Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching.

Label-free homogeneous optical detection of low concentration of oligonucleotides using graphene oxide in complex solutions containing proteins remains difficult. We used a colloidal graphene oxide (GO) as a fluorescent probe quencher to detect microRNA-21 spiked-in cell culture medium, overcoming previously reported problematic aspects of protein interference with graphene oxide. We used a "turn off" assay for specific quenching-based detection of oligo DNA-microRNA hybridization in solution. A fluorescein conjugated 30-mer single-stranded DNA (ssDNA) probe was combined with a complementary synthetic microRNA (18 nucleotides) target. The probe-target hybridization was detected by specific quenching due to photoinduced electron transfer (PET). On the next step, GO captures and quenches the unhybridized probe by fluorescence resonance energy transfer (FRET) in the presence of cell culture medium supplemented with platelet lysate, 0.1% sodium dodecyl sulfate (SDS), 0.1% Triton X-100 and 50% formamide. This resulted in sensitive measurement of the specific probe-target complexes remaining in solution. The detection is linear in the range of 1 nM and 8 nM in a single 100 µL total volume assay sample containing 25% cell culture medium supplemented with platelet lysate. We highlight a general approach that may be adopted for microRNA target detection within complex physiological media.

Phenotypic assessment of liver-derived cell cultures during in vitro expansion.

Background: Liver cells represent an attractive source of cells for autologous regenerative medicine. The present study assesses the liver cells' stability during in vitro expansion, as a prerequisite for therapeutic use. Results: The human liver cell cultures in this study were propagated efficiently in vitro for at least 12 passages. No significant changes in morphology, intracellular ultrastructures and characteristic markers expression were found during in vitro expansion of cells from all analyzed donors. However, expanded cells derived from male donors of >60 years old, lost the Y chromosome. Conclusion: Liver-derived cell cultures adopt a proliferative, stable mesenchymal phenotype, through an epithelial to mesenchymal transition process. The molecular and phenotypic changes of the cells during propagation are uniform, despite the heterogeneity of the different donors. Loss of Y chromosome occurs after cells' propagation in elder male donors.

Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential.

Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3'-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes.

Plasma Small Extracellular Vesicles Derived miR-21-5p and miR-92a-3p as Potential Biomarkers for Hepatocellular Carcinoma Screening.

INTRODUCTION: Liquid biopsy using circulating microvesicles and exosomes is emerging as a new diagnostic tool that could improve hepatocellular carcinoma (HCC) early diagnosis and screening protocols. Our study aimed to investigate the utility of plasma exosomal miR-21-5p and miR-92-3p for HCC diagnosis during screening protocols. METHODS: The study group included 106 subjects: 48 patients diagnosed with HCC during screening, who underwent a potentially curative treatment (surgical resection or liver transplantation), 38 patients with liver cirrhosis (LC) on the waiting list for liver transplantation, and 20 healthy volunteers. The exosomes were isolated by precipitation with a reagent based on polyethylene glycol and were characterized based on morphological aspects (i.e., diameter); molecular weight; CD63, CD9, and CD81 protein markers; and exosomal miR-21-5p and miR-92a-3p expression levels. RESULTS: We first demonstrate that the exosome population isolated with the commercially available Total Exosome Isolation kit respects the same size ranging, morphological, and protein expression aspects compared to the traditional ultracentrifugation technique. The analysis of the expression profile indicates that miR-21-5p was upregulated (p = 0.017), and miR-92a-3p was downregulated (p = 0.0005) in plasma-derived exosomes from HCC subjects, independently from the patient's characteristics. AUROC for HCC diagnosis based on AFP (alpha-fetoprotein) was 0.72. By integrating AFP and the relative expression of exosomal miR-21-5p and miR-92a-3p in a logistic regression equation for HCC diagnosis, the combined AUROC of the new exosomal miR HCC score was 0.85-significantly better than serum AFP alone (p = 0.0007). CONCLUSION: Together with serum AFP, plasma exosomal miR-21-5p and miR-92a-3p could be used as potential biomarkers for HCC diagnosis in patients with LC subjected to screening and surveillance.

Senescence-induced immunophenotype, gene expression and electrophysiology changes in human amniocytes.

The aim of the study was to evidence replicative senescence-induced changes in human amniocytes via flow cytometry, quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) and automated/manual patch-clamp. Both cryopreserved and senescent amniocytes cultured in BIO-AMF-2 medium featured high percentages of pluripotency cell surface antigens SSEA-1, SSEA-4, TRA1-60, TRA1-81 (assessed by flow cytometry) and expression of pluripotency markers Oct4 (Pou5f1) and Nanog (by qRT-PCR). We demonstrated in senescent vs cryopreserved amniocytes decreases in mesenchymal stem cell surface markers. Senescence-associated ß-galactosidase stained only senescent amniocytes, and they showed no deoxyuridine incorporation. The gene expression profile revealed a secretory phenotype of senescent amniocytes (increased interleukin (IL)-1α, IL-6, IL-8, transforming growth factor ß, nuclear factor κB p65 expression), increases for cell cycle-regulating genes (p16INK4A ), cytoskeletal elements (ß-actin); HMGB1, c-Myc, Bcl-2 showed reduced changes and p21, MDM2 decreased. Via patch-clamp we identified five ion current components: outward rectifier K+ current, an inactivatable component, big conductance Ca2+ -dependent K+ channels (BK) current fluctuations, Na+ current, and inward rectifier K+ current. Iberiotoxin 100 nmol/L blocked 71% of BK fluctuations, and lidocaine 200 µmol/L exerted use-dependent Na+ current block. Transient receptor potential (TRP)M7-like current density at -120 mV was significantly increased in senescent amniocytes. The proinflammatory profile acquired by senescent amniocytes in vitro may prevent their use in clinical therapies for immunosuppression, antiapoptotic and healing effects.

