Sequence-selective DNA recognition and enhanced cellular up-take by peptide–steroid conjugates

Several GCN4 bZIP TF models have previously been designed and synthesized. However, the synthetic routes towards these constructs are typically tedious and difficult. We here describe the substitution of the Leucine zipper domain of the protein by a deoxycholic acid derivative appending the two GCN4 binding region peptides through an optimized double azide–alkyne cycloaddition click reaction. In addition to achieving sequence specific dsDNA binding, we have investigated the potential of these compounds to enter cells. Confocal microscopy and flow cytometry show the beneficial influence of the steroid on cell uptake. This unique synthetic model of the bZIP TF thus combines sequence specific dsDNA binding properties with enhanced cell-uptake. Given the unique properties of deoxycholic acid and the convergent nature of the synthesis, we believe this work represents a key achievement in the field of TF mimicry

Specific dsDNA recognition by a mimic of the DNA binding domain of the c-Myc/Max transcription factor

We here report on the synthesis of the first mimic of the DNA binding domain of the c-Myc/Max-bHLH-ZIP transcription factor able to selectively recognize its cognate E-box sequence 50'-CACGTG-3' through the major groove of the double-stranded DNA. The designed peptidosteroid conjugate was shown to be effective as DNA binder in the presence of excess competitor DNA

Cyclodextrin-peptide conjugates for sequence specific DNA binding

Synthetic models of bZIP transcription factors have been developed with the capability of specific DNA recognition. Our design is based on the CuAAC mediated conjugation of basic region Leucine Zipper peptides to different derivatives of alpha,beta and gamma-cyclodextrins equipped with azide functionalities. Thorough optimization of reaction conditions allowed convergent and simultaneous conjugation of two long unprotected cationic peptides to cyclodextrin-bis azide derivatives. The resulting constructs were shown to specifically recognize their cognate DNA sequence with nM affinities. In comparison with previously developed TF models, the derivatives described here combine the enhanced DNA binding capabilities with an easy and convergent synthetic route

LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures

Abstract The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3′-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3′-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs

Role of Pseudoisocytidine Tautomerization in Triplex-Forming Oligonucleotides: In Silico and in Vitro Studies

Pseudoisocytidine (^ΨC) is a synthetic cytidine analogue that can target DNA duplex to form parallel triplex at neutral pH. Pseudoisocytidine has mainly two tautomers, of which only one is favorable for triplex formation. In this study, we investigated the effect of sequence on ^ΨC tautomerization using λ-dynamics simulation, which takes into account transitions between states. We also performed in vitro binding experiments with sequences containing ^ΨC and furthermore characterized the structure of the formed triplex using molecular dynamics simulation. We found that the neighboring methylated or protonated cytidine promotes the formation of the favorable tautomer, whereas the neighboring thymine or locked nucleic acid has a poor effect, and consecutive ^ΨC has a negative influence. The deleterious effect of consecutive ^ΨC in a triplex formation was confirmed using in vitro binding experiments. Our findings contribute to improving the design of ^ΨC-containing triplex-forming oligonucleotides directed to target G-rich DNA sequences

Intravenous NPA for the treatment of infarcting myocardium early: InTIME-II, a double-blind comparison on of single-bolus lanoteplase vs accelerated alteplase for the treatment of patients with acute myocardial infarction

Aims to compare the efficacy and safety of lanoteplase, a single-bolus thrombolytic drug derived from alteplase tissue plasminogen activator, with the established accelerated alteplase regimen in patients presenting within 6 h of onset of ST elevation acute myocardial infarction. Methods and Results 15 078 patients were recruited from 855 hospitals worldwide and randomized in a 2:1 ratio to receive either lanoteplase 120 KU. kg-1 as a single intravenous bolus, or up to 100 mg accelerated alteplase given over 90 min. The primary end-point was all-cause mortality at 30 days and the hypothesis was that the two treatments would be equivalent. By 30 days, 6.61% of alteplase-treated patients and 6.75% lanoteplase-treated patients had died (relative risk 1.02). Total stroke occurred in 1.53% alteplase- and 1.87% lanoteplase-treated patients (ns); haemorrhagic stroke rates were 0.64% alteplase and 1.12% lanoteplase (P=0.004). The net clinical deficit of 30-day death or non-fatal disabling stroke was 7.0% and 7.2%, respectively. By 6 months, 8.8% of alteplase-treated patients and 8.7% of lanoteplase-treated patients had died. Conclusion Single-bolus weight-adjusted lanoteplase is an effective thrombolytic agent, equivalent to alteplase in terms of its impact on survival and with a comparable risk-benefit profile. The single-bolus regimen should shorten symptoms to treatment times and be especially convenient for emergency department or out-of-hospital administration. (C) 2000 The European Society of Cardiology

