Polyprenol-Based Lipofecting Agents for In Vivo Delivery of Therapeutic DNA to Treat Hypertensive Rats.

Development of efficient vectors for transfection is one of the major challenges in genetic engineering. Previous research demonstrated that cationic derivatives of polyisoprenoids (PTAI) may serve as carriers of nucleic acids. In the present study, the effectiveness of two PTAI-based formulations (PTAI-6-8 and 10-14) was investigated and compared to the commercial reagents. The purpose of applied gene therapy was to enhance the expression of vascular endothelial growth factor (VEGF-A) in the renal medulla of spontaneously hypertensive rats (SHR) and to test its potential as a novel antihypertensive intervention. In the first part of the study (in vitro), we confirmed that PTAI-based lipoplexes efficiently transfect XC rat sarcoma cells and are stable in 37 °C for 7 days. In the in vivo experiments, we administered selected lipoplexes directly to the kidneys of conscious SHR (via osmotic pumps). There were no blood pressure changes and VEGF-A level in renal medulla was significantly higher only for PTAI-10-14-based formulation. In conclusion, despite the promising results, we were not able to achieve VEGF-A expression level high enough to verify VEGF-A gene therapy usefulness in SHR. However, results of our study give important indications for the future development of PTAI-based DNA carriers and kidney-targeted gene delivery.

Synthesis of ß-lactams via diastereoselective, intramolecular Kinugasa reactions.

Intramolecular Kinugasa reactions on in situ generated carbohydrate-derived alkynylnitrones are described. The effects of the length of chains, their mutual configuration, influence of experimental conditions on product distribution and feasibility of the ß-lactam ring construction were studied. Intramolecular reactions proceed with high stereoselectivity to provide in each case one product only. The cycloadducts from tartaric acid were converted into the corresponding non-racemic 4-acetoxy azetidinones in good yields.

Diastereoisomerically Pure, (S)-O-1,2-O-isopropyli dene-(5-O-α-d-glucofuranosyl) t-butanesulfinate: Synthesis, Crystal Structure, Absolute Configuration and Reactivity.

The reaction of t-butylmagnesium chlorides with diastereomerically pure (R)-1,2-O-isopropylidene-3,5-O-sulfinyl-α-d-glucofuranose (R)-4 was found to be stopped at the stage of the corresponding, diastereoisomerically pure 1,2-O-isopropylidene-(5-O-α-d-glucofuranosyl) t-butanesulfinate (S)-10 for which the crystal structure and the (S)-absolute configuration was determined by X-ray crystallography. Comparison of the absolute configurations of the starting sulfite (R)-4, and t-butanesulfinate (S)-10 (which crystallizes in the orthorhombic system, space group P212121, with the single compound molecule present in the asymmetric unit), clearly indicates that the reaction of nucleophilic substitution at the stereogenic sulfur atom in the sulfite (R)-4 occurs with the full inversion of configuration via the trigonal bipyramidal sulfurane intermediate 4c in which both the entering and leaving groups are located in apical positions.

Bypassing the stereoselectivity issue: transformations of Kinugasa adducts from chiral alkynes and non-chiral acyclic nitrones.

An approach to ß-lactams via a reaction between chiral copper acetylides and acyclic nitrones is reported. Electronic circular dichroism (ECD) in combination with NMR spectroscopy was used to determine the absolute configuration of all components of complex mixtures of azetidinones. Stereochemical preferences observed in the studied reactions are discussed and a model of a new reaction pathway supported by DFT conformational analysis is proposed. Subsequent transformations of the synthesized Kinugasa adducts followed by epimerization at the C-3 carbon atom led to trans-substituted azetidinones with improved stereoselectivity, mimicking a variety of important ß-lactam structures. On the other hand, the oxidation of the furyl residue to the carboxylic group followed by oxidative decarboxylation with lead tetraacetate afforded the more thermodynamically stable trans-substituted 4-acetoxy azetidinone. The latter strategy is particularly attractive for the diastereomeric mixture in which isomers with the same configuration at the C-3 carbon atom dominate.

Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus.

BACKGROUND: Cationic derivatives of polyprenols (trimethylpolyprenylammonium iodides - PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine. METHODS: Several vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. RESULTS: The mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid. CONCLUSION: The animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent.

Efficient and non-toxic gene delivery by anionic lipoplexes based on polyprenyl ammonium salts and their effects on cell physiology.

