Synthesis and Biological Properties of Polyphenol-Containing Linear and Dendrimeric Cationic Peptides.

Natural polyphenols are promising compounds for the pharmacological control of oxidative stress in various diseases. However, low bioavailability and rapid metabolism of polyphenols in a form of glycosides or aglycones have stimulated the search for the vehicles that would provide their efficient delivery to the systemic circulation. Conjugation of polyphenols with cationic amphiphilic peptides yields compounds with a strong antioxidant activity and ability to pass through biological barriers. Due to a broad range of biological activities characteristic of polyphenols and peptides, their conjugates can be used in the antioxidant therapy, including the treatment of viral, oncological, and neurodegenerative diseases. In this work, we synthesized linear and dendrimeric cationic amphiphilic peptides that were then conjugated with gallic acid (GA). GA is a non-toxic natural phenolic acid and an important functional element of many flavonoids with a high antioxidant activity. The obtained GA-peptide conjugates showed the antioxidant (antiradical) activity that exceeded 2-3 times the antioxidant activity of ascorbic acid. GA attachment had no effect on the toxicity and hemolytic activity of the peptides. GA-modified peptides stimulated the transmembrane transfer of the pGL3 plasmid encoding luciferase reporter gene, although GA attachment at the N-terminus of peptides reduced their transfection activity. Several synthesized conjugates demonstrated the antibacterial activity in the model of Escherichia coli Dh5α growth inhibition.

Natural human Bet v 1-specific IgG antibodies recognize non-conformational epitopes whereas IgE reacts with conformational epitopes.

BACKGROUND: The nature of epitopes on Bet v 1 recognized by natural IgG antibodies of birch pollen allergic patients and birch pollen-exposed but non-sensitized subjects has not been studied in detail. OBJECTIVE: To investigate IgE and IgG recognition of Bet v 1 and to study the effects of natural Bet v 1-specific IgG antibodies on IgE recognition of Bet v 1 and Bet v 1-induced basophil activation. METHODS: Sera from birch pollen allergic patients (BPA, n = 76), allergic patients without birch pollen allergy (NBPA, n = 40) and non-allergic individuals (NA, n = 48) were tested for IgE, IgG as well as IgG1 and IgG4 reactivity to folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments comprising the N-terminal (F1) and C-terminal half of Bet v 1 (F2) and unfolded peptides spanning the corresponding sequences of Bet v 1 and the apple allergen Mal d 1 by ELISA or micro-array analysis. The ability of Bet v 1-specific serum antibodies from non-allergic subjects to inhibit allergic patients IgE or IgG binding to rBet v 1 or to unfolded Bet v 1-derivatives was assessed by competition ELISAs. Furthermore, the ability of serum antibodies from allergic and non-allergic subjects to modulate Bet v 1-induced basophil activation was investigated using rat basophilic leukaemia cells expressing the human FcεRI which had been loaded with IgE from BPA patients. RESULTS: IgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG1 and IgG4 antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes. IgG competition studies show that IgG specific for unfolded/sequential Bet v 1 epitopes is not inhibited by folded Bet v 1 and hence the latter seem to represent cryptic epitopes. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 peptides and therefore does not seem to be a result of primary sensitization to PR10 allergen-containing food. Natural Bet v 1-specific IgG antibodies inhibited IgE binding to Bet v 1 only poorly and could even enhance Bet v 1-specific basophil activation. CONCLUSION: IgE and IgG antibodies from BPA patients and birch pollen-exposed non-sensitized subjects recognize different epitopes. These findings explain why natural allergen-specific IgG do not protect against allergic symptoms and suggest that allergen-specific IgE and IgG have different clonal origin.

Treatment of COVID-19 patients with a SARS-CoV-2-specific siRNA-peptide dendrimer formulation.

BACKGROUND: Severe acute respiratory syndrome corona virus (SARS-CoV-2) infection frequently causes severe and prolonged disease but only few specific treatments are available. We aimed to investigate safety and efficacy of a SARS-CoV-2-specific siRNA-peptide dendrimer formulation MIR 19® (siR-7-EM/KK-46) targeting a conserved sequence in known SARS-CoV-2 variants for treatment of COVID-19. METHODS: We conducted an open-label, randomized, controlled multicenter phase II trial (NCT05184127) evaluating safety and efficacy of inhaled siR-7-EM/KK-46 (3.7 mg and 11.1 mg/day: low and high dose, respectively) in comparison with standard etiotropic drug treatment (control group) in patients hospitalized with moderate COVID-19 (N = 52 for each group). The primary endpoint was the time to clinical improvement according to predefined criteria within 14 days of randomization. RESULTS: Patients from the low-dose group achieved the primary endpoint defined by simultaneous achievement of relief of fever, normalization of respiratory rate, reduction of coughing, and oxygen saturation of >95% for 48 h significantly earlier (median 6 days; 95% confidence interval [CI]: 5-7, HR 1.75, p = .0005) than patients from the control group (8 days; 95% CI: 7-10). No significant clinical efficacy was observed for the high-dose group. Adverse events were reported in 26 (50.00%), 25 (48.08%), and 28 (53.85%) patients from the low-, high-dose and control group, respectively. None of them were associated with siR-7-EM/KK-46. CONCLUSIONS: siR-7-EM/KK-46, a SARS-CoV-2-specific siRNA-peptide dendrimer formulation is safe, well tolerated and significantly reduces time to clinical improvement in patients hospitalized with moderate COVID-19 compared to standard therapy in a randomized controlled trial.

Evaluation of the efficacy of permethrin- and cypermethrin-based textile against taiga tick, Ixodes persulcatus.

A comparison of the efficacy of permethrin- and cypermethrin-based textile against taiga ticks (Ixodes persulcatus) was carried out in a tick-borne viral encephalitis hotspot in the Irkutsk Region (Russia) using model samples of impregnated textiles. We demonstrated that permethrin- and cypermethrin-treated model samples have similar protective parameters in terms of maximum height reached by the tick when climbing up the treated textile (20.9-38.7 cm for cypermethrin, 27.6-39.3 cm for permethrin, depending on concentration) and knockdown time (i.e., the time until a female tick falls off the treated textile; 3.52-4.31 min for cypermethrin, 5.02-8.25 min for permethrin, depending on concentration). In contrast, when evaluating the 'biting speed' index (which is the ratio of the average attaching time of ticks contacting untreated textiles and ticks contacting treated textiles), it has been shown that permethrin-treated textiles accelerate biting. So, using permethrin-treated protective clothing against the taiga tick could be risky because it increases the likelihood of being bitten and thus getting infected. In contrast, cypermethrin-treated textiles appear to block the ability of ticks to attack warm-blooded animals and humans - after contact with cypermethrin-treated textiles none of the ticks attached to a rabbit. So cypermethrin-based textiles could be an alternative to permethrin for tick-bite protection clothing production if there is no toxic effect on humans of textile materials based on it.

Low-dose dobutamine stress gated blood pool SPECT assessment of left ventricular contractile reserve in ischemic cardiomyopathy: a feasibility study.

PURPOSE: The purpose of the present study was to evaluate the feasibility of gated blood pool single-photon emission computed tomography (GBPS) with low-dose dobutamine (LDD) stress test, performed on a single-photon emission computed tomography (SPECT) camera equipped with cadmium-zinc-telluride (CZT) solid-state detectors, in assessing of left ventricle (LV) contractile reserve in patients with ischemic cardiomyopathy (ICM). METHODS: A total of 52 patients (age 59 ± 7.2 years, 47 men and 5 women) with ICM and a control group of 10 patients without obstructive coronary artery lesion underwent GBPS and transthoracic echocardiography (TTE) at rest and during LDD stress test (5, 10, 15 µg/kg/min). The duration of each GBPS step was 5 min. Stress-induced changes in LV ejection fraction (ΔLVEF), peak ejection rate, LV volumes, and mechanical dyssynchrony (phase histogram standard deviation, phase histogram bandwidth and entropy) obtained with GBPS were estimated. RESULTS: All GBPS indices except end-diastolic volume showed significant dynamics during stress test in both groups. The majority of parameters in ICM patients showed significant changes at a dobutamine dose of 10 µg/kg/min as compared to the rest study. Seventeen percent of ICM patients, but none from the control group, showed a decrease in LVEF during stress, accompanied by a significant increase in entropy. The intra- and inter-observer reproducibility was excellent for both rest and stress studies. There was a moderate correlation (r = 0.5, p = 0.01) between GBPS and TTE, with a mean difference value of - 1.7 (95% confidence interval - 9.8; 6.4; p = 0.06) in ΔLVEF. CONCLUSION: Low-dose dobutamine stress GBPS performed with high-efficiency CZT-SPECT cameras can be performed for evaluating stress-induced changes in LV contractility and dyssynchrony with lower acquisition time. A dobutamine dose of 10 µg/kg/min can potentially suffice to detect stress-induced changes in patients with ICM during GBPS. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04508608 (August 7, 2020).

The diagnostic value of SPECT CZT quantitative myocardial blood flow in high-risk patients.

