Studies of Genetic and Proteomic Risk Factors of Amyotrophic Lateral Sclerosis Inspire Biomarker Development and Gene Therapy.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease affecting the upper and lower motor neurons, leading to muscle weakness, motor impairments, disabilities and death. Approximately 5-10% of ALS cases are associated with positive family history (familial ALS or fALS), whilst the remainder are sporadic (sporadic ALS, sALS). At least 50 genes have been identified as causative or risk factors for ALS. Established pathogenic variants include superoxide dismutase type 1 (SOD1), chromosome 9 open reading frame 72 (c9orf72), TAR DNA Binding Protein (TARDBP), and Fused In Sarcoma (FUS); additional ALS-related genes including Charged Multivesicular Body Protein 2B (CHMP2B), Senataxin (SETX), Sequestosome 1 (SQSTM1), TANK Binding Kinase 1 (TBK1) and NIMA Related Kinase 1 (NEK1), have been identified. Mutations in these genes could impair different mechanisms, including vesicle transport, autophagy, and cytoskeletal or mitochondrial functions. So far, there is no effective therapy against ALS. Thus, early diagnosis and disease risk predictions remain one of the best options against ALS symptomologies. Proteomic biomarkers, microRNAs, and extracellular vehicles (EVs) serve as promising tools for disease diagnosis or progression assessment. These markers are relatively easy to obtain from blood or cerebrospinal fluids and can be used to identify potential genetic causative and risk factors even in the preclinical stage before symptoms appear. In addition, antisense oligonucleotides and RNA gene therapies have successfully been employed against other diseases, such as childhood-onset spinal muscular atrophy (SMA), which could also give hope to ALS patients. Therefore, an effective gene and biomarker panel should be generated for potentially "at risk" individuals to provide timely interventions and better treatment outcomes for ALS patients as soon as possible.

Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time.

Recently, increasing numbers of cell culture experiments with 3D spheroids presented better correlating results in vivo than traditional 2D cell culture systems. 3D spheroids could offer a simple and highly reproducible model that would exhibit many characteristics of natural tissue, such as the production of extracellular matrix. In this paper numerous cell lines were screened and selected depending on their ability to form and maintain a spherical shape. The effects of increasing concentrations of doxorubicin (DXR) on the integrity and viability of the selected spheroids were then measured at regular intervals and in real-time. In total 12 cell lines, adenocarcinomic alveolar basal epithelial (A549), muscle (C2C12), prostate (DU145), testis (F9), pituitary epithelial-like (GH3), cervical cancer (HeLa), HeLa contaminant (HEp2), embryo (NIH3T3), embryo (PA317), neuroblastoma (SH-SY5Y), osteosarcoma U2OS, and embryonic kidney cells (293T), were screened. Out of the 12, 8 cell lines, NIH3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U2OS formed regular spheroids and the effects of DXR on these structures were measured at regular intervals. Finally, 5 cell lines, A549, HeLa, SH-SY5Y, U2OS, and 293T, were selected for real-time monitoring and the effects of DXR treatment on their behavior were continuously recorded for 5 days. A potential correlation regarding the effects of DXR on spheroid viability and ATP production was measured on days 1, 3, and 5. Cytotoxicity of DXR seemed to occur after endocytosis, since the cellular activities and ATP productions were still viable after 1 day of the treatment in all spheroids, except SH-SY5Y. Both cellular activity and ATP production were halted 3 and 5 days from the start of the treatment in all spheroids. All cell lines maintained their spheroid shape, except SHSY-5, which behaved in an unpredictable manner when exposed to toxic concentrations of DXR. Cytotoxic effects of DXR towards SH-SY5Y seemed to cause degradation of the extracellular matrix, since all cells were dismantled from the spheroid upon cell death. On the other hand, 293T spheroids revealed retarded cellular activity and ATP productions upon DXR treatment throughout the experiment. Since 293T was the embryonic kidney cells, the fast clearance or neutralizations could have made them resistant towards DXR. In conclusion, the same degree of sensitivity from the 2D system did not translate to a 3D culture system, resulting in higher IC50 values than the 2D system. The varying sensitivities and tolerances to drugs could be better understood with a 3D cell culture system.

Real-time monitoring of cisplatin cytotoxicity on three-dimensional spheroid tumor cells.