BACKGROUND: One of the major challenges limiting the development of gene therapy is an absence of efficient and safe gene carriers. Among the nonviral gene delivery methods, lipofection is considered as one of the most promising. In the present study, a set of cationic polyprenyl derivatives [trimethylpolyprenylammonium iodides (PTAI)] with different lengths of polyprenyl chains (from 7, 8 and 11 to 15 isoprene units) was suggested as a component of efficient DNA vehicles. METHODS: Optimization studies were conducted for PTAI in combination with co-lipid dioleoylphosphatidylethanolamine on DU145 human prostate cancer cells using: size and zeta potential measurements, confocal microscopy, the fluorescein diacetate/ethidium bromide test, cell counting, time-lapse monitoring of cell movement, gap junctional intercellular coupling analysis, antimicrobial activity assay and a red blood cell hemolysis test. RESULTS: The results obtained show that the lipofecting activity of PTAI allows effective transfection of plasmid DNA complexed in negatively-charged lipoplexes of 200-500 nm size into cells without significant side effects on cell physiology (viability, proliferation, morphology, migration and gap junctional intercellular coupling). Moreover, PTAI-based vehicles exhibit a potent bactericidal activity against Staphylococcus aureus and Escherichia coli. The developed anionic lipoplexes are safe towards human red blood cell membranes, which are not disrupted in their presence. CONCLUSIONS: The developed carriers constitute a group of promising lipofecting agents of a new type that can be utilized as effective lipofecting agents in vitro and they are also an encouraging basis for in vivo applications.

Approach to Monobactams and Nocardicins via Diastereoselective Kinugasa Reaction.

A Kinugasa reaction between copper(I) acetylides and cyclic nitrones derived from chiral amino alcohols and glyoxylic acid is reported. The stereochemical preferences observed in this reaction are discussed. The alkyne molecule approaches the nitrone exclusively anti to the large substituent next to the nitrogen atom to provide the cis-substituted ß-lactam ring preferentially. The six-membered oxazinone ring can be opened by reduction with lithium borohydride. Deprotection of the ß-lactam nitrogen atom can be achieved by lithium in liquid ammonia reduction or by CAN oxidation, depending on the substituents attached to the four-membered azetidinone ring. The adducts obtained by the Kinugasa reaction provide an attractive entry to a variety of monocyclic ß-lactam structures related to monobactams and nocardicins.

Glycosyl hydroperoxides: a new class of potential antimalarial agents.

Motivated by the antimalarial properties observed in organic peroxides, an extensive series of glycosyl hydroperoxides was prepared with the aim of identifying new bioactive molecules. Selected compounds were tested against a Plasmodium falciparum culture (chloroquine-susceptible strain D10 and chloroquine-resistant strain W2). Screening results indicated that the factors critical for antimalarial activity were the presence of a hydroperoxide moiety and solubility in water at pH 5.0. Moreover, the ability to inhibit ß-hematin formation in vitro has been evaluated (BHIA Assay).

The Oral Cavity-Another Reservoir of Antimicrobial-Resistant Staphylococcus aureus?

Staphylococcus aureus is one of the most common potentially pathogenic bacteria that may asymptomatically colonize many sites of healthy carriers. Non-nasal carriage, especially in the oral cavity, and its role in transmitting antimicrobial-resistant S. aureus strains in the healthcare community, is poorly understood. This study aimed to assess the prevalence and antimicrobial susceptibility of S. aureus in both oral and nasal cavities among preclinical dentistry students. A total of 264 oral and nasal swabs were taken from 132 participants, and all specimens were cultured using standard diagnostic procedures and antimicrobial susceptibility testing (EUCAST). The prevalence of S. aureus exclusively in the nasal (11.4%) or oral (9.1%) cavity was comparable, while concurrent oral and nasal carriage was present in 27.3% of participants. Although antibiotic resistance rates observed in both oral and nasal isolates were similar (ranging from 2.7% to 95.5%), 16.7% of carriers exhibited distinct antibiotic resistance profiles between oral and nasal isolates. Three (2.7%) methicillin-resistant S. aureus (MRSA) were isolated from the mouth and nose but multidrug resistance (27.3%) was more frequent in the oral than in the nasal isolates: 34% and 21.1%, respectively. This study demonstrated that preclinical dentistry students have a similar rate of oral S. aureus carriage as the nasal carriage rate, and that the oral cavity can be colonized by antimicrobial-resistant strains that do not originate from the nose. Consequently, the oral cavity seems to be an unjustly overlooked body site in screening for S. aureus carriage.

Total synthesis of ezetimibe, a cholesterol absorption inhibitor.

