The Problem of Wrongly Identified and Nonverifiable Nucleotide Sequences and Cell Lines in Research Papers, or How to Study Things That May Not Exist.

Established cell lines are widely used in research, however an appealing question is the comparability of the cells between various laboratories, their characteristics and stability in time. Problematic is also the cell line misidentification, genetic and phenotypic shift or Mycoplasma contamination which are often forgotten in research papers. The monocyte/macrophage-like cell line RAW 264.7 has been one of the most commonly used myeloid cell line for more than 40 years. Despite its phenotypic and functional stability is often discussed in literature or at various scientific discussion panels, their stability during the consecutive passages has not been confirmed in any solid study. So far, only a few functional features of these cells have been studied, for example their ability to differentiate into osteoclasts. Therefore, in the present paper we have investigated the phenotype and functional stability of the RAW 264.7 cell line from passage no. 5 till passage no. 50. We found out that the phenotype (expression of particular macrophage-characteristic genes and surface markers) and functional characteristics (phagocytosis and NO production) of RAW 264.7 cell line remains stable through passages: from passage no. 10 up to passage no. 30. Overall, our results indicated that the RAW 264.7 cell line should not be used after the passage no. 30 otherwise it may influence the data reliability.

The RBL-2H3 cell line is a commonly used histamine-releasing cell line used in inflammation, allergy and immunological research. Quite commonly, it is referred to in research papers as a mast cell line, despite the fact that it is derived from basophils. There is also a lack of consistency, both between different research groups using the same cell line and with both mast cell and basophil physiology. The review follows the development of the RBL-2H3 cell line from its inception and then goes on to assess the nature of the cell line in terms of its characteristics and its response to various stimuli. The relationship of this cell line to the various mast cell subtypes and basophils is discussed and it is concluded that while the RBL-2H3 cell line shares some characteristics with both mast cells and basophils, it is not fully representative of either.

Design, synthesis, DNA binding studies and evaluation of anticancer potential of novel substituted biscarbazole derivatives against human glioma U87 MG cell line.

Although the field of induced pluripotent stem (iPS) cells is a very new, hundreds of research papers regarding them have been published over the past three years. This chapter concentrates on the medical relevance of iPS cells and where the research regarding iPS cells has reached in such a short period time. The reprogramming of cells using the “stemness” genes and the resultant populations similarity to human embryonic stem (ES) cells has allowed for another source of pluripotent stem cells to be generated which have fewer ethical ramifications then ES cells. We have compared other forms of reprogramming somatic cells to pluripotent cells and explain that even though generating iPS cell lines using the “stemness” factors is slow and inefficient it is far superior in generating pluripotent stem cells then the other methods. This relatively new technology has enabled pluripotent cell lines to be generated from various animal species such as pig, which as yet has a no counterpart in ES cell lines. One of the biggest advantages to using iPS cells is the ability to generate patient specific cells that can be used to treat patients without the complications of rejection and immunosuppression associated with using allogeneic ES cells. However, the ability to generate the correct cell type appropriate for treating the disease and, in the case of patients with genetic disorders, generating iPS cells that do not contain the mutation, are problems that must be overcome for the technology to be useful. On the other hand, using iPS cells generated from various disease types could help unfold the stages of development of the disease and enable drug testing on the diseased cells, which ultimately could be applied to treat the disease in patients. There are still some hurdles that need to be overcome; the most crucial is the safety issues associated with the generation of iPS cell lines. At the moment somatic cells are reprogrammed with vectors that integrate the DNA into the host genome in a manner not fully controlled, which could result in unfavorable insertion sites. In addition, there is the fear that the transgene might reactivate oncogenes; MYC, for instance, one of the reprogramming factors, is also known to be an oncogenes. Overall, the ability to reprogram somatic cells using stemness genes to generated iPS cells is a breakthrough whose full potential is still hard to estimate.

Reproducible laboratory research relies on correctly identified reagents. We have previously described gene research papers with wrongly identified nucleotide sequence(s), including papers studying miR-145. Manually verifying reagent identities in 36 recent miR-145 papers found that 56% and 17% of papers described misidentified nucleotide sequences and cell lines, respectively. We also found 5 cell line identifiers in miR-145 papers with misidentified nucleotide sequences and cell lines, and 18 cell line identifiers published elsewhere, that did not represent indexed human cell lines. These 23 identifiers were described as non-verifiable (NV), as their identities were unclear. Studying 420 papers that mentioned 8 NV identifier(s) found 235 papers (56%) that referred to 7 identifiers (BGC-803, BSG-803, BSG-823, GSE-1, HGC-7901, HGC-803, and MGC-823) as independent cell lines. We could not find any publications describing how these cell lines were established. Six cell lines were sourced from cell line repositories with externally accessible online catalogs, but these cell lines were not indexed as claimed. Some papers also stated that short tandem repeat (STR) profiles had been generated for three cell lines, yet no STR profiles could be identified. In summary, as NV cell lines represent new challenges to research integrity and reproducibility, further investigations are required to clarify their status and identities.

