and HbA1c 8.04% [64.4 mmol/mol]). HbA1c reduction was significantly greater with iGlarLixi (-1.40% [-15.3 mmol/mol]) than with iGlar (-0.76% [-8.3 mmol/mol]). Significantly more iGlarLixi patients reached HbA1c <7% at week 26 (71.5% vs 38.5%, P < .0001), with significantly lower weight gain (LS mean difference -1.06 kg, P < .0001). Documented symptomatic hypoglycemia (plasma glucose ≤3.9 mmol/L [70 mg/dL]) was recorded in 14.2% of patients with iGlarLixi and 12.3% with iGlar. No severe hypoglycemia was reported in either group. Other than the expected gastrointestinal issues associated with glucagon-like peptide 1 receptor agonists, we found no major difference in the incidence of TEAEs. CONCLUSIONS: HbA1c reduction was significantly greater with iGlarLixi than with iGlar; significantly more patients achieved HbA1c <7%, with no additional risk of hypoglycemia and without weight gain. iGlarLixi (1:1) provided an effective treatment option for Japanese patients with T2DM inadequately controlled on OADs. Clinical Trial Number: NCT02752828.

Immunogenicity and safety of high-dose quadrivalent influenza vaccine in Japanese adults ≥65 years of age: a randomized controlled clinical trial.

A trivalent high-dose inactivated influenza vaccine has been licensed in healthy adults ≥65 years of age and provides better protection against influenza infection and related complications than trivalent standard-dose vaccine. This phase I/II clinical trial (NCT03233217), conducted at two sites in Japan, examined the safety and immunogenicity of a quadrivalent formulation of the high-dose inactivated influenza vaccine (IIV4-HD). Healthy adults ≥65 years of age were randomized to receive IIV4-HD by intramuscular injection (n = 60), IIV4-HD by subcutaneous injection (n = 60), or a quadrivalent standard-dose inactivated influenza vaccine (IIV4-SD) by subcutaneous injection (n = 55). Irrespective of administration route, post-vaccination (day 28-35) hemagglutination inhibition geometric mean titers and seroconversion rates were higher for IIV4-HD than for IIV4-SD. Hemagglutination inhibition geometric mean titers and seroconversion rates were also higher for intramuscular than subcutaneous administration of IIV4-HD. Solicited reactions were more common in participants who received IIV4-HD administered subcutaneously than in those who received IIV4-HD administered intramuscularly or IIV4-SD administered subcutaneously. Unsolicited adverse events were similar between the vaccine groups, and no safety signals were detected. This study showed that IIV4-HD administered by either intramuscular or subcutaneous injection was well tolerated and highly immunogenic in healthy Japanese adults ≥65 years of age. Although this study was descriptive, the results add to the evidence that high-dose inactivated influenza vaccines are more immunogenic than standard-dose vaccines in this age group and that intramuscular administration provides greater immunogenicity and lower reactogenicity than subcutaneous administration.

Enzymatic Synthesis of Cu(II)-Responsive Deoxyribozymes through Polymerase Incorporation of Artificial Ligand-Type Nucleotides.

Metal-mediated artificial base pairs, consisting of ligand-type nucleotides and a bridging metal ion, have shown promise as functional units to develop stimuli-responsive DNA materials. Although a variety of metal-mediated base pairs have been constructed with artificial ligand-type nucleotides and various metal ions, the application of such metal-mediated base pairs has been relatively poorly explored mainly due to the cumbersome chemical synthesis of artificial DNA strands. Herein we report a facile enzymatic method to synthesize DNA strands containing a ligand-type hydroxypyridone (H) nucleotide, which forms a CuII-mediated base pair (H-CuII-H). A two-step primer extension reaction using two commercially available polymerases enabled the incorporation of a H nucleotide at an internal position of oligonucleotides. The polymerase synthesis was subsequently applied to the development of metal-responsive deoxyribozymes (DNAzymes), whose catalytic activity was regulated by the formation of a single H-CuII-H base pair in its stem region. The DNAzyme activity was reversibly switched by the alternate addition and the removal of CuII ions. Furthermore, metal-dependent orthogonal activation of a CuII-responsive H-DNAzyme and a HgII-responsive T-DNAzyme was experimentally demonstrated by utilizing both H-CuII-H as well as widely explored T-HgII-T base pairs. These results suggest that the incorporation of H-CuII-H base pairs would facilitate the rational design of metal-responsive functional DNAs. Accordingly, the facile enzymatic synthesis of artificial ligand-bearing DNAs developed in this study would significantly expand the toolbox of DNA-based supramolecular chemistry and DNA nanotechnology.