Electrophysiology, immunophenotype, and gene expression characterization of senescent and cryopreserved human amniotic fluid stem cells.

We characterized human amniotic fluid stem cells (AFSC) in senescent cultures (6 weeks) versus cryopreserved cells using whole-cell patch-clamp, immunophenotyping, and differential gene expression profiling for senescence genes. We evidenced five ion current components (outward rectifier, A-type, inward rectifier, and big conductance Ca2+-dependent K+ currents, fast voltage-dependent Na+ currents). Senescent AFSC showed reduced expression of CD90, CD44, CD133, over 500-fold increase of interferon gamma and telomerase reverse transcriptase genes, increased cycle-dependent kinase 4 inhibitors, p53-binding protein 1, and decreased calreticulin and CD44. HLA-ABC immune expression was similar, and HLA-DR expression very low in both cell types. A subset of cryopreserved AFSC featured large inward rectifier K+ currents, voltage-dependent Na+ currents, and neural progenitor markers evidenced by immunophenotyping and RT-PCR. In all AFSC, in both culture conditions, at patch rupture the outward currents were very low, and they increased progressively over several minutes upon cytoplasm dialysis with pipette solution.

Prenatal screening for the 35delG GJB2, del (GJB6-D13S1830), and del (GJB6-D13S1854) mutations in the Romanian population.

OBJECTIVE: In this study, the aim of prenatal screening was to estimate the carrier frequency of the three mutations 35delG, del (GJB6-D13S1830), and del (GJB6-D13S1854), which are known to be the leading mutations of hereditary hearing loss in European populations. METHODS: We performed a prenatal screening to assess the carrier frequency of the most common mutations at the DFNB1 gene locus in the general population. Samples of amniotic fluid (n=339) and chorionic villi (n=11) were taken from an unselected group of 350 unrelated pregnant women with normal hearing. Genomic fetal DNA was extracted and analyzed by PCR multiplex assays. RESULTS: The rate of carriers for the 35delG GJB2 mutation was 3.14%, comparable to that of most Southeastern European populations. All samples were negative for GJB6-D13S1830 and GJB6-D13S1854 deletions. The genetic tests were considered for carrier detection and early diagnosis rather than termination of pregnancy. CONCLUSIONS: Our study suggests a need for detecting the carriers. This is the first step for the construction of a national database and provides information for health planners and policy makers to help them in planning programs and allocation resources. The molecular testing was well received by pregnant women and appears to be feasible and highly acceptable.

Detection of human papilloma viruses using nanostructurated silicon support in microarray technology.

In a typical microarray experiment, DNA is arrayed on a solid substrate as spots, the array being probed with a sample or a capture molecule of interest and the interaction monitored through different detection methods. The present study evaluates the possibility to use micro-array technology to genotype samples with Human Papilloma Viruses (HPV). The performance of DNA microarrays strongly depend on their surface properties. The efficiency of DNA immobilization in terms of sensitivity and specificity is one of the most important step in obtaining a microarray chip for diagnosis of HPV family viruses. Here we report the preparation and evaluation of nano-porous silicon surfaces for HPV detection based on DNA micro-array technique. Two different surfaces based on similar porous structure chemically modified in order to efficiently immobilize ss-DNA specific for HPV viruses were investigate.

Clinical improvement after treatment with VEGF(165) in patients with severe chronic lower limb ischaemia.

The present study focuses on the application of a therapeutic strategy in patients with chronic severe lower limb ischaemia using a plasmid vector encoding the vascular endothelial growth factor (phVEGF(165)). It has been shown that VEGF promotes neo-vascularization and blood vessel network formation and thus might have the ability to improve blood-flow at the level of the affected limbs. However, little information is available regarding the necessary level of expression of VEGF and its possible related adverse effects. We have subcloned VEGF ( 165 )isoform into pCMV-Script expression vector (Stratagene) under the control of the CMV promoter. Three patients with chronic ischaemia of the lower limb, considered as not suitable for surgical re-vascularization, received intramuscular injection with 0.5 ml saline solution containing 10(11) copies of VEGF ( 165 ) plasmid. The clinical evolution has been monitored by angiography and estimated by walking time on the rolling carpet (Gardner protocol). Two months after therapy, all three patients showed complete relief of rest pain, improvement of ischaemic ulcer lesions and increased walking distance on the rolling carpet most probably due to appearance of newly formed collateral vessels.