Ezetimibe (1), a strong ß-lactamic cholesterol absorption inhibitor, was synthesized from (R)-6-(4-fluorophenyl)-5,6-dihydro-2H-pyran-2-one 7. Independent pathways were analyzed in order to select the optimal one, which involved 1,3-dipolar cycloaddition with C-(4-benzyloxyphenyl)-N-(4-fluorophenyl)-nitrone (8), intramolecular nucleophilic displacement at the benzylic position of the lactone, cleavage of the N-O bond, elimination of a water molecule, hydrogenation of the double bond, rearrangement of the six-membered lactone ring into a ß-lactam moiety, and final deprotection of the phenolic hydroxyl group. Highly stereoselective Sc(OTf)3-catalyzed 1,3-dipolar cycloaddition was the most crucial step of the synthesis. Owing to the rigid transition state of the cycloaddition, the absolute configuration of the starting lactone controlled the formation of other stereogenic centers of the final molecule 1.

Current Molecular, Cellular and Genetic Aspects of Peri-Implantitis Disease: A Narrative Review.

(1) Background: Peri-implantitis is a multi-factorial disease with an inflammatory background that occurs in both soft and hard tissues surrounding implants. In recent years, the understanding of the cellular, molecular and genetic background of peri-implantitis has broadened. This study aims to summarize the currently available articles on the subject and highlight the most recent advances over the last 20 years. (2) Methods: For this study, the Embase and PubMed libraries were searched using the keywords: ("peri-implantitis" AND "cytokine" OR "genetics" OR "cellular") and ("peri-implantitis" AND "cytokine" OR "genetics" OR "cellular" AND "risk factors"). The search revealed a total of 3013 articles (992 from PubMed, 2021 from Embase). Following screening of the titles and abstracts and full-text reads, 55 articles were included. (3) Results: In peri-implantitis IL-6, IL-1ß, TNF-α, MMP-8 and their genetic variations appear to be the most important cytokines in relation to not only pathogenesis, but also their potential diagnostic capabilities. Epithelial and inflammatory cells, along with those of the bone lineage, are prime cellular elements found in peri-implantitis. (4) Conclusions: A wide array of cells stand behind peri-implantitis, as well as cytokines and their genetic variations that take part in the process. However, the growing interest in this topic has led to the introduction of specific new diagnostic tools to enable a better understanding of patients' responses to treatment and, in turn, to even enable prediction of the risk of developing peri-implant disease.

Increased oxytocinergic system activity in the cardiac muscle in spontaneously hypertensive SHR rats.

Introduction: The present study aimed to determine whether the presence of cardiac hypertrophy due to arterial hypertension is associated with a change in the activity of the oxytocinergic system in cardiomyocytes. Material and methods: The experiments were performed on male, spontaneously hypertensive rats (SHR, n = 10) and normotensive Wistar-Kyoto rats (WKY, n = 12). Blood samples were collected from both SHR and WKY animals to asses plasma oxytocin (OT) concentration; the rats were sacrificed by decapitation. Samples of the left and right ventricles were harvested for the analysis of the OT and oxytocin receptor (OTR) protein by ELISA, and OT and OTR mRNA expression by RT-PCR. Immunohistopathological studies were performed to confirm the presence of OTR receptors in the cardiac muscle of the ventricles. Results: Plasma OT concentration did not differ between SHR and WKY rats. In the SHR rats, the expression of OT mRNA and the OT protein level was higher in the left and the right ventricle, while OTR mRNA expression was significantly lower in both the left and the right ventricle. However, the level of OTR protein was higher only in the left ventricle of the SHR rats. The presence of OTR receptors was confirmed by immunohistochemical analysis in the muscle of the right and left ventricle. Conclusions: The presence of arterial hypertension is associated with increased activity of the oxytocinergic system in the heart, especially in the area of the left ventricle. These findings support the important role of this system in the maintenance of cardiovascular homeostasis.

A formal synthesis of ezetimibe via cycloaddition/rearrangement cascade reaction.

A formal synthesis of a powerful cholesterol inhibitor, ezetymibe 1, is described. The crucial step of the synthesis is based on Cu(I)-mediated Kinugasa cycloaddition/rearrangement cascade reaction between terminal acetylene derived from acetonide of L-glyceraldehyde and suitable C,N-diarylnitrone. The adduct with (3R,4S) configuration at the azetidinone ring, obtained with high stereoselectivity, was subsequently subjected to deprotection of the diol side chain followed by glycolic cleavage and base-induced isomerization at the C3 carbon atom to afford the (3S,4S) aldehyde, which has been already transformed into ezetimibe by the Schering-Plough group.

Myocardial infarction caused by Graves' disease.

Not required for Clinical Vignette.

COVID-19 in dental care: What do we know?

