Synthesis, internalization and visualization of n-(4-carbomethoxy) pyrrolidone terminated PAMAM [G5:G3-TREN] Tecto(dendrimers) in mammalian cells

Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

Integrative systematics and ecology of a new deep-sea family of tanaidacean crustaceans

A new family of paratanaoidean Tanaidacea – Paranarthrurellidae fam. nov. – is erected to accommodate two genera without family classification (Paratanaoidea incertae sedis), namely Armatognathia Kudinova-Pasternak, 1987 and Paranarthrurella Lang, 1971. Seven new species of Paranarthrurella and two of Armatognathia are described from material taken in different deep-sea areas of the Atlantic and Pacific oceans. The type species of Paranarthrurella — P. caudata (Kudinova-Pasternak, 1965) — is redescribed based on the paratype. The genus Cheliasetosatanais Larsen and Araújo-Silva, 2014 originally classified within Colletteidae is synonymised with Paranarthrurella, and Arthrura shiinoi Kudinova-Pasternak, 1973 is transferred to Armatognathia. Amended diagnoses of Armatognathia and Paranarthrurella genera are given. Choosing characters for distinguishing and defining both genera was supported by Principal Component Analysis. Designation of the new family is supported by molecular phylogenetic analysis of COI and 18S datasets. The distribution of all species currently included in the new family was visualised and their bathymetric distribution analysed

Screening the Expression of ABCB6 in Erythrocytes Reveals an Unexpectedly High Frequency of Lan Mutations in Healthy Individuals

Lan is a high-incidence blood group antigen expressed in more than 99.9% of the population. Identification of the human ABC transporter ABCB6 as the molecular basis of Lan has opened the way for studies assessing the relation of ABCB6 function and expression to health and disease. To date, 34 ABCB6 sequence variants have been described in association with reduced ABCB6 expression based on the genotyping of stored blood showing weak or no reactivity with anti-Lan antibodies. In the present study we examined the red blood cell (RBC) surface expression of ABCB6 by quantitative flow cytometry in a cohort of 47 healthy individuals. Sequencing of the entire coding region of the ABCB6 gene in low RBC ABCB6 expressors identified a new allele (IVS9+1G>A, affecting a putative splice site at the boundary of exon 9) and two nonsynonymous SNPs listed in the SNP database (R192Q (rs150221689) and G588 S (rs145526996)). The R192Q mutation showed co-segregation with reduced RBC ABCB6 expression in a family, and we found the G588 S mutation in a compound heterozygous individual with undetectable ABCB6 expression, suggesting that both mutations result in weak or no expression of ABCB6 on RBCs. Analysis of the intracellular expression pattern in HeLa cells by confocal microscopy indicated that these mutations do not compromise overall expression or the endolysosomal localization of ABCB6. Genotyping of two large cohorts, containing 235 and 1039 unrelated volunteers, confirmed the high allele frequency of Lan-mutations. Our results suggest that genetic variants linked to lower or absent cell surface expression of ABCB6/Langereis may be more common than previously thought.This work was supported by the Lendulet Program of the Hungarian Academy of Sciences (GS), OTKA 83533 and by the Polish POIG grant 01.01.02-10-005/08 TESTOPLEK, supported by the EU through the European Regional Development Fund. Hajnalka Andrikovics is a recipient of the Janos Bolyai Research Scholarship from the Hungarian Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Dr. Camilo Toro and Dr. William Gahl of the NIH Undiagnosed Diseases Program for an affected patient specimen; that work was supported by the Intramural Research Program of the National Human Genome Research Institute and the Office of the Director of the NIH. We thank Lionel Arnaud (National Institute of Blood Transfusion (INTS), Paris, France) for helpful discussions

Mesalazine–PAMAM Nanoparticles for Transporter-Independent Intracellular Drug Delivery: Cellular Uptake and Anti-Inflammatory Activity

Michal Gorzkiewicz,1,2 Monika Marcinkowska,1 Maciej Studzian,3,4 Iwona Karwaciak,4,5 Lukasz Pulaski,3,4 Barbara Klajnert-Maculewicz1 1Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland; 2Department of Molecular Medicine II, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany; 3Department of Oncobiology and Epigenetics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland; 4Laboratory of Transcriptional Regulation, Institute of Medical Biology PAS, Lodz, Poland; 5Laboratory of Epigenetics, Institute of Medical Biology PAS, Lodz, PolandCorrespondence: Michal Gorzkiewicz, Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/143 Pomorska Street, Lodz, 90-236, Poland, Tel +48 42 635 41 47, Email [email protected]: Mesalazine is one of the main drugs used to treat inflammatory bowel diseases. However, its applicability is limited by its rapid inactivation and removal from the organism, as well as the need for its membrane transporter-dependent cellular uptake to exert therapeutic effect. The present study involved the development of an innovative nanocarrier, based on poly(amidoamine) (PAMAM) dendrimer of the 4th generation, to obtain higher concentrations of the drug in the intestinal epithelial cells, thus increasing its anti-inflammatory potential. The work involved synthesis and in vitro characterization of covalent PAMAM-mesalazine conjugate with succinic linker.Results: PAMAM-mesalazine conjugate was synthesized and characterized by 1H NMR, 13C NMR, FTIR and MALDI-TOF MS. This allowed to confirm the purity of the obtained compound and intermediates. Based on the analyses, it was found that ~45 drug molecules were successfully attached to one molecule of PAMAM dendrimer. The conjugate was then characterized in terms of hydrodynamic diameter, zeta potential, spectral properties, drug release from the carrier, as well as cellular uptake and cytotoxicity in two in vitro models of gastrointestinal epithelium (CaCo-2 and HT-29 human cell lines). Analyzing cellular parameters related to the specific mechanism of action of mesalazine (inhibition of NF-κB signaling, decrease in interleukin and prostaglandin synthesis, and ROS scavenging), we showed that such a dendrimer-based carrier may enhance cellular uptake of the drug, which translated into its improved anti-inflammatory efficacy.Conclusion: The use of PAMAM macromolecule as a carrier for mesalazine increases the bioavailability of the drug, ensuring enhanced cellular uptake and bypassing the need to utilize mesalazine-specific membrane transporters. All these characteristics translate into an improved anti-inflammatory activity of mesalazine in vitro. In conjunction with appropriately designed in vivo studies, such a compound may prove to be a promising alternative to the therapeutics currently used in inflammatory bowel diseases.Graphical Abstract: Keywords: PAMAM dendrimer, mesalazine, inflammatory bowel disease, drug deliver

Reduced corneal sensitivity with neuronal degeneration is a novel clinical feature in Wolfram Syndrome

PURPOSE To evaluate corneal sensitivity and corneal nerve morphology among patients with Wolfram syndrome (WFS). DESIGN An observational clinical case series with confirmatory experiments. METHODS We included a group of 12 patients with biallelic mutations in the WFS1 gene and a control group composed of 30 individuals with type 1 diabetes (T1D). All participants (n = 42) underwent a complete ophthalmic examination, esthesiometry, and retinal nerve fiber layer assessment using optical coherence tomography. Morphologic assessment of corneal neuropathy by in vivo corneal confocal microscopy was conducted in 11 patients with WFS (both eyes) and 1 WFS patient (1 eye) as well as in 24 patients with T1D (both eyes in 6 patients and 1 eye in 18 patients). Additionally, corneas from Wfs1KO mice and their wild-type littermates were subjected to laser scanning confocal microscopy. RESULTS Corneal sensitivity was significantly reduced in patients with WFS compared with patients with T1D (4.50 cm [interquartile range, 3.50–5.50 cm] vs 6.00 cm [interquartile range, 6.00–6.00 cm]; P < 10–5). Additionally, corneal nerve fiber and branch density as well as nerve fiber length were low among patients with WFS. Corneal sensitivity correlated with macular average thickness (R = 0.6928; P = .039) and best-corrected visual acuity (R = –0.61; P = .002) in the WFS group. Similarly, Wfs1 knockout mice also presented corneal neurodegeneration changes when corneal nerve fiber density and length were measured using laser scanning confocal microscopy. CONCLUSIONS Decreased corneal sensitivity and corneal nerve degeneration are observed in WFS. Corneal sensitivity is linked with the degree of disease progression as measured by visual acuity and retinal thinning

Synthesis, Internalization and Visualization of N-(4-Carbomethoxy) Pyrrolidone Terminated PAMAM [G5:G3-TREN] Tecto(dendrimers) in Mammalian Cells

Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article

Fludarabine-Specific Molecular Interactions with Maltose-Modified Poly(propyleneimine) Dendrimer Enable Effective Cell Entry of the Active Drug Form: Comparison with Clofarabine

Fludarabine is an anticancer antimetabolite essential for modern chemotherapy, but its efficacy is limited due to the complex pharmacokinetics. We demonstrated the potential use of maltose-modified poly(propyleneimine) dendrimer as drug delivery agent to improve the efficiency of therapy with fludarabine. In this study, we elaborated a novel synthesis technique for radioactively labeled fludarabine triphosphate to prove for the first time the direct ability of nucleotide-glycodendrimer complex to enter and kill leukemic cells, without the involvement of membrane nucleoside transporters and intracellular kinases. This will potentially allow to bypass the most common drug resistance mechanisms observed in the clinical setting. Further, we applied surface plasmon resonance and molecular modeling to elucidate the properties of the drug-dendrimer complexes. We showed that clofarabine, a more toxic nucleoside analogue drug, is characterized by significantly different molecular interactions with poly(propyleneimine) dendrimers than fludarabine, leading to different cellular outcomes (decreased rather than increased treatment efficiency). The most probable mechanistic explanation of uniquely dendrimer-enhanced fludarabine toxicity points to a crucial role of both an alternative cellular uptake pathway and the avoidance of intracellular phosphorylation of nucleoside drug form