Blockchain Papers

Huidong Huang, Zhenzhen Guo, Chunjuan Zhang, Cheng Cui · 7 authors

Engineering cell-derived nanovesicles with active-targeting ligands is an important strategy to enhance the targeting efficiency. However, the enhanced binding capability to targeting cells also leads to the binding with nontarget cells that share the same biomarkers. DNA-based logic gate is a kind of molecular system that responds to chemical inputs by generating output signals, and the relationship between the input and the output is based on a certain logic. Thus, the DNA-based logic gate could provide a new approach to improve the delivery efficiency of the nanovesicle. In this work, we developed a DNA logic-gated module that coupled two tumor cell-targeting factors (e.g., low pH and a tumor cell biomarker) in a Boolean manner. Immobilization of this module on the surface of the nanovesicle enables the nanovesicle to sense tumor cell-targeting factors and regard these cues as inputs AND logic gate. With the guide of DNA-based logic gate, gold carbon dots (GCDs) encapsulated within nanovesicles were delivered into target cells, and then the intracellular redox status variation was reflected by fluorescence change of GCDs. Overall, we developed DNA logic-gated nanovesicles that contract different targeting factors into a unique tag for target cells. This facile functionalization strategy can pave the way for constructing smart nanovesicles and would broaden their application in the field of precision medicine and personalized treatment.

Anton Jan van Zonneveld, Ton J. Rabelink, Roel Bijkerk

No abstract is available for this record.

Roderick A.F. MacLeod, Wilhelm G. Dirks, Hans G. Drexler

The key role played by continuous cell lines in biomedical research is often taken for granted: on the Highwire Press server, ‘HeLa’ as keyword currently scores over 0.5 Mio bibliographic hits compared to just 1.26 Mio for ‘cancer’ alone (http://highwire.stanford.edu/). Although peerlessly cited, HeLa (cervical carcinoma) is only one of roughly 104 cancer cell lines. Keeping track of these is scarcely practicable, and the chronic problem of cell line cross-contamination (CLCC)1, 2—first signalled 3 decades ago for HeLa,3 one of few human cancer cell line then available—is now a major issue. Research using cancer cell lines covers all areas, whether as models for specific diseases, as illustrated by the early development of imatinib using the K-562 chronic myeloid leukemia model,4 or for eukaryotic cells, as with the first demonstration of RNA-interference in vertebrates which was performed using HeLa,5 or as oncogenomic resources, e.g. the Cancer Genome Project which lists data for hundreds of examples drawn from all available tumour types (http://www.sanger.ac.uk/genetics/CGP/Census/). It is nigh impossible to think of any major biological research field where cell lines are not widely used. Hence it would be tragic were investigators forced to boycott cell lines for fear of CLCC. It is sometimes argued that one eukaryotic cell is very much like another, rendering misidentification a side-issue. Good practice aside, use of misidentified examples frustrates independent data replication. Furthermore, postgenomic research highlights ever finer differences between tumour types. Usefulness of cell models assumes authenticity and deep characterization. For intractable primary tumors, such as neuroblastoma or Hodgkin lymphoma, cell lines often serve as prototypes where misidentification would be catastrophic. As Cicero famously observed, ‘One falsehood leads easily to another’. Although acknowledging the importance of good technique and awareness, we and others have urged intervention at other levels of the scientific process, for example by those reviewing grant applications, or by journal editors and referees who could, respectively, encourage use of authentic cell lines or sanction users of false examples. Few non-specialists are aware of the problem—a knowledge gap most acute among those likely to suffer most, namely starting scientists. We, therefore welcome this opportunity to air proposals which might serve to combat CLCC. Reports describing CLCC have increased of late, reflecting both technical advances and hightened awareness. With DNA profiling it is now possible unequivocally to distinguish individual cell lineages. The application of forensic DNA STR-profiling speeds and simplifies authentication.6 Previously, detection of untoward matches between supposedly unrelated cell lines required laborious cytogenetic analyses and good memories. Nevertheless, cytogenetics still has a role to play, notably when reference karyotypes of individual cell lines have been previously described enabling downstream identification. The long-overdue attention by high-profile journals has benefited awareness,2 meanwhile leading specialist journals now award single instances of CLCC a degree of priority previously unthinkable.7 Hitherto, most instances of CLCC have been reported by cell repositories. The reasons for this are twofold: first, unlike originators, it is in their own interest for cell banks rapidly to uncover CLCC, if only to avoid recall actions; second, only suitably equipped facilities holding cell lines en masse are equipped to perform meaningful authentication. Using cytogenetics combined with traditional DNA multilocus VNTR-profiling, we found that 18% purportedly ‘new’ cell lines are false, mainly due to CLCC involving classic cell lines.8 This estimate is conservative, given the limited database of <500 cell lines then available for comparison. Furthermore, we found that 29% of all originators depositing cell lines had included at least one false example. The current method of choice for cell line authentication involves analysis of short tandem repeat (STR) markers and is based on forensic profiling which, unlike previous DNA profiling techniques, has been standardized across major cell banks worldwide. Both STR profiling data6 and the Sanger Institute's parallel SNP/genotyping survey of ∼3,500 mainly repository-sourced cell lines (http://www.sanger.ac.uk/genetics/CGP/Genotyping/synlinestable.shtml) confirm that CLCC is rife. Unexpectedly, we found that CLCC among leukemia-lymphoma cell lines (the single most identifiable group) occurs at about the same frequencies (∼16%) whether obtained directly from originators or from indirect sources.9 This implies that CLCC occurs primarily during ‘establishment’, presumably by contamination of fledgling cultures of new cell lines. Although ‘downstream’ CLCC among established cell lines undoubtedly occurs, its effects are likely to be localized. CLCC is likely to occur as a result of sharing reagents or pipettes under the cleanbench. We found no evidence linking CLCC to fraud, at least among freely available cell lines. Immortalization of some cell types may be unfeasible; undue success with intractable cell types warrants consideration of CLCC before rushing into print. The precise classifications of early ‘classic’ cell lines remain subject to revision. Their identities are articles of faith given the sparse documentation and the non-viability or inaccessibility of early cryopreserved material. Many classic cell lines go under multiