Функциональная активность CHO-клеточных линий, экспрессирующих различные мутации ATP7B гена (Изучение цитотоксичности определенных концентраций Меди и Цинка).

Institute of Mother and Child Health Care, Chisinau, Moldova, Klinik und Poliklinik für Transplantationsmedizin, Universitatsklinikum Munster, GermanyBoala Wilson este un bun exemplu modern pentru dezvoltarea ascensivă a ştinţei medicale. Cercetările moderne în domeniul biologiei moleculare şi celulare încearca să descopere multe mistere a bolii Wilson. În timpul actual există un interes crescut în cercetarile legate cu fi ziologia metalelor. În timpul de faţă, noi observăm discuţii fierbinţi în cercurile ştiinţifi ce cu privire la utilizarea preparatelor ce conţin Zn la tratarea bolii Wilson. Lucrarea dată decernează studiile activităţii funcţionale a liniilor de celule CHO, ce se expresează în diferite mutaţii ATP7B prin influenţa anumitor soluţii cu concentraţie determinată de Cu şi Zn şi determinarea citotoxicitaţii legăturilor date. Material şi metode. Au fost utilizate culturile celulare din colecţia germană de culturi celulare (DSMZ). Folosind metoda transducţiei retrovirale, sau obţinut 12 tulpini celulare CHO, ce se expreseaza în diferite mutaţii ATP7B. Mutaţiile se localizează în domeniul de legatură cu Cu (n=2), TM (n=4), TGE bucle (n=1) legături de bucle ATP (n=5) gene ATP7B. Pentru aprecierea citotoxicităţii se foloseşte testul MTT. Rezultate. În rezultatul cercetărilor noastre, noi am obţinut următoarele date: concentraţia soluţiei de Zn 500mikroM reprezintă o concentraţie toxică procent de moarte a celulelor se echivalează cu 0,22%. Concentraţia soluţiei de 50microM stimulează diviziunea celulară. Procentul de celule CHO ce au supravieţuit, şi ce expresează gena ATP7B, în soluţie de 1mM de Cu creşte pîna la 20% la adăugarea în soluţia dată 50microM de soluţie cu Zn. Celulele CHO, ce nu expresează gena ATP7B îşi măresc cu 57% capacitatea de supravieţuire în concentraţia de 0,5mM CuCl2 şi 50 Mm ZnCl2 în mediul de cultură. Concluzii. Ionii de Zn preîntîmpină toxicitatea ionilor de Cu fără dependenţă de faptul dacă se expresează sau nu gena ATP7B şi independent faţă de ce fel de mutaţie-gravă sau medie după forma clinică.Введение. Болезнь Вильсона является отличным примером современного поступательного развития медицинской науки. Современные исследования в области клеточной и молекулярной биологии пытаются раскрыть многие загадки болезни Вильсона. В настоящее время существует повышенный интерес к исследованиям, связанных с физиологией металлов. В настоящее время, мы наблюдаем горячие дебаты в научных кругах вокруг применения препаратов цинка при лечении болезни Вильсона. Данная работа посвящена исследованию функциональной активности СНО клеточных линий, экстрессирующих различные мутации ATP7B при воздействии определенных концентраций растворов Cu и Zn и определение цитотоксичности данных соединений. Материал и методы. Культуры клетки были использованы из Немецкой Коллекции Культур Тканей (DSMZ). Используя метод ретровирусной трансдукции, были получены 12 СНО клеточных линий, экспрессирующие различные мутации ATP7B. Мутации находятся в медь-связывающем домене (n = 2), TM (n = 4), TGE петли (n = 1), АТФ-связывающей петли (п = 5) гена ATP7B. Для оценки цитотоксичности использовался МТТ-тест

GdxFe3-xO4 Nanoparticles with Silane Shell as Potential Theranostic Agent for Cancer Treatment

In this study of GdxFe3-xO4 compound core silane shell nanoparticles method is represented. Samples were analyzed with SEM, EDA and FTIR spectroscopy. Further attaching of carborane compounds is discussed. Products can be used in biomedical applications for MRI imaging and drug delivery for NCT, so can be considered as potential theranostic agents for cancer treatment. © 2022 Institute of Physics Publishing. All rights reserved.This study was funded by the Ministry of Education and Science of the Republic of Kazakhstan (grant No AP08051954)

Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene, predominantly expressed in the liver. Two compounds that knockdown TTR, comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx), are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background, this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown, siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (>80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds

Boron and Gadolinium Loaded fe3o4 Nanocarriers for Potential Application in Neutron Cancer Therapy

In this article, a novel method of simultaneous carborane-and gadolinium-containing compounds as efficient agents for neutron capture therapy (NCT) delivery via magnetic nanocarriers is presented. The presence of both Gd and B increases the efficiency of NCT and using nanocarriers enhances selectivity. These factors make NCT not only efficient, but also safe. Superparamagnetic Fe3O4 nanoparticles were treated with silane and then the polyelectrolytic layer was formed for fur-ther immobilization of NCT agents. Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), energy dispersive X-ray (EDX), ultraviolet–visible (UV-Vis) and Mössbauer spectroscopies, dynamic light scattering (DLS), scanning electron microscopy (SEM), vibrating-sample magnetometry (VSM) were applied for the characterization of the chemical and element composition, structure, mor-phology and magnetic properties of nanocarriers. The cytotoxicity effect was evaluated on different cell lines: BxPC-3, PC-3 MCF-7, HepG2 and L929, human skin fibroblasts as normal cells. average size of nanoparticles is 110 nm; magnetization at 1T and coercivity is 43.1 emu/g and 8.1, respectively; the amount of B is 0.077 mg/g and the amount of Gd is 0.632 mg/g. Successful immobilization of NCT agents, their low cytotoxicity against normal cells and selective cytotoxicity against cancer cells as well as the superparamagnetic properties of nanocarriers were confirmed by analyses above. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.This study was funded by the Ministry of Education and Science of the Republic of Kazakhstan (grant No. AP08051954 "Synthesis and modification of magnetic nanoparticles for targeted delivery of drugs"), Joint Institute for Nuclear Research-Republic of Kazakhstan cooperation program (Order No. 391, 20 July 2020) and grant №M20MC-024 of The Belarusian Republican Foundation for Fundamental Research

LIPH Expression in Skin and Hair Follicles of Normal Coat and Rex Rabbits

Natural mutations in the LIPH gene were shown to be responsible for hair growth defects in humans and for the rex short hair phenotype in rabbits. In this species, we identified a single nucleotide deletion in LIPH (1362delA) introducing a stop codon in the C-terminal region of the protein. We investigated the expression of LIPH between normal coat and rex rabbits during critical fetal stages of hair follicle genesis, in adults and during hair follicle cycles. Transcripts were three times less expressed in both fetal and adult stages of the rex rabbits than in normal rabbits. In addition, the hair growth cycle phases affected the regulation of the transcription level in the normal and mutant phenotypes differently. LIPH mRNA and protein levels were higher in the outer root sheath (ORS) than in the inner root sheath (IRS), with a very weak signal in the IRS of rex rabbits. In vitro transfection shows that the mutant protein has a reduced lipase activity compared to the wild type form. Our results contribute to the characterization of the LIPH mode of action and confirm the crucial role of LIPH in hair production

Plasmin Plays an Essential Role in Amplification of Psoriasiform Skin Inflammation in Mice

BACKGROUND: Although increased levels of plasminogen activators have been found in psoriatic lesions, the role of plasmin converted from plasminogen by plasminogen activators in pathogenesis of psoriasis has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: Here we examined the contribution of plasmin to amplification of inflammation in patients with psoriasis. We found that plasminogen was diminished, but that the amount and activity of its converted product plasmin were markedly increased in psoriasis. Moreover, annexin II, a receptor for plasmin was dramatically increased in both dermis and epidermis in psoriasis. Plasmin at sites of inflammation was pro-inflammatory, eliciting production of inflammatory factors, including CC chemokine ligand 20 (CCL20) and interleukin-23 (IL-23), that was mediated by the nuclear factor-kappaB (NF-κB) signaling pathway and that had an essential role in the recruitment and activation of pathogenic C-C chemokine receptor type 6 (CCR6)+ T cells. Moreover, intradermal injection of plasmin or plasmin together with recombinant monocyte/macrophage chemotactic protein-1 (MCP-1) resulted in induction of psoriasiform skin inflammation around the injection sites with several aspects of human psoriasis in mice. CONCLUSIONS/SIGNIFICANCE: Plasmin converted from plasminogen by plasminogen activators plays an essential role in amplification of psoriasiform skin inflammation in mice, and targeting plasmin receptor--annexin II--may harbor therapeutic potential for the treatment of human psoriasis

Long-term evolution of the hypervariable region of hepatitis C virus in a common-source-infected cohort

The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed