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Extracellular vesicles (EV) are lipid-bilayer enclosed vesicles in submicron size that are released from cells. A variety of molecules, including proteins, DNA fragments, RNAs, lipids, and metabolites can be selectively encapsulated into EVs and delivered to nearby and distant recipient cells. In tumors, through such intercellular communication, EVs can regulate initiation, growth, metastasis and invasion of tumors. Recent studies have found that EVs exhibit specific expression patterns which mimic the parental cell, providing a fingerprint for early cancer diagnosis and prognosis as well as monitoring responses to treatment. Accordingly, various EV isolation and detection technologies have been developed for research and diagnostic purposes. Moreover, natural and engineered EVs have also been used as drug delivery nanocarriers, cancer vaccines, cell surface modulators, therapeutic agents and therapeutic targets. Overall, EVs are under intense investigation as they hold promise for pathophysiological and translational discoveries. This comprehensive review examines the latest EV research trends over the last five years, encompassing their roles in cancer pathophysiology, diagnostics and therapeutics. This review aims to examine the full spectrum of tumor-EV studies and provide a comprehensive foundation to enhance the field. The topics which are discussed and scrutinized in this review encompass isolation techniques and how these issues need to be overcome for EV-based diagnostics, EVs and their roles in cancer biology, biomarkers for diagnosis and monitoring, EVs as vaccines, therapeutic targets, and EVs as drug delivery systems. We will also examine the challenges involved in EV research and promote a framework for catalyzing scientific discovery and innovation for tumor-EV-focused research., Graphical abstract Image 1, Highlights • The full spectrum of small extracellular vesicles (sEV) based cancer fundamental and clinical studies is introduced. • It is one of very few comprehensive reviews that systematically introduce/summarize sEV in cancer research. • The reviewed topics will advance the frontiers of science, highlight challenges, and support higher education.

Desirable features of exosomes have made them a suitable manipulative platform for biomedical applications, including targeted drug delivery, gene therapy, cancer diagnosis and therapy, development of vaccines, and tissue regeneration. Although natural exosomes have various potentials, their clinical application is associated with some inherent limitations. Recently, these limitations inspired various attempts to engineer exosomes and develop designer exosomes. Mostly, designer exosomes are being developed to overcome the natural limitations of exosomes for targeted delivery of drugs and functional molecules to wounds, neurons, and the cardiovascular system for healing of damage. In this review, we summarize the possible improvements of natural exosomes by means of two main approaches: parental cell-based or pre-isolation exosome engineering and direct or post-isolation exosome engineering. Parental cell-based engineering methods use genetic engineering for loading of therapeutic molecules into the lumen or displaying them on the surface of exosomes. On the other hand, the post-isolation exosome engineering approach uses several chemical and mechanical methods including click chemistry, cloaking, bio-conjugation, sonication, extrusion, and electroporation. This review focuses on the latest research, mostly aimed at the development of designer exosomes using parental cell-based engineering and their application in cancer treatment and regenerative medicine. Graphic Abstract

In this study, we explored the relation between metastatic states vs the capacity of confined migration, amoeboid transition, and cellular stiffness. We compared across an isogenic panel of human breast cancer cells derived from MDA-MB-231 cells. It was observed that cells after lung metastasis have the fastest migration and lowest stiffness, with a significantly higher capacity to transition into an amoeboid mode. Our findings illustrate that metastasis is a selective process favoring motile and softer cells. Moreover, the observation that circulating tumor cells resemble the parental cell line, but not lung-metastatic cells, suggests that cells with higher deformability and motility are likely selected during extravasation and colonization.

Microvesicles are formed through shedding from the plasma membrane, a process shared by almost all human cells. Microvesicles are highly abundant and have been detected in blood, urine, cerebrospinal fluid, and saliva. They contain a library of cargo derived from their parental cell during formation, including proteases, micro-RNAs and lipids and delivery of this parental cell-derived cargo to other cells can alter target cell function and drive disease. Cell specific molecules on the surface of microvesicles, obtained during microvesicle formation, allows their parental cell to be identified and populations of microvesicles to be investigated for roles in the pathogenesis of various diseases. For instance, recent work by our group has identified a role for neutrophil microvesicles in atherosclerosis. Microvesicle profiles could in future be associated with certain diseases and act as a biomarker to allow for earlier diagnosis. This short review will discuss some of the processes central to all microvesicles before focusing on neutrophil microvesicles, their potential role in cardiovascular disease and the mechanisms that may underpin this.

Over recent decades, cell lineage tracing, clonal analyses, molecular barcoding, and single cell “omic” analysis methods have proven to be valuable tools for research and discovery. Here, we report a clonal molecular barcoding method, which we term SunCatcher, that enables longitudinal tracking and retrieval of live barcoded cells for further analysis. Briefly, single cell-derived clonal populations are generated from any complex cell population and each is infected with a unique, heritable molecular barcode. One can combine the barcoded clones to recreate the original parental cell population or generate custom pools of select clones, while also retaining stocks of each individual barcoded clone. We developed two different barcode deconvolution methods: a Next-Generation Sequencing method and a highly sensitive, accurate, rapid, and inexpensive quantitative PCR-based method for identifying and quantifying barcoded cellsin vitroandin vivo. Because stocks of each individual clone are retained, one can analyze not only the positively selected clones but also the negatively selected clones result from any given experiment. We used SunCatcher to barcode individual clones from mouse and human breast cancer cell lines. Heterogeneous pools of barcoded cells reliably reproduced the original proliferation rates, tumor-forming capacity, and disease progression as the original parental cell lines. The SunCatcher PCR-based approach also proved highly effective for detecting and quantifying early spontaneous metastases from orthotopic sites that would otherwise have not been detected by conventional methods. We envision that SunCatcher can be applied to any cell-based studies and hope it proves a useful tool for the research community.

Human induced pluripotent stem cells (hiPSC) are considered promising sources of pancreatic islet organoids to treat diabetes. Due to that epigenetic memory predisposes iPSCs to enhanced differentiation into the parental cell type, we sought to generate iPSC lines from islet cells isolated from healthy adult pancreata. Islet cells were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vivo teratoma formation. This cell line will be a valuable resource for drug screening, and potential source for high-efficient generation of insulin-producing islets for treating diabetes.

Purpose of Review: The field of extracellular vesicles is growing exponentially because of the important role that these extracellular organelles had on cell to cell communication, triggering a large number of review compilations focusing on different aspects of their biology. Although their importance as effectors or potential biomarkers is well covered, the highlight of extracellular vesicles as carriers of active enzymes which have the capability to transform the surrounding media is less covered by bibliographic studies. In the present review, we focus our attention on enzymatic activity carried by vesicles, with special attention on their contribution to liver conditions. Recent Findings: Extracellular vesicles are circulating membrane-bound entities, characterized by a specific cargo. This cargo depends on the parental cell and the stimulus that triggers their release. Interestingly, the cargo includes active enzymes which had the ability of transforming the extracellular environment. Among them, extracellular vesicles derived from hepatocytes harbour specific liver enzymes that may cause an impact in the surrounds and target cells. Summary In this review: we summarize different active enzymes described in extracellular vesicles and we focus on enzymatic activities associated to liver damage. Since their release increases under liver damage conditions, their activity impact could play a role in the pathogenesis of liver and liver-associated diseases. Numerous examples in different liver conditions provided evidence of the potential of extracellular vesicles as therapeutic targets.

Extracellular vesicles (EVs) receive special attention from oncologists due to their assumed usefulness as prognostic markers, vaccines to induce anti-cancer immune response, and physiological delivery tools. The latter application, which supports the reduction of side effects of treatment, is still fraught with many challenges, including established methods for loading EVs with selected cargo and directing them towards target cells. EVs could be loaded with selected cargo either in vitro using several physicochemical techniques, or in vivo by modification of parental cell, which may have an advantage over in vitro procedures, since some of them significantly influence EVs’ properties. Otherwise, our research findings suggest that EVs could be passively supplemented with micro RNAs (miRNAs) or miRNA antagonists to induce expected biological effect. Furthermore, our observations imply that antigen-specific antibody light chains could coat the surface of EVs to increase the specificity of cell targeting. Finally, the route of EVs’ administration also determines their bioavailability and eventually induced therapeutic effect. Besides, EV membrane lipids may possibly possess immune adjuvant activity. The review summarizes the current knowledge on the possibilities to manipulate EVs to use them as a delivery tool, with the special emphasis on anti-cancer therapy.

Stem cell transplantation, as used clinically, suffers from low retention and engraftment of the transplanted cells. Inspired by the ability of platelets to recruit stem cells to sites of injury on blood vessels, we hypothesized that platelets might enhance the vascular delivery of cardiac stem cells (CSCs) to sites of myocardial infarction injury. Here, we show that CSCs with platelet nanovesicles fused onto their surface membranes express platelet surface markers that are associated with platelet adhesion to injury sites. We also find that the modified CSCs selectively bind collagen-coated surfaces and endothelium-denuded rat aortas, and that in rat and porcine models of acute myocardial infarction the modified CSCs increase retention in the heart and reduce infarct size. Platelet-nanovesicle-fused CSCs thus possess the natural targeting and repairing ability of their parental cell types. This stem cell manipulation approach is fast, straightforward and safe, does not require genetic alteration of the cells, and should be generalizable to multiple cell types.

Exosomes are nanovesicles secreted from various types of cells and can be isolated from various bodily fluids, such as blood and urine. The number and molecular contents, including proteins and RNA of exosomes, have been shown to reflect their parental cell origins, characteristics and biological behaviors. An increasing number of studies have demonstrated that exosomes play a role in the course of tumorigenesis, diagnosis, treatment and prognosis, although its precise functions in tumors are still unclear. Moreover, owing to a lack of a standard approach, exosomes and its contents have not yet been put into clinical practice successfully. This review aims to summarize the current knowledge on exosomes and its contents in esophageal cancer as well as the current limitations/challenges in its clinical application, which may provide a basis for an all-around understanding of the implementation of exosomes and exosomal contents in the surveillance and therapy of esophageal cancer.

Exosomes are nano-sized biovesicles of endocytic origin physiologically released by nearly all cell types into surrounding body fluids. They carry cell-specific cargos of protein, lipids, and genetic materials and can be selectively taken up by neighboring or distant cells. Since the intrinsic properties of exosomes are strictly influenced by the state of the parental cell and by the cellular microenvironment, the analysis of exosome origin and content, and their cell-targeting specificity, make them attractive as possible diagnostic and prognostic biomarkers. While the possible role of exosomes as messengers and a regenerative tool in cardiovascular diseases (CVDs) is actively investigated, the evidence about their usefulness as biomarkers is still limited and incomplete. Further complications are due to the lack of consensus regarding the most appropriate approach for exosome isolation and characterization, both important issues for their effective clinical translation. As a consequence, in this review, we will discuss the few information currently accessible about the diagnostic/prognostic potential of exosomes in CVDs and on the methodologies available for exosome isolation, analysis, and characterization.

Ultrasensitive detection of low-abundance DNA point mutations is a challenging molecular biology problem, because nearly identical mutant and wild-type molecules exhibit crosstalk. Reliable ultrasensitive point mutation detection will facilitate early detection of cancer and therapeutic monitoring of cancer patients. The objective of this study was to develop a method to correct errors in low-level cell line mixes. We tested sample mixes with digital-droplet PCR (ddPCR) and next-generation sequencing. We introduced two corrections: baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation; and haplotype counting was used to correct errors in cell counting and pipetting. We found ddPCR to have better correlation for detecting low-level mutations without applying any correction (R2 = 0.80) and be more linear after introducing both corrections (R2 = 0.99). The VAF correction was found to be more significant than haplotype correction. It is imperative that various technologies be evaluated against each other and laboratories be provided with defined quality control samples for proficiency testing.

Despite the high prevalence and the enormous impact the uterine fibroids has on the healthcare system and the economy, there is no effective medical treatment available to eliminate fibroids. This is due in part to the fact that very little is known about their pathophysiology; in order to look for and potentially find treatments capable of eliminating fibroids, we must gain a deep understanding of the mechanisms behind their initiation, stimulation, and growth. In this article, we comprehensively review current knowledge of the pathogenesis of uterine fibroids, with the aim of better understanding their origin, as well as discussing the action of already existing medical treatments in order to highlight ways that potential future therapies may be able to target the disease. A literature search was performed in PubMed to find articles related to the etiopathogenesis of fibroids and their treatments. Myomas comprise areas of disordered smooth muscle fascicles characterized by an excess of acellular extracellular matrix (ECM) with a dysregulated phenotype. Although the initial event is believed to be smooth muscle cell proliferation, it is thought that a complex signaling system is also necessary. Non-hormonal factors may be responsible for initiating the development of uterine fibroids, although hormonal stimulation is an essential factor for their growth Recent studies have confirmed that the different cell types contained in fibroids are all clonally derived from a parental cell with implied multipotent stem cell properties. Ethnicity also strongly influences the development and clinical severity of fibroids. Many Growth factors and their respective receptors have been implicated in fibroid growth. The available evidence continues to reinforce the importance of progesterone in the development of fibroids. The Ulipristal Acetate mediated volume reduction is due to a series of multifactorial and successive events that lead to a low proliferation rate.

Background & Aim Induced pluripotent stem cell (iPSC) research is rapidly moving towards translational and clinical applications.These applications require robust and consistent workflows that utilize high quality reagents that are xeno-free.When commonly used animal-origin components are replaced with xeno-free alternatives, performance often suffers,thereby necessitating thorough qualification of reagents and the process.Methods that enhance consistency will minimize extra effort and costs associated with clones that fail to expand,or do not meet quality standards for downstream use. Methods, Results & Conclusion Previously,we reported the first off-the-shelf reprogramming kit specifically designed for clinical and translational research.To streamline iPSC generation and ensure the consistent creation of high quality iPSCs,xeno-free workflows for T cells were optimized to minimize the effect of donor to donor variability.Upfront phenotyping of the T-cells prior to reprogramming was performed,along with optimization of a combination of conditions including hypoxia,matrix,and seeding density.These optimizations were shown to offer consistent iPSC generation from difficult to reprogram donor cells,albeit with varying efficiencies.To confirm the parental cell type,T-cell derived iPSC clones were analyzed for immune repertoire,leveraging a next-generation sequencing based assay.The resulting iPSC lines were also confirmed to be foot-print free,authenticated to be derived from the corresponding donor cells,HLA profiled,and were further subjected to comprehensive characterization methods to assess the quality and safety profile.Pluripotency and differentiation potential of iPSC clones was confirmed using ScoreCard,a focused qPCR panel,and PluriTest,an array-based global gene expression platform.Clones qualified to be pluripotent and possess trilineage differentiation potential were thoroughly investigated for genomic stability.Each of the clones tested showed a normal karyotype using both traditional G-banding as well as array-based methods KaryoStat and KaryoStat HD.Further assessment of oncogenic hotspot mutations that are known to occur at a higher frequency in ESC and iPSC,including TP53,indicated the absence of oncogenic structural variants in all the clones tested.The combination of qualified reagents and defined xeno-free workflows, in combination with comprehensive and predictive characterization assays aids in easy transition of early investigational work towards translational and clinical research.

© 2016 The Authors. Prostate cancer is the second leading cancer in men world-wide. Due to its heterogeneous nature, a considerable amount of research effort has been dedicated in identifying effective clinical biomarkers with a focus on proteins, messenger RNA and microRNAs [1]. However, there is limited data on the role and expression of long noncoding RNAs (lncRNAs) in prostate cancer exosomes [2]. This array dataset which is linked to our publication describes the profiling of human lncRNAs in prostate cancer and their exosomes from five different cell lines [3]. From this dataset, we identified a list of statistically significant prostate cancer lncRNAs which are differentially expressed in the exosomes compared to their parent cell lines. This dataset has been deposited into Gene Expression Omnibus (GSE81034).

The FDA-approval of potent targeted therapies has led to great changes in the therapeutic landscape of chronic lymphocytic leukemia (CLL). As a key example, venetoclax, a first-in-class BCL-2 inhibitor, leads to response in about 80% of patients with relapsed/refractory (R/R) CLL. Disease progression on venetoclax, however, has been increasingly observed, and better biologic understanding of resistance mechanisms to this agent is needed. To systematically discover the potential mechanisms of resistance to venetoclax, we performed both genome-scale loss- (LOF) and gain-of-function (GOF) genetic modifier screens in the BCL-2-driven OCI-Ly1 lymphoma cell line using CRISPR-Cas9 sgRNA and ORF libraries, respectively. Significant hits from both screens included the BCL-2 family: the LOF screen with pro-apoptotic genes (PMAIP1, BAX, BAK1, BCL-2L11) and the GOF screen with anti-apoptotic genes (BCL2L1, BCL2L2, BCL2, MCL1). In addition, the LOF screen uncovered genes in pathways relevant to lymphoid biology (i.e, NFKBIA) and lymphoid transcription factors and modulators (IKZF5, ID3, EP300, NFIA). The GOF screen also uncovered components of the energy-stress sensor PKA/AMPK signaling pathways (ADIPOQ, PRKAR2B, PRKAA2) and regulators of mitochondrial metabolism. In parallel, we performed an integrated transcriptome, whole proteome and functional characterization of an OCI-Ly1 cell line rendered resistant to venetoclax (OCI-Ly1-R) from the parental cell line (OCI-Ly1-S). RNA-seq and spectrometry-based proteomics revealed coordinated dysregulation of transcripts and proteins in the resistant line originating from genes critical to cellular metabolism, cell cycle, B-cell biology and autophagy. Of the transcripts and proteins significantly associated with the resistant cell line, only MCL-1 overlapped with the gene hits from the genome-scale screens. Treatment of the OCI-Ly-R cells with the MCL-1 inhibitor S63845 synergized with venetoclax. Given the dysregulation of proteins critical to metabolism in both the GOF screen and in OCI-Ly1-R cells, we also evaluated the role of metabolic reprogramming in venetoclax resistance. We first assessed mitochondrial respiration by measuring the oxygen consumption rate. Compared to OCI-Ly-S cells, OCI-Ly1-R cells demonstrated markedly higher respiration levels, suggesting a state of higher oxidative phosphorylation (OXPHOS). More directly, we measured oxygen consumption following venetoclax exposure. Consistent with impairment of OXPHOS by venetoclax, we observed both an immediate decrease in oxygen consumption and an immediate burst of glycolysis following venetoclax in the OCI-Ly1-S cells, but not in the OCI-Ly1-R cells. In line with these findings, the AMPK inhibitor dorsomorphin and mitochondrial electron transport chain (mETC) inhibitors synergized with venetoclax in OCI-Ly1-S cells. Transcriptome related to ID3 (identified as one of the LOF screen targets) was characterized in isogenic ID3-knockout OCI-Ly1 lines. It revealed PRKAR2B overexpression as a key effect, suggesting a role for ID3, and perhaps of other lymphoid transcription factors in regulating metabolic reprogramming associated with resistance. Indeed, exposure of ID3 knockout lines to mETC inhibitors overcame resistance to venetoclax. To determine if there is a genetic basis for the drug resistance seen in OCI-Ly1-R cells, we compared whole-exome sequencing (WES) results of DNA isolated from the OCI-Ly1-R and OCI-Ly1-S cell lines. A clear region was amplified on chromosome 1q23, which includes MCL1 and PRKAB2 (the regulatory subunit of AMPK). Similarly, a WES-based analysis of paired CLL DNA samples isolated from 6 R/R CLL patients just prior to venetoclax initiation and at time of progression on venetoclax was performed. We did not identify any non-silent somatic single nucleotide in BCL2 or its family members at baseline or at progression, despite marked clonal shifts in all patients. We confirmed the presence of the amp(1q23) as acquired at relapse after venetoclax in 3 out of 6 patients. Our study reveals that venetoclax resistance implicates changes not only for outer mitochondrial membrane (MCL-1 expression) but also for inner membrane (oxydative metabolism). Such mitochondrial reprogramming represents a new vulnerability that can potentially be exploited through combinatorial therapy with metabolic modulators to overcome resistance. Disclosures Guieze: abbvie: Honoraria; janssen: Honoraria; gilead: Honoraria. Thompson:Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Adaptive Biotechnologies: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Davids:Merck: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; MEI Pharma: Consultancy, Research Funding; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Surface Oncology: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Brown:Sun Pharmaceutical Industries: Research Funding; Abbvie: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy; Janssen: Consultancy; Sunesis: Consultancy; Roche/Genentech: Consultancy; Verastem: Consultancy, Research Funding; Boehringer: Consultancy; Loxo: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy; Genentech: Consultancy. Wierda:AbbVie, Inc: Research Funding; Genentech: Research Funding. Letai:AstraZeneca: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Vivid Biosciences: Equity Ownership. Wu:Neon Therapeutics: Equity Ownership.

Objective To discuss the difference between multi-drug resistant cell line A549/Gem and its parental cell A549 on the basis of establishment of human gemcitabine-resistant cell line A549/Gem so as to elaborate the possible mechanisms of gemcitabine resistance.

Evidence for a new HLA class II specificity is presented. It is recognized by LE serum, which reacts with most DR1 and/or DR4 individuals (r = 0.86). Its frequency in the French population is 0.33. Absorption-elution experiments showed that the serum reactivity was not due to a mixture of anti-DR1 and anti-DR4 antibodies, but to a single antibody population which could be absorbed on and eluted from both DR1(+) or DR4(+) cells. LE specificity seemed to be expressed on DR but not on DQ molecules since the serum reacted with and could be absorbed by DR+,DQw- cells; it did not react with a DR-,DQw+ mutant cell, but did react with the DR+,DQw+ parental cell. The relationship between LE specificity and MC1 and Te23 specificities remains to be determined.

Objective To discuss the difference between multi-drug resistant cell line H460/Gem and its parental cell NCI-H460 on the basis of establishment of human gemcitabine-resistant cell line H460/Gem so as to elaborate the possible mechanisms of gemcitabine resistance.

SUMMARY A new red alga Meristotheca imbricata Faye et Masuda (Solieriaceae, Gigartinales) was described on the basis of specimens collected from southern Japan. Although this species might have been for a long time included in the concept of M. coacta Okamura, until recently a very poorly known alga, it is distinguished from the latter by the following features: (i) each thallus consists of one primary blade and several secondary blades, the latter arising from the apical or subapical portion of terminal segments of the primary blade; (ii) the individual blades are repeatedly dichotomously divided into linear segments of which margins are neither undulate nor crispate, and are fleshy, imbricate, frequently anastomosing and often acervate; (iii) each tetrasporangial initial is attached by a basal pit-connection to the parental cell, and the position of the pit-connection changes from basal to lateral by inward growth of the tetrasporangium; and (iv) the carpogonial branches sometimes have a one-celled sterile lateral.

A new species of a chlorarachniophyte alga, Bigelowiella longifila sp. nov., is described. It is classified as a member of Bigelowiella as flagellate cells constitute the main stage of the life cycle. However, this alga is different from the only described species of the genus, B. natans Moestrup, in having a unique amoeboid stage in the life cycle. We observed an interesting behavior of amoeboid daughter cells after cell division: One of the two daughter cells inherits the long filopodium of the parental cell, and it subsequently transports its cell contents through the filopodium to develop at its opposite end. The other daughter cell forms a new filopodium. This unequal behavior of daughter cells may have evolved before the chlorarachniophytes and some colorless cercozoans diverged.

International audience; DNA is not the sole medium by which parents transmit information to their offspring. Epigenetic inheritance, in particular , is based on the partial transmission of the cellular state of the parental cell to its descendants. Although the reality of epigenetic inheritance is now firmly established, whether it has an influence on the long term evolutionary process is still subject to debate. To address this question, we used RAevol, an in silico experimental evolution platform, and defined 4 scenarios with static or dynamic environments and with or without epigenetic inheritance. Simulations in dynamic environments show that protein inheritance indeed increases the rate of evolution on the long term. But they also show that it impedes evolution in its very first stages. This negative effect can be explained by instabilities generated by the interference between the two inheritance mediums. On the opposite, the long term gain can be explained by protein inheritance reducing the constraints on the genetic regulation network.

Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. Since the αVβ6 integrin is a principal receptor for FMDV, we transduced a bovine kidney cell line to stably express both the αV and β6 bovine integrin subunits. This stable cell line (LFBK-αVβ6) showed β6 expression and enhanced susceptibility to FMDV infection for ≥ 100 cell passages. LFBK-αVβ6 cells were highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophilic FMDV strain O/TAW/97. In comparison to other cell types that are currently used for virus isolation, LFBK-αVβ6 cells were more effective at detecting FMDV in clinical samples, supporting their use as a more sensitive tool for virus isolation.

Structural chromosomal rearrangements are commonly observed in tumor karyotypes and in radiation-induced genomic instability. Here we report the effects of TP53 deficiency on karyotypic stability before and after irradiation using related cells and clones differing in cellular TP53 status. The parental cell line, TK6, is a TP53 wild-type human B-lymphoblastoid line with a highly stable karyotype. In the two TK6 derivatives used here, TP53 has been inactivated by biochemical means (expression of HPV16 E6; TK6-5E) or genetic means (allelic inactivation; NH32). Biochemical inactivation of TP53 (TK6-5E) had little effect on the spontaneous karyotype, whereas allelic inactivation of TP53 (NH32) resulted in a modest increase in spontaneous karyotypic instability. After 2 Gy gamma irradiation, the number of unstable clones derived from TP53-deficient cells was significantly elevated compared to the TP53 wild-type counterpart. Extensively destabilized clones were common after irradiation in the set of clones derived from NH32 cells, and one was observed in the set of TK6-5E clones; however, they were never observed in TK6-derived clones. In two of the irradiated NH32 clones, whole chromosomes or chromosome bands were preferentially involved in alterations. These results suggest that genomic instability may differ both quantitatively and qualitatively as a consequence of altered TP53 expression. Some of the results showing repeated and preferential chromosome involvement in aberrations support a model in which instability may be driven by cis mechanisms.

Chlorinated organic chemicals are widely used in industry and are present in the environment. Five chlorinated aliphatic hydrocarbons, namely 1-2-dichloroethane, 1,1,2-trichloroethane, trichloroethylene, 2,3-dichlorobutane and 1-chlorohexane were investigated to determine their influence upon the fidelity of cell division in cultured mammalian cells. In order to determine the influence of these chemical compounds upon the fidelity of cell division, a technique known as differential staining of chromosomes and spindle was performed with one genetically engineered cell line and its parental cell line. The genetically engineered cell line used in this study expressed a human P450 enzyme, CYP2E1. Four chemicals, 1-2-dichloroethane, trichloroethylene, 2,3-dichlorobutane and 1-chlorohexane required metabolic bioactivations in order to induce spindle damage in cultured mammalian cells whereas 1,1,2-trichloroethane was a direct-acting spindle poison.

Isolate cell lines with improved stability or expression properties from a parental cell line.

Summary The new oral infraciliature comes from the posterior longitudinal proliferation of numerous somatic kineties of the parental perizonal zone during the bipartition of Caenomorpha medusula . The new perizonal zone of the opisthe originates from proliferation of the anterior extremes of all the somatic kineties that make up the parental perizonal zone. The proter retains both, the oral infraciliature and the perizonal zone of the parental cell. Conjugation in C. medusula presents three maturation divisions and three postzygotic divisions. During conjugation the oral infraciliature of each conjugant degenerates to be replaced by a new one.

Introduction: Ultrasensitive point mutation detection can facilitate minimal residual disease detection, early detection of cancer, and therapeutic monitoring of cancer patients. However, nearly identical mutant and wildtype molecules exhibit crosstalk with most technologies, thus producing high background levels. To evaluate technologies against each other for accurate ultrasensitive point mutation detection it is crucial to supply laboratories with reference samples of various dilutions. Methods: We tested a series of sample mixes with digital-droplet PCR and next generation sequencing. Using linearity and accuracy as the gold standard, we empirically introduced two corrections to generate improved quality control reagents. Baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation of variant, while haplotype counting was used to correct technical errors (cell counting and pipetting). Results: Correlation between the level of dilution and the measured VAF was relatively good (R2 = 0.80) when measured using ddPCR. However, correcting for the parental VAFs substantially improved the correlation (R2 = 0.97). In contrast, correlation of the cell count dilution and the measured VAF, as determined by next generation sequencing (AmpliSeq), was initially relatively poor (R2 = 0.63). However, substantial improvement in correlation was achieved by correcting for the parental VAFs (R2 = 0.94). In addition, correcting by haplotype counting alone only slightly improved accuracy in both technologies (ddPCR, from R2 = 0.80 to R2 = 0.81; AmpliSeq, from R2 = 0.63 to R2 = 0.64). Furthermore, correcting for both factors resulted in the best correlation for ddPCR-generated data (R2 = 0.99); however, introducing both corrections for AmpliSeq data did not prove more beneficial than correcting for parental VAFs alone (both corrections, R2 = 0.94 compared to parental VAF correction only, R2 = 0.94). Conclusions: Validation of ultrasensitive variant detection requires standardized reference samples. These will also permit assessing various ultrasensitive detection technologies against one another. Citation Format: Marija Debeljak, Lisa Haley, Derek A. Anderson, Emily M. Adams, Masaya Suenaga, Katie Beierl, Ming-Tseh Lin, Michael Goggins, Christopher D. Gocke, James R. Eshleman. Generating quality control reference standards to evaluate ultrasensitive variant detection. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1378.

Division of Schizosaccharomyces pombe by medial fission produces identically sized daughter cells that grow by tip extension until their own division is prompted by reaching the same critical size for division as the parental cell. The fidelity of this size control in the absence of perturbation means that cells of the same size are at the same point in the cell cycle. Size selection of small cells from an asynchronous culture by centrifugal elutriation permits generation of synchronous cultures large enough for biochemical analysis. The changes observed in the synchronized cell cycle progression of such cultures are representative of those that accompany cell cycle progression of individual cells. Here, we describe how size selection with the Beckman Coulter JE-5.0 rotor can be used to generate synchronized cultures. Because of the continuous passage of medium through the rotor throughout the procedure, elutriation is considered to have less impact on the integrity of the cell cycle than other approaches. Two protocols are presented here: The first generates a 2-L culture ideal for detailed biochemical analysis, whereas the second allows rapid generation and simultaneous analysis of three smaller (200-mL) cultures.

represents apromising production system for biopharmaceuticals,particularly for those needing human-type post-transla-tional modifications [1,2]. For further rational improve-ment of the cell line and the cultivation process adetailed understanding of the metabolism and metabolicchanges during glycoprotein production is desirable.Metabolism and metabolic burden upon production ofa

In functional assays, parental cell lines are commonly used as a negative control for cell lines expressing recombinant proteins. Since the apparent primary difference between the two lines is the ...

The evolutionary origin of the egg stage of animal development presents several difficulties for conventional developmental and evolutionary narratives. If the egg's internal organization represents a template for key features of the developed organism, why can taxa within a given phylum exhibit very different egg types, pass through a common intermediate morphology (the so-called "phylotypic stage"), only to diverge again, thus exemplifying the embryonic "hourglass"? Moreover, if different egg types typically represent adaptations to different environmental conditions, why do birds and mammals, for example, have such vastly different eggs with respect to size, shape, and postfertilization dynamics, whereas all these features are more similar for ascidians and mammals? Here, I consider the possibility that different body plans had their origin in self-organizing physical processes in ancient clusters of cells, and suggest that eggs represented a set of independent evolutionary innovations subsequently inserted into the developmental trajectories of such aggregates. I first describe how "dynamical patterning modules" (DPMs) associations between components of the metazoan developmental-genetic toolkit and certain physical processes and effects may have organized primitive animal body plans independently of an egg stage. Next, I describe how adaptive specialization of cells released from such aggregates could have become "proto-eggs," which regenerated the parental cell clusters by cleavage, conserving the characteristic DPMs available to a lineage. Then, I show how known processes of cytoplasmic reorganization following fertilization are often based on spontaneous, self-organizing physical effects ("egg-patterning processes": EPPs). I suggest that rather than acting as developmental blueprints or prepatterns, the EPPs refine the phylotypic body plans determined by the DPMs by setting the boundary and initial conditions under which these multicellular patterning mechanisms operate. Finally, I describe how this new perspective provides a resolution to the embryonic hourglass puzzle.

A hepatic invasive human colorectal xenograft model was derived in nude mice by selection through the liver of the parental cell line, C170. Following intraperitoneal injection, tumours selectively grew on the liver in > 80% of the animals within 15–20 days. The liver-invading xenograft line, renamed C170HM2, had a significantly greater expression of the LewisX antigen compared to C170 (mean linear fluorescence per cell > 1000 compared with 500 for C170, P < 0.02). C170HM2 had significantly elevated proliferation (when compared with C170) in the presence of epidermal (P < 0.001) and basic fibroblast growth factor (P < 0.001). C170HM2 also mitogenically responded to type I collagen (derived from rat tails), unlike C170. C170HM2 tumours when invading the liver expressed both interstitial collagenase and gelatinase activity at the invading edge.

To better characterize differences in expression and secretion capacity, we quantitatively analyzed production of Green Fluorescent Protein (GFP) and secretion of recombinant human IgG in transiently transfected CHOK1SV, ECACC K1 and CHO DG44 parental cell lines. By analyzing these production trends in a transient transfection system, we were able to compare recombinant protein production and secretion between the different CHO parental cell lines independent of integration site effect.

GS-NS/0 recombinant antibody production cell line was adapted to cholesterol-free and animal protein free (APF) medium. Lower peak viable cell density (VCD) and higher productivities were seen in the cholesterol-independent cells than in parental cell line while the quality of the antibodies was similar. The cholesterol-independent cell line were then selected and characterized.

The morphology and morphogenesis of two species of the genus Lembadion, L. lucens and L. bullinum, are described. In both species, left and right ventral kineties converge behind the mouth forming a postoral suture. Buccal infraciliature is formed by one polykinety and two very close paroral kineties (inner and outer). During stomatogenesis, the new oral structures originate from the paroral kineties. The inner paroral kinety forms the new adoral polykinety and regenerates the outer paroral kinety of the proter, while the paroral kineties of the opisthe originate from the outer paroral kinety of the parental cell. Somatic proliferation starts before the stomatogenesis at the equatorial level of the cell, and extends towards the poles forming an equatorial band. Two large invariant zones, anterior and posterior, remain in the dividing cell. Moreover, the kinetodesmal fibers disappear in the proliferation band during the bipartition (fission) process.

Under the constitutive control of c-Myc over-expression the CHO cultures showed an increase in growth rate and maximum cell number accompanied by a similar decrease in specific glucose consumption rate. Additionally overexpression of c-Myc appeared to induce morphological transformation and partial anchorage-independence. Although, the c-myc transfected cell line exhibited apoptosis at much lower rates than it is widely reported and associated with the over-expression of c-Myc, it was nevertheless apparent that c-Myc was responsible for the induction of higher apoptotic rates when compared with the control cell line. Hence, the anti-apoptotic gene bcl-2 was also used to transfect the c-Myc CHO cell line, in order to reduce cell death. Over-expression of both oncoproteins resulted in a cell line that exhibited higher proliferation rates and maximum cell numbers, with a decrease in apoptosis when compared to the parental cell line.

Retroviral vectors are powerful tools for gene transfer that are useful in the context of experimental as well as clinical applications. Defective recombinant retroviruses allow for efficient gene transfer into a broad range of mammalian cells derived from different species and different tissues. Safe gene transfer can be achieved using helper-free viral stocks, owing to the development of vectors and packaging cell lines that minimize the probability of hazardous recombination events. Retrovirus-mediated gene transfer leads to stable proviral integration in the target-cell genome, a key feature when the foreign genetic material must be faithfully transmitted to the progeny of the transduced parental cell, murine leukemia virus (MLV)-based vectors accommodate numerous modifications, thus providing a plastic tool that can be tailored for very diverse applications.

We designed a new separable bilayer (SB) microfiltration device for the viable enrichment of circulating tumor cells (CTCs) independent of antigen expression. Bilayer pore structures at the micro scale decrease cell damage to preserve viability, while maintaining throughput to allow rapid enrichment. The large pore size (40 um) of the upper layer facilitates the CTCs proliferation on chip. The separability enables the efficient release of the CTCs after successful on chip culture of captured CTCs. We characterized the device performances including capture efficiency, enrichment against leukocytes, viability and proliferability. The SB device can enrich tumor cells with 80% capture efficiency, higher than 103 enrichment, and better than 70% viability from 1 mL whole blood samples on a 1 cm2 device. Multiple cell lines were shown to be able to proliferate after captured on SB device. Furthermore, using CTCs derived from an in vivo model system, cell cultures were established by releasing the captured CTCs from the chip. The cultured CTCs and their corresponding parental cell lines were injected into mice. Tumors were established with no discernable differences in volume and rate of growth, demonstrating similar tumorigenicity of viable CTCs recovered by the SB device. In a feasibility study, we successfully detected CTCs from 1 mL of clinical whole blood sample enriched via SB device. Citation Format: Ming-Da Zhou, Sijie Hao, Anthony J. Williams, Bo Lv, Jiyue Zhu, Richard J. Cote, Ram H. Datar, Yu-Chong Tai, Siyang Zheng. Separable bilayer microfiltration device for viable label-free enrichment of circulating tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-193. doi:10.1158/1538-7445.AM2014-LB-193

We evaluated the transfer to and from Escherichia coli of endogenously isolated plasmid material from the River Butrón during the growth of three donor strains and two recipient strains as well as after the survival of these parental cells in river water. Transfer frequency varied greatly during the growth of donor cells, with minimum values in the exponential phase; frequency remained constant, however, during the growth of recipient strains. After survival in river water, donor cells lost their ability for plasmid transfer before any other physiological variations in the cells caused by environmental stress were detected. Under the same conditions and during equal periods, however, no variation in the ability of recipient cells to receive and express plasmid material was observed.

In order to investigate the molecular pathogenesis of human osteosarcoma, a reliable metastatic osteosarcoma animal model is required. Here we report the development of a series of metastatic sublines (LM1-LM4) derived from a human osteosarcoma 143B parental cell line expressing GFP in the nucleus and RFP in the cytoplasm. The sublines have increasing ability to form pulmonary metastases in nude mice after intravenous injection. In vitro proliferation rates of all sublines were similar. When the 143B parental and the metastatic sublines were transplanted orthotopically in the tibia of nude mice, all cell lines were tumorigenic. Primary tumor growth and metastases were imaged with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan). LM2 and LM3 had higher metastatic capability than the parental 143B. LM1 had higher metastatic capability than LM2 or LM3. LM4 cells resulted in the most pulmonary micrometastases. The number of pulmonary metastases was 8-fold higher in LM4 cells than that with 143B parental cells. This series of metastatic sublines should be a powerful tool to investigate genetic and other factors that promote or inhibit osteosarcoma metastasis and for evaluation of novel anti-metastatic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5230. doi:10.1158/1538-7445.AM2011-5230

A major impediment to the use of Tumor Necrosis Factor-Like Apoptosis Inducing Ligand (TRAIL) Receptor targeted drugs is that many tumors evolve resistance to these agents. On the other hand however, multiple different kinds of drugs are reported to be able to synergize with TRAIL. This raises the question of whether it is possible to overcome resistance using the same drugs that cause synergy. We tested this idea by creating a series of isogenic tumor cells with acquired TRAIL resistance or defined mechanisms of resistance that are known to occur in human tumors then compared these cells to the TRAIL-sensitive parental cell line. Although diverse classes of anti-cancer drug were all able to synergize with TRAIL most of these agents were unable to overcome resistance and there was no correlation between the amount of synergy that is seen with a particular agent and its ability to overcome acquired resistance. Proteosome inhibitors were however able to overcome diverse resistance mechanisms suggesting that one should select drugs for TRAIL R agonist combination therapy based not just on their ability to synergize but rather on their ability to both overcome resistance and synergy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 207.