Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1

Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods.

Improvement of Cell Culture Methods for the Successful Generation of Human Keratinocyte Primary Cell Cultures Using EGF-Loaded Nanostructured Lipid Carriers

Human skin keratinocyte primary cultures can be established from skin biopsies with culture media containing epithelial growth factor (EGF). Although current methods are efficient, optimization is required to accelerate the procedure and obtain these cultures in less time. In the present study, we evaluated the effect of novel formulations based on EGF-loaded nanostructured lipid carriers (NLC). First, biosafety of NLC containing recombinant human EGF (NLC-rhEGF) was verified in immortalized skin keratinocytes and cornea epithelial cells, and in two epithelial cancer cell lines, by quantifying free DNA released to the culture medium. Then we established primary cell cultures of human skin keratinocytes with basal culture media (BM) and BM supplemented with NLC-rhEGF, liquid EGF (L-rhEGF), or NLC alone (NLC-blank). The results showed that cells isolated by enzymatic digestion and cultured with or without a feeder layer had a similar growth rate regardless of the medium used. However, the explant technique showed higher efficiency when NLC-rhEGF culture medium was used, compared to BM, L-rhEGF, or NLC-blank. Gene expression analysis showed that NLC-rhEGF was able to increase EGFR gene expression, along with that of other genes related to cytokeratins, cell–cell junctions, and keratinocyte maturation and differentiation. In summary, these results support the use of NLC-rhEGF to improve the efficiency of explant-based methods in the efficient generation of human keratinocyte primary cell cultures for tissue engineering use.

P13.19 Bi-modular G-quadruplex DNA-crypto-aptamers diminish viability of glioma primary cell cultures of patients

BACKGROUND G-quadruplex oligonucleotides (GQs) exhibit specific anti-survival activity in human cancer cell lines; they can selectively inhibit the viability/proliferation. The most studied, AS1411, had been in clinical trials. This anti-proliferative ability of GQs could be translated into glioma, which currently has poor prognosis and low-efficiency therapeutic treatments for glioblastoma multiform (GBM). Set of GQs have been designed, synthesized, and tested: they have different amount of Q-quartets, they have dimers of different GQ modules: either covalent dimers or non-covalent ones; all of them could be coined as ‘twins’. MATERIAL AND METHODS Folding of synthetic DNA oligonucleotides into GQs and thermal stability have been studied by circular dichroism, melting with unfolding-folding regimes, oligomerization was followed by original SE-HPLC. Conventional human cell lines U87 and fibroblasts from human embryo (HEF) were provided from the collection of the Centre of Neurosurgery (Moscow, Russia). GBM primary cell cultures N1, G11, Sus/fP2, G22, G23, and G01 were developed in Burdenko National Medical Research Centre of Neurosurgery (NMRCN) from the surgery samples of patients (PCC_SSP). All samples had WT IDH1. This study was approved by the Ethics Committee of Burdenko NMRCN, Russian Academy of Medical Sciences (№_12/2020, 15.12.2020). All subjects gave written informed consent in accordance with the guidelines of Declaration of Helsinki. Cell viability was tested by conventional MTT-test. RESULTS Novel bi-modular GQ, bi-(AID-1-T), twin of three-quartet AID-1-T, was designed by covalent conjugation of two AID-1-Ts via three thymidine link, TTT; and linking did not interfere with its GQ structure. Comparison of bi-(AID-1-T) with mono-modular AID-1-T, mono-modular two-quartet HD1, bi-modular bi-HD1, and two-quartet AS1411, was made. Among five GQs, bi-(AID-1-T) had the highest anti-survival activity for U87, while not affecting the control, HEF. GQs, for the first time, were tested on several PCC_SSP. Sensitivity of PCC_SSP toward GQs varied, with apparent IC50 of less than 1 μM for bi-(AID-1-T) toward the most sensitive G11 (glioma, Grade III). CONCLUSION GQs as anti-proliferative crypto-aptamers with moderate activity due to restricted functioning of apparent GQ-binding proteins could be applied toward real glioma PCC_SSP. Variety of effects reflects glioma inter-tumor heterogeneity.Research was funded by Ministry of Education and Science of Russia, grant number № 075-15-2020-809 (13.1902.21.0030) and by Russian Foundation for Basic Research, grants number №18-29-01047

RNA sequencing and functional studies of patient-derived cells reveal that neurexin-1 and regulators of this pathway are associated with poor outcomes in Ewing sarcoma

Purpose The development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets. Methods Thirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods. By RNA-sequencing we analysed the transcriptome of ES patient-derived cells (n = 24) and ES-CSCs (n = 11) to identify the most abundant and differentially expressed genes (DEGs). Expression of the top DEG(s) in ES-CSCs compared to ES cells was validated at both RNA and protein levels. The functional and prognostic potential of the most significant gene (neurexin-1) was investigated using knock-down studies and immunohistochemistry of two independent tumour cohorts. Results ES-CSCs were isolated from all primary cell cultures, consistent with the premise that ES is a CSC driven cancer. Transcriptional profiling confirmed that these cells were of mesenchymal origin, revealed novel cell surface targets for therapy that regulate cell-extracellular matrix interactions and identified candidate drivers of progression and relapse. High expression of neurexin-1 and low levels of regulators of its activity, APBA1 and NLGN4X, were associated with poor event-free and overall survival rates. Knock-down of neurexin-1 decreased viable cell numbers and spheroid formation. Conclusions Genes that regulate extracellular interactions, including neurexin-1, are candidate therapeutic targets in ES. High levels of neurexin-1 at diagnosis are associated with poor outcome and identify patients with localised disease that will relapse. These patients could benefit from more intensive or novel treatment modalities. The prognostic significance of neurexin-1 should be validated independently.

Ecotoxicology assessments in avian species using cell-based models: A review

Cell-based models in avian species have historically focused on virology due to the demands of animal agriculture and vaccine production industries. Recent years have witnessed a gradual rise in the use of these models ( in ovo, cell lines, primary cell cultures, organ slices, and organ-on-a-chip) in ecotoxicological studies as scientists and governments begin the shift to new approach methodologies, a shift validated by the recent memo by the Environmental Protection Agency announcing the end of mammalian testing in the next two decades. This rise has been hindered by the limited standards available for avian species and the unknowns surrounding cell-based assay applicability in extrapolation to in vivo. Toxicologists have incorporated these models in many different studies, including maternal deposition, mechanistic, metabolic, and non-target analysis methods, demonstrating the broad utility of cell-based assays. In ovo methods are ideal for reproductive and early life stage development studies, primary cell cultures for metabolic analysis, cell lines for long term studies requiring culture, organ slices for metabolic research, and organ-on-a-chip models for predictive analysis. These models all have their limitations that researchers need to consider when choosing which is most appropriate for the intended research, however. The current indications are that future avian cell-based model testing would benefit from expanding the species diversity available in cell lines and increasing metabolic conservation in full replacement methods. In ovo and primary cell culture methods should also be examined to increase efficiency and further reduce animal usage. This review examines the use, limitations, and published applications of these models in an ecotoxicological context to understand the current state of avian cell-based models to explain what future directions should be taken and how best to apply the methods available to current problems that avian researchers are approaching.

Author Correction: Novel methods to establish whole‑body primary cell cultures for the cnidarians Nematostella vectensis and Pocillopora damicornis

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cnidarian Cell Cryopreservation: A Powerful Tool for Cultivation and Functional Assays

Cnidarian primary cell cultures have a strong potential to become a universal tool to assess stress-response mechanisms at the cellular level. However, primary cell cultures are time-consuming regarding their establishment and maintenance. Cryopreservation is a commonly used approach to provide stable cell stocks for experiments, but it is yet to be established for Cnidarian cell cultures. The aim of this study was therefore to design a cryopreservation protocol for primary cell cultures of the Cnidarian Anemonia viridis, using dimethyl sulfoxide (DMSO) as a cryoprotectant, enriched or not with fetal bovine serum (FBS). We determined that DMSO 5% with 25% FBS was an efficient cryosolution, resulting in 70% of post-thaw cell survival. The success of this protocol was first confirmed by a constant post-thaw survival independently of the cell culture age (up to 45 days old) and the storage period (up to 87 days). Finally, cryopreserved cells displayed a long-term recovery with a maintenance of the primary cell culture parameters and cellular functions: formation of cell aggregates, high viability and constant cell growth, and unchanged intrinsic resistance to hyperthermal stress. These results will further bring new opportunities for the scientific community interested in molecular, cellular, and biochemical aspects of cnidarian biology.

Nanostructured Lipid Carriers Made of Ω-3 Polyunsaturated Fatty Acids: In Vitro Evaluation of Emerging Nanocarriers to Treat Neurodegenerative Diseases

Neurodegenerative diseases (ND) are one of the main problems of public health systems in the 21st century. The rise of nanotechnology-based drug delivery systems (DDS) has become in an emerging approach to target and treat these disorders related to the central nervous system (CNS). Among others, the use of nanostructured lipid carriers (NLCs) has increased in the last few years. Up to today, most of the developed NLCs have been made of a mixture of solid and liquid lipids without any active role in preventing or treating diseases. In this study, we successfully developed NLCs made of a functional lipid, such as the hydroxylated derivate of docohexaenoic acid (DHAH), named DHAH-NLCs. The newly developed nanocarriers were around 100 nm in size, with a polydispersity index (PDI) value of <0.3, and they exhibited positive zeta potential due to the successful chitosan (CS) and TAT coating. DHAH-NLCs were shown to be safe in both dopaminergic and microglia primary cell cultures. Moreover, they exhibited neuroprotective effects in dopaminergic neuron cell cultures after exposition to 6-hydroxydopamine hydrochloride (6-OHDA) neurotoxin and decreased the proinflammatory cytokine levels in microglia primary cell cultures after lipopolysaccharide (LPS) stimuli. The levels of the three tested cytokines, IL-6, IL-1β and TNF-α were decreased almost to control levels after the treatment with DHAH-NLCs. Taken together, these data suggest the suitability of DHAH-NLCs to attaining enhanced and synergistic effects for the treatment of NDs.