Efficient delivery of oncolytic enterovirus by carrier cell line NK-92

Data S1: Supporting information. Table S1 Primers for RT-PCR of MEF2D fusion oncogenes. Table S2. Source of agents used in in vitro drug sensitivity analyses Figure S1 Structure of MEF2D fusion in BCP-ALL cell lines. Upper figures indicate the structure of wild-type MEF2D and the fusion partner proteins. Lower figures indicate predicted protein structure (left) and nucleotide sequence (right) in each MEF2D fusion. Names of cell lines are indicated on the left end Figure S2 Duration of cell culture until cell line establishment in BCP-ALL cell lines with precise record. Precise records of cell line establishment were available in six YA series cell lines (YAMN73, YAMN74, YAMN90R, YAMN91, YACL95, and YAMN96) and in two KCB series cell lines (KCB1 and KCB4). YAMN74 and YAMN96 were MEF2D fusion-positive ALL cell lines. YAMN73, YAMN91, and KCB1 were BCR-ABL1-positive cell lines, YAMN90R was TCF3-PBX1-positive cell line, YACL95 was KMT2A fusion-positive cell line, and KCB4 was negative for known fusion genes. Vertical line indicates duration (days) of cell culture until cell line establishment. P value in Mann-Whitney U test is shown Figure S3 Genetic and clinical characteristics of human BCP-ALL cell lines Figure S4 The percentages of BCP-ALL cell lines derived from the samples at diagnosis and those derived from the samples at relapse. Basic records of cell line establishment were available in 16 MEF2D fusion-positive (MEF2D+) ALL cell and in 57 MEF2D fusion-negative (MEF2D−) BCP-ALL cell lines. Blue columns indicate the percentage of cell lines established from the samples at diagnosis, while orange columns indicate the percentage of cell lines established from the samples at relapse. P value in Fisher's exact test is shown Figure S5 Immunophenotypic characteristics of MEF2D fusion + ALL cell lines. A, The percentage of cell lines with cytoplasmic μ-chain positivity over 40% in each BCP-ALL subgroup is indicated. B, Surface expression level of CD10 in each cell line is plotted as a scatter diagram. P values in chi-square test (cytoplasmic μ-chain) and Mann-Whitney U test (CD10) are shown Figure S6 Cell cycle analyses in BCP-ALL cell lines. A, MEF2D fusion-positive (MEF2D+) (indicated by red circles) and MEF2D fusion-negative (MEF2D−) (indicated by gray circles) BCPALL cell lines were cultured with fresh media at a density of 0.5 × 105/mL for 24 hours, and then cell cycle analyses using flow cytometry after PI staining were performed. The median percentage of G0/G1 dormancy phase in three independent analyses in each cell line is plotted as a scatter diagram. P value in Mann-Whitney U test is shown. B, The percentage of cell lines with a high proportion of dormant cells (G0/G1 > 66.7%). Green columns indicate the cell lines (G0/G1 > 66.7%). P value in Fisher's exact test is shown Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Anaplastic thyroid carcinoma (ATC) is one of the most lethal human malignancies, with a median overall survival of less than six months. While most of the genetic mutations occurring in papillary thyroid carcinoma (PTC) were recently discovered through an integrated genomic approach, molecular genetics of ATC is still in part unknown. ATC is refractory to conventional therapies, including surgery, chemotherapy, radiotherapy, and radioiodine therapy. New target agents are currently being evaluated in clinical trials. Under this frame, there is an urgent need to investigate the molecular biology and the therapeutic opportunities of this understudied, rare cancer. The aim of this special issue is to gather a collection of papers focusing on molecular tools for diagnosis and therapy of anaplastic thyroid carcinoma. The issue contains eight papers including four review articles and four research papers. The review article of C. S. Fuziwara and E. T. Kimura analyzes the role of miRNA in anaplastic thyroid carcinoma. miRNAs are a class of small noncoding RNAs that regulate posttranscriptional gene expression. Increasing evidences show that they may drive oncogenesis. The article underscores the families of miRNA that are deregulated in ATC, namely, the miR-200 family, miR-30 family, let-7 family, miR 17–92 cluster, miR-146a/b, and miR 221/222. Modulation of miRNA levels, using miRNA mimics or anti-miRs, may open new therapeutic options for ATC. In the review article of M. Ragazzi et al. the focus is on the histopathology of ATC. The authors discussed the typical molecular features of ATC cells, with an elegant description of the main histological subtypes and the differential diagnostic criteria. The review included also an update of the molecular genetics of ATC. The other two review articles included in the special issue focus on new proteins overexpressed in ATC, which may represent novel therapeutic targets, that is, Aurora kinases and transferrin receptor. In detail, the work of E. Baldini et al. focuses on Aurora kinases in ATC. The review is a comprehensive update of the structure, expression, localization, and functions of Aurora proteins and their role in human cancers. Further, the review summarizes the findings, mainly made by the authors group, of Aurora kinases in thyroid cancers. Importantly, preclinical data indicate that Aurora kinase inhibitors may have a therapeutic potential for ATC treatment. The review of R. Parenti et al. focuses on type 1 transferrin receptor (TfR1/CD71) in ATC. The TfR1/CD71 is a cell membrane glycoprotein involved in iron homeostasis and cell growth. The authors showed immunohistochemical data demonstrating the overexpression of TfR1/CD71 in ATC. They discuss the opportunities to target TfR1/CD71 by monoclonal or recombinant antibodies or transferrin-gallium-TfR1/CD71 molecular complexes or small interfering RNAs (siRNAs). Four research articles are included in the special issue. Two of these articles are from the group of Sipetic. In the first research paper, V. Zivaljevic et al. analyze the risk factors for ATC. The study, done on 126 ATC patients, showed that independent risk factors for ATC are low education level, type B blood group, and goitre. A similar study, included in the special issue, from the same group of authors, addresses the importance of age in patient survival. The study demonstrates that the best prognosis for ATC is in patients younger than 50 years old. In the research article by F. Baldan et al. the synergy between HDAC inhibitors and PARP inhibitors has been investigated in an ATC-derived cell line SW1736. The authors showed that both compounds synergize in activation of apoptosis and induction of thyroid-specific gene expression. Thus, this work suggests that the combined use of HDAC and PARP inhibitors may be a useful strategy for treatment of ATC. Finally, W. Arancio and et al. conducted a competing endogenous RNA analysis to investigate the role of the stem cell factor SOX2 in ATC cell lines. The authors identified a functional network of SOX2 interactors including genes involved in the biogenesis of microRNAs (DICER1, RNASEN, and EIF2C2), in the control cell cycle (TP53 and CCND1), and in mitochondrial activity (COX8A). In conclusion, within this special issue, we gather articles underlining several aspects of molecular biology and therapeutic options for ATC. We hope that this special issue could contribute to the advancement of the knowledge of this rare and aggressive cancer.

The end stage renal disease is one of the central problems in nephrology. The worldwide prevalence of chronic kidney disease is rising annually. The process of renal fibrogenesis is thereby characterised by a relatively uniform and irreversible progression, independent from the underlying disease. The result is a fibrosis process, which could be thought of as a dysregulated reparation process. The clinical correlate in the process illustrates the progressive insufficiency of the affected organ. The aim of the therapeutic approach is therefore to stop the progression, or ideally to reach a regression of the progressing fibrosis. However, no therapy concept fullfils this aspiration until today. IL-1 as a proinflammatory cytocine is significantly involved in the pathogenesis of the chronic renal disease. IL-1Ra is already successfully used to treat rheumatoid arthritis. In the actual research paper we examined the effect of IL-1 and IL-1-Ra on proximal and distal tubular epithelial cells in vitro with regard to a potential therapeutic use as an agent for end stage renal disease. Regarding the methodology we used BrdU-incorporation and cell counting to determine the proliferation activity of the cells. The expression and synthesis of the extracellular matrix protein fibronectin we analyzed by ELISA and western blot. The matrix degradation was investigated using gel zymography and, finally, to detect the IL-1 receptors, we used PCR. We could prove the expression of the IL-1 receptors in both cell lines and observed a TNF-α and TGF-β1 dependent reduction of the expression of IL-1RI and IL-1RAcP, which may illustrate a negative regulatory process. However, the absence of IL-1α and IL-1β inside the cells does not indicate autocrine stimulation by tubular epithelial cells. Under stimulation with IL-1α and IL-1β we could study a low-grade inhibition of proliferation in proximal tubular epithelial cells, yet we could not demonstrate an effect on fibronectin synthesis. In contrast we found an induction of matrix degradation (MMP-2 and -9) by IL-1 in both cell lines. In no experiment we could prove a significant effect of IL-1Ra, which is contradictory to preliminary findings and reveals further need for clarifying studies. The effects of IL-1 we could study in this research paper are similar to the mechanism of EMT. However, the missing effect on fibronectin synthesis supports the theory that furhter cytocines, especially TGF-β1, who could act as mediators for IL-1 are relevant. However, the exact clarification of these interactions remains to be reserved for furhter studies.