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is primarily transmitted by airborne droplets and its spread is favored by close human contact, thus the COVID-19 pandemic is the new challenge in dental practice. The oral cavity was considered as a SARS-CoV-2 reservoir, the viruses were detected in the saliva and periodontal pockets of infected persons. Therefore, aside from the most common symptoms, COVID-19 can manifest as lesions in the oral cavity. Due to the high risk of cross-contamination in the dental office, new precautionary measures were implemented in professional dental care to ensure safety for both, dental staff and patients. Given the dynamically changing situation, dental practitioners should follow local guidelines and implement them according to current needs and available resources. The key to success is to reduce the risk of cross-infection with SARS-CoV-2 at no cost to the good oral health of the population.

Structure-chiroptical properties relationship of carbapenams by experiment and theory.

The present work examines the relationship between the molecular structure and chiroptical properties of carbapenams through use of electronic circular dichroism spectroscopy (ECD). The applicability of the helicity rule that correlates the molecular structures of various ß-lactam analogues and their ECD spectra is examined against a set of differently substituted carbapenams. It is demonstrated that the studied compounds conform to the rule. The rule can be also applied to the carbapenams with an additional chromophoric unit interfering with the amide chromophore. For the representative carbapenams, the experimental curves are compared to the ECD spectra computed using time-dependent density functional theory (TDDFT) in order to validate the experimental data. The study reveals a high effectiveness of the ECD spectroscopy for the configurational assignment at the bridgehead carbon atom and demonstrates a strong dependence of the molecular conformation on substitution of the five-membered ring and side-chain flexibility of investigated carbapenams.

Direct, catalytic synthesis of carbapenams via cycloaddition/rearrangement cascade reaction: unexpected acetylenes' structure effect.

Reactions of acetylenes derived from glyceraldehyde and propargyl aldehyde show remarkable reactivity in Kinugasa cycloaddition/rearrangement cascade process catalyzed by Cu(I) ion. Reactions proceed by formation of a rigid dinuclear copper(I) complex in which each copper ion is coordinated to one or both oxygen atoms in the acetylene molecule and to both triple bonds. It has been demonstrated that one oxygen atom can be replaced by the phenyl ring, which is able to coordinate the copper ion by the aromatic sextet. Kinugasa reactions that proceed in a high yield can also be performed in the presence of a catalytic amount of the copper salt to provide products in an acceptable yield without a decrease of diastereoselectivity.

Ferrier-Petasis rearrangement of 4-(vinyloxy)azetidin-2-ones: an entry to carbapenams and carbacephams.

Trimethylsilyl triflate promotes Ferrier-Petasis rearrangement of 4-(vinyloxy)-, 4-(propenyloxy)-, and 4-(isopropenyloxy)azetidin-2-ones to corresponding 4-(carbonylmethyl)azetidin-2-ones. The latter compounds may serve as attractive intermediates in the synthesis of carbapenem antibiotics. To illustrate the potential of this reaction, selected rearrangement products have been transformed into carbapenams.

Boost of serum resistance and storage stability in cationic polyprenyl-based lipofection by helper lipids compositions.

Lipofection is a widely used molecular biology technique and one of the most promising non-viral gene therapy strategies. However, one of the main drawbacks of using cationic lipids-based lipoplexes in DNA/RNA delivery is serum-associated inhibition of transfection. We have addressed this issue using PTAI (trimethylpolyprenylammonium iodides)-based lipofection model. To overcome serum-sensitivity we used 100 different formulations based on different PTAI, various helper lipids compositions, lipoplex surface modifications with polyethylene glycol (PEG), and precondensation of DNA with poly-L-lysine (PLL). Multicomponent helper lipids compositions boosted serum resistance and largely improved long-term storage of PTAI-based reagents. This was observed, in particular, for PTAI with longer isoprenoid chains. Additionally, our PTAI-based carriers were efficient for DNA and RNA delivery and safe for human red blood cells (RBC). Moreover, a broad array of the modifications used resulted in an important observation - a diverse susceptibility of various cell types to different compositions was noted. Overall, our results show that helper lipids composition mediates efficient serum-resistant DNA/RNA lipofection. Additionally, multicomponent PTAI-based reagents are promising gene delivery carriers both, at the cellular and organismal level.

Asymmetric Kinugasa reaction of cyclic nitrones and nonracemic acetylenes.

Kinugasa reactions between chiral acetylenes and five-membered nitrones, achiral and bearing a stereogenic center in both enantiomeric forms, proceed in moderate to good yield with high diastereoselectivity affording mostly one dominant product. The first step of the reaction is controlled by the configuration of the nitrone, whereas the protonation of intermediate enolate in the second step depends mainly on the configuration of the bridgehead carbon atom formed in the first step. In the case of the mismatched pair, the configuration at the C-6 center of the carbapenam skeleton may also be affected by the configuration of the stereogenic center in the acetylene portion.