Impact of COVID-19 on Diagnostic Cardiac Procedural Volume in Oceania: The IAEA Non-Invasive Cardiology Protocol Survey on COVID-19 (INCAPS COVID)

Objectives: The INCAPS COVID Oceania study aimed to assess the impact caused by the COVID-19 pandemic on cardiac procedure volume provided in the Oceania region. Methods: A retrospective survey was performed comparing procedure volumes within March 2019 (pre-COVID-19) with April 2020 (during first wave of COVID-19 pandemic). Sixty-three (63) health care facilities within Oceania that perform cardiac diagnostic procedures were surveyed, including a mixture of metropolitan and regional, hospital and outpatient, public and private sites, and 846 facilities outside of Oceania. The percentage change in procedure volume was measured between March 2019 and April 2020, compared by test type and by facility. Results: In Oceania, the total cardiac diagnostic procedure volume was reduced by 52.2% from March 2019 to April 2020, compared to a reduction of 75.9% seen in the rest of the world (p<0.001). Within Oceania sites, this reduction varied significantly between procedure types, but not between types of health care facility. All procedure types (other than stress cardiac magnetic resonance [CMR] and positron emission tomography [PET]) saw significant reductions in volume over this time period (p<0.001). In Oceania, transthoracic echocardiography (TTE) decreased by 51.6%, transoesophageal echocardiography (TOE) by 74.0%, and stress tests by 65% overall, which was more pronounced for stress electrocardiograph (ECG) (81.8%) and stress echocardiography (76.7%) compared to stress single-photon emission computerised tomography (SPECT) (44.3%). Invasive coronary angiography decreased by 36.7% in Oceania. Conclusion: A significant reduction in cardiac diagnostic procedure volume was seen across all facility types in Oceania and was likely a function of recommendations from cardiac societies and directives from government to minimise spread of COVID-19 amongst patients and staff. Longer term evaluation is important to assess for negative patient outcomes which may relate to deferral of usual models of care within cardiology

Reduction of cardiac imaging tests during the COVID-19 pandemic: The case of Italy. Findings from the IAEA Non-invasive Cardiology Protocol Survey on COVID-19 (INCAPS COVID)

Background: In early 2020, COVID-19 massively hit Italy, earlier and harder than any other European country. This caused a series of strict containment measures, aimed at blocking the spread of the pandemic. Healthcare delivery was also affected when resources were diverted towards care of COVID-19 patients, including intensive care wards. Aim of the study: The aim is assessing the impact of COVID-19 on cardiac imaging in Italy, compare to the Rest of Europe (RoE) and the World (RoW). Methods: A global survey was conducted in May–June 2020 worldwide, through a questionnaire distributed online. The survey covered three periods: March and April 2020, and March 2019. Data from 52 Italian centres, a subset of the 909 participating centres from 108 countries, were analyzed. Results: In Italy, volumes decreased by 67% in March 2020, compared to March 2019, as opposed to a significantly lower decrease (p < 0.001) in RoE and RoW (41% and 40%, respectively). A further decrease from March 2020 to April 2020 summed up to 76% for the North, 77% for the Centre and 86% for the South. When compared to the RoE and RoW, this further decrease from March 2020 to April 2020 in Italy was significantly less (p = 0.005), most likely reflecting the earlier effects of the containment measures in Italy, taken earlier than anywhere else in the West. Conclusions: The COVID-19 pandemic massively hit Italy and caused a disruption of healthcare services, including cardiac imaging studies. This raises concern about the medium- and long-term consequences for the high number of patients who were denied timely diagnoses and the subsequent lifesaving therapies and procedures