Remarkable advances have been made in genetics and genomics over the last decade due to the rapid technology innovation in microarray and sequencing. Thanks to biomedical discoveries, it is feasible now to improve the diagnosis and treatment by genetic and genomic tests, for paving the way for an era of personalized/precision medicine in health care. However, it remains challenging to identify causal mutations from massive amounts of genomic data. There is an unprecedented demand for novel computational methods and analytical strategies to improve the accuracy of variants identification and the power of association tests. This special issue aims to publish applications of innovative analysis pipelines and algorithms to find the better solutions of complex genetic and genomic problems in a time efficient manner. We look for original research findings and practical applications that contribute to the diagnosis and management of human disorders. In this special issue, we selected ten papers from dozens of submissions after in-depth review. We summarize their key contributions and findings as follows. In the field of medical genetics, we collected three research papers and one review paper. S. Perez-Alvarez et al. developed a statistical algorithm (FARMS) for variable selection aiming to identify variables with the optimal predication performance for a specific outcome. The authors further applied FARMS to a high-dimensional dataset of over 800 individuals and showed that the proposed method is more efficient than other approaches such as regression based method. Y. Wang et al., in another methodology paper, proposed a novel method (LRSDec) to identify gene modules and genetic interactions between them. It is based on regularized low-rank approximation and enjoys nearly optimal error bounds. We expect its wide applications in the field of genetic interaction data analysis, image processing, and so on. Using a statistical genomic approach T. Du et al. reported the discovery of the association between FSHR polymorphisms and polycystic ovary morphology in women with polycystic ovary syndrome. In a review article, R. de Vlaming and P. J. F. Groenen surveyed the use of ridge regression for prediction in quantitative genetics by genotyping data. They also performed a suite of simulations to estimate the effect of sample size, the number of SNPs, and trait heritability on the accuracy of the results. In the field of medical genomics, we collected six research articles. Four of them employed network-based computational strategies to investigate the molecular mechanisms of complex diseases including cancer, obesity, and Type 2 Diabetes (T2D). By computing coexpression gene pairs in two types of lung cancers and the normal lung tissues, F. Long et al. identified molecular biomarkers that distinguish small-cell lung cancer and non-small-cell lung cancer. In another article, Q. Zou et al. proposed a network-based method to predict the association between microRNAs and diseases and further developed it into a web server (http://datamining.xmu.edu.cn/~jinjinli/MircoDAP.html.) for use by the community. In “Network-Based Association Study of Obesity and Type 2 Diabetes with Gene Expression Profiles,” S. Zhang et al. integrated multiple omics data of obesity and T2D to construct a comprehensive biological network. Their novel strategy revealed the pathways associated with both obesity and T2D. Another article by S. Park et al. explored the impact of inflammatory responses to the risk of colorectal cancer and Alzheimer's disease in the view of systems biology. Y. Sun and Q. Liu presented a computational framework for deciphering the correlation between breast tumor samples and cell lines. They proposed to integrate both copy-number changes and gene expression profiles. Their investigation can serve as a useful guide to bridge the gap between cell lines and tumors and help to select the most suitable cell line models for personalized cancer studies. The article by T. Liu et al. conducted a gene coexpression and evolutionary conservation analysis of the human preimplantation embryos. Their study demonstrated the patterns of evolutionary constraints that were imposed on different stages of human preimplantation embryos. We believe the publications in this collection will attract and benefit readers from multiple disciplines, not only bioinformaticians and statistical geneticists for methodology development, but also researchers and clinicians in using the novel tools for their science discovery.

Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines: Role of cytokines

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.

Absence of BCL10 Mutations in Human Malignant Mesothelioma

CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients.

Bafilomycin A1 is a specific inhibitor of the vacuolar type H+-ATPase (V-ATPase) in cells, and inhibits the acidification of organelles containing this enzyme, such as lysosomes and endosomes. Recently, while editing and reviewing chapters on autophagy for Methods in Enzymology, we noticed repeated references to the effect of bafilomycin A1 in blocking the fusion of autophagosomes with lysosomes. Of course we have seen this in various research papers as well, but reading this routinely in chapters written by various people over a short period of time really caused this to stand out. Every one of these chapters referred to the paper by Yamamoto et al. In that paper, treatment with 100 nM bafilomycin A1 for 1 h blocks the fusion of autophagosomes with lysosomes in the rat hepatoma H-4-II-E cell line, based on data from electron microscopy. However, data from one of our labs noted an apparently different result in a relatively recent manuscript. Therefore, we decided to look into this more carefully.