Metal-responsive structural transformation between artificial DNA duplexes and three-way junctions.

DNA three-way junctions (3WJs) are essential structural motifs for DNA nanoarchitectures and DNA-based materials. We report herein a metal-responsive structural transformation between DNA duplexes and 3WJs using artificial oligonucleotides modified with a 2,2'-bipyridine (bpy) ligand. A mixture of bpy-modified DNA strands and natural complementary strands were self-assembled exclusively into duplexes without any transition metal ions, while they formed 3WJs in the presence of NiII ions. This transformation was induced by the formation of an interstrand NiII(bpy)3 complex, which served as a template for the 3WJ assembly. Altering the amount and identity of the metal ion regulated the 3WJ induction efficiency. Removal of the metal using EDTA quantitatively regenerated the duplexes. The metal-dependent structural conversion shown here has many potential applications in the development of stimuli-responsive DNA materials.

High incidence of community-acquired pneumonia among rapidly aging population in Japan: a prospective hospital-based surveillance.

The age-group-specific incidence and etiological patterns of community-acquired pneumonia (CAP) have not been fully established in Japan. A 2-year prospective surveillance was conducted in Kochi city, Western Japan. All CAP patients aged ≥15 years who visited a community-based hospital were enrolled in the study. Clinical samples were examined by conventional bacterial culture and urinary antigen tests, and 6 bacterial pathogens and 16 respiratory viruses were identified from sputum samples by multiplex polymerase chain reaction assays. The age-group-specific incidence of CAP was estimated using a population-based data set of the total number of outpatients in the whole city. Ninety of the 131 enrolled patients, 68.7% were positive for respiratory pathogens. Streptococcus pneumoniae was the leading bacterial pathogen identified (28.2%). Respiratory viruses were identified in 36 patients (27.5%), and human entero-rhinovirus was the most common (13.3%) among them. The estimated overall incidence of adult CAP in Kochi was 9.6 per 1,000 person-years (PY); the estimated age group-specific incidence was 3.4, 10.7, and 42.9 per 1,000 PY for those aged 15-64, 65-74, and ≥75 years, respectively. The high incidence of CAP in these rural city of Japan, probably reflects the substantial aged population. S. pneumoniae and respiratory viruses play important roles in CAP in all age groups.

[An autopsied case of pulmonary clear cell adenocarcinoma].

We report a case of a 72-year-old woman who died of primary lung clear cell adenocarcinoma. She was an active smoker, but with no other significant previous medical abnormalities. She visited our hospital with complaining of hemoptysis lasting for a month. Her sputum production and coughing had also increased. Chest X-ray films showed obstructive pneumonia in the left lower lobe. Chest computed tomography (CT) showed a tumor shadow and collapsed portion in the left hilar area. Sputum cytology and further diagnostic tests revealed stage IV lung adenocarcinoma. Chemotherapy with carboplatin and paclitaxel was initiated, but no improvement was obtained. She died from progressive cancer invasion into the airway. An autopsy revealed that more than 90% of the cancer cells were clear cells. These cancer calls were positive for PAS and occasionally showed Alcian blue-positive intracytoplasmic mucin and glandular structures. They were immunocytochemically stained with cytokeratin 7 but not with cytokeratin 20. According to previous reports in the literature, cases of primary lung clear cell adenocarcinoma are very rare.

Idiopathic localized bronchostenosis in an adult man with frequent recurring pneumonia.

The case of a 48-year-old Japanese man with idiopathic bronchostenosis in the right lower lobe is reported. The patient had fourteen episodes of pneumonia in two years and therefore surgical resection of the right lower lobe was performed for both diagnosis and treatment. Histopathology demonstrated no evidence of malignancy, tuberculosis, sarcoidosis or amyloid deposition. Despite an exhaustive evaluation, a specific etiology was never determined. The patient was given the diagnosis of acquired idiopathic localized bronchostenosis with frequent recurrence of pneumonia.