Three-dimensional (3D) cell cultivation is a powerful technique for monitoring and understanding diverse cellular mechanisms in developmental cancer and neuronal biology, tissue engineering, and drug development. 3D systems could relate better to in vivo models than two-dimensional (2D) cultures. Several factors, such as cell type, survival rate, proliferation rate, and gene and protein expression patterns, determine whether a particular cell line can be adapted to a 3D system. The 3D system may overcome some of the limitations of 2D cultures in terms of cell-cell communication and cell networks, which are essential for understanding differentiation, structural organization, shape, and extended connections with other cells or organs. Here, the effect of the anticancer drug cisplatin, also known as cis-diamminedichloroplatinum (II) or CDDP, on adenosine triphosphate (ATP) generation was investigated using 3D spheroid-forming cells and real-time monitoring for 7 days. First, 12 cell lines were screened for their ability to form 3D spheroids: prostate (DU145), testis (F9), embryonic fibroblast (NIH-3T3), muscle (C2C12), embryonic kidney (293T), neuroblastoma (SH-SY5Y), adenocarcinomic alveolar basal epithelial cell (A549), cervical cancer (HeLa), HeLa contaminant (HEp2), pituitary epithelial-like cell (GH3), embryonic cell (PA317), and osteosarcoma (U-2OS) cells. Of these, eight cell lines were selected: NIH-3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U-2OS; and five underwent real-time monitoring of CDDP cytotoxicity: HeLa, A549, 293T, SH-SY5Y, and U-2OS. ATP generation was blocked 1 day after addition of 50 µM CDDP, but cytotoxicity in HeLa, A549, SH-SY5Y, and U-2OS cells could be visualized only 4 days after treatment. In 293T cells, CDDP failed to kill entirely the culture and ATP generation was only partially blocked after 1 day. This suggests potential CDDP resistance of 293T cells or metabolic clearance of the drug. Real-time monitoring and ATP measurements directly confirmed the cytotoxicity of CDDP, indicating that CDDP may interfere with mitochondrial activity.

Spotlight on nano-theranostics in South Korea: applications in diagnostics and treatment of diseases.

From the synergistic integration and the multidisciplinary strengths of the BioNano Sensor Research Center, Gachon Bionano Research Institute, and Lee Gil Ya Cancer and Diabetes Institute, researchers, students, and faculties at Gachon University in collaboration with other institutions in Korea, Australia, France, America, and Japan have come together to produce a special issue on the diverse applications of nano-theranostics in nanomedicine. This special issue will showcase new research conducted by various scientific groups in Gyonggi-do and Songdo/Incheon, South Korea. The objectives of this special issue are as follows: 1) to bring together and demonstrate some of the latest research results in the field, 2) to introduce new multifunctional nanomaterials and their applications in imaging and detection methods, and 3) to stimulate collaborative interdisciplinary research at both national and international levels in nanomedicine.

Current applications of graphene oxide in nanomedicine.

Graphene has attracted the attention of the entire scientific community due to its unique mechanical and electrochemical, electronic, biomaterial, and chemical properties. The water-soluble derivative of graphene, graphene oxide, is highly prized and continues to be intensely investigated by scientists around the world. This review seeks to provide an overview of the currents applications of graphene oxide in nanomedicine, focusing on delivery systems, tissue engineering, cancer therapies, imaging, and cytotoxicity, together with a short discussion on the difficulties and the trends for future research regarding this amazing material.

Association analysis of the lymphocyte-specific protein tyrosine kinase (LCK) gene in type 1 diabetes.

Prior data associating the expression of lymphocyte-specific protein tyrosine kinase (LCK) with type 1 diabetes, its critical function in lymphocytes, and the linkage of the region to diabetes in the nonobese diabetic (NOD) mouse model make LCK a premier candidate for a susceptibility gene. Resequencing of LCK in 32 individuals detected seven single nucleotide polymorphisms (SNPs) with allele frequencies >3%, including four common SNPs previously reported. These and six other SNPs from dbSNP were genotyped in a two-stage strategy using 2,430 families and were all shown not to be significantly associated with type 1 diabetes. We conclude that a major role for the common LCK polymorphisms in type 1 diabetes is unlikely. However, we cannot rule out the possibility of there being a causal variant outside the exonic, intronic, and untranslated regions studied.

Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease.

Genes and mechanisms involved in common complex diseases, such as the autoimmune disorders that affect approximately 5% of the population, remain obscure. Here we identify polymorphisms of the cytotoxic T lymphocyte antigen 4 gene (CTLA4)--which encodes a vital negative regulatory molecule of the immune system--as candidates for primary determinants of risk of the common autoimmune disorders Graves' disease, autoimmune hypothyroidism and type 1 diabetes. In humans, disease susceptibility was mapped to a non-coding 6.1 kb 3' region of CTLA4, the common allelic variation of which was correlated with lower messenger RNA levels of the soluble alternative splice form of CTLA4. In the mouse model of type 1 diabetes, susceptibility was also associated with variation in CTLA-4 gene splicing with reduced production of a splice form encoding a molecule lacking the CD80/CD86 ligand-binding domain. Genetic mapping of variants conferring a small disease risk can identify pathways in complex disorders, as exemplified by our discovery of inherited, quantitative alterations of CTLA4 contributing to autoimmune tissue destruction.