BACKGROUND: The objective of this study was to evaluate the accuracy of global MBF and MFR quantitation performed by myocardial perfusion scintigraphy (MPS) for the detection of multivessel coronary artery disease (CAD). METHODS: 52 CAD patients underwent CZT MPS, with the evaluation of MBF and MFR, followed by invasive coronary angiography (ICA). According to MPS and ICA results, all patients were divided into three groups: (1) non-obstructive CAD and normal MPS scan (control group) (n = 7), (2) one vessel disease (1VD) (n = 16), (3) multivessel disease (MVD) (n = 29). RESULTS: Global absolute MBF and MFR were significantly reduced in MVD patients as compared to those with 1VD [0.93 (IQR 0.76; 1.39) vs 1.94 (1.37; 2.21) mL·min-1·g-1, P = .00012] and [1.4 (IQR 1.02; 1.85) vs 2.3 (1.8; 2.67), P = . 0 004], respectively. The Syntax score correlated with global stress MBF (ρ = - 0.64; P < .0001) and MFR (ρ = - 0.53; P = .0003). ROC analysis showed higher sensitivity and specificity for stress MBF and MFR compared with semiquantitative MPS stress evaluation. Multivariate regression analysis showed that only stress MBF [OR (95% CI) 0.59 (0.42-0.82); P < .0003] was an independent predictor of MVD. CONCLUSIONS: Quantitative myocardial blood flow values assessed with the use of CZT camera may identify high-risk patients, such as those with multivessel disease.

Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation.

BACKGROUND: First vaccines for prevention of Coronavirus disease 2019 (COVID-19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti-SARS-CoV-2 effect of siRNA both in vitro and in vivo. METHODS: To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS-CoV-2 genes fused with the firefly luciferase reporter gene and SARS-CoV-2-infected cells was used. The most potent siRNA, siR-7, was modified by Locked nucleic acids (LNAs) to obtain siR-7-EM with increased stability and was formulated with the peptide dendrimer KK-46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation - siR-7-EM/KK-46. Using the Syrian Hamster model for SARS-CoV-2 infection the antiviral capacity of siR-7-EM/KK-46 complex was evaluated. RESULTS: We identified the siRNA, siR-7, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro. Moreover, we showed that LNA-modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR-7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR-7-EM/KK-46 in vivo. CONCLUSIONS: Thus, we developed a therapeutic strategy for COVID-19 based on inhalation of a modified siRNA-peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID-19 patients.

Chromosome Folding Promotes Intrachromosomal Aberrations under Radiation- and Nuclease-Induced DNA Breakage.

The long-standing question in radiation and cancer biology is how principles of chromosome organization impact the formation of chromosomal aberrations (CAs). To address this issue, we developed a physical modeling approach and analyzed high-throughput genomic data from chromosome conformation capture (Hi-C) and translocation sequencing (HTGTS) methods. Combining modeling of chromosome structure and of chromosomal aberrations induced by ionizing radiation (IR) and nuclease we made predictions which quantitatively correlated with key experimental findings in mouse chromosomes: chromosome contact maps, high frequency of cis-translocation breakpoints far outside of the site of nuclease-induced DNA double-strand breaks (DSBs), the distinct shape of breakpoint distribution in chromosomes with different 3D organizations. These correlations support the heteropolymer globule principle of chromosome organization in G1-arrested pro-B mouse cells. The joint analysis of Hi-C, HTGTS and physical modeling data offers mechanistic insight into how chromosome structure heterogeneity, globular folding and lesion dynamics drive IR-recurrent CAs. The results provide the biophysical and computational basis for the analysis of chromosome aberration landscape under IR and nuclease-induced DSBs.

A novel peptide dendrimer LTP efficiently facilitates transfection of mammalian cells.

One of the urgent problems of gene therapy is the search for effective transfection methods. Synthetic cationic peptides (CPs) are considered to be one of the most promising approaches for intracellular transport of oligonucleotides. Almost unlimited possibilities of the architectural design of CPs (linear and cyclic structures with a variation of chirality as well as dendrimers) make CPs an effective tunable carrier in this field. Cationic peptide dendrimers (PDs), as a relatively new direction, have significant advantages as gene delivery vehicles by virtue of non-natural ε-amide bonds that significantly increase their resistance to proteolysis. Moreover they also possess much lower cytotoxicity than linear peptides, which is crucial for the potential clinical application of CPs. In a further development of oligonucleotide delivery systems, we have synthesized a collection of 14 CPs, including linear peptides, lipopeptides and PDs. Their activity was evaluated by transfection of 293T cells with plasmids containing reporter genes encoding luciferase or a green fluorescent protein. The obtained results demonstrated that the greatest activity was exhibited by PDs, particularly LTP, an arginine-rich peptide dendrimer, which possesses low cytotoxic and hemolytic activity. The peptide exhibited high cell-penetrating activity, confirmed by fast dissipation of the membrane potential of cells probed by dis-C3-(5). The quantitative analysis of labelled LTP in tissue samples of mice revealed that the Cy5-LTP/siRNA complexes have a reasonable tropism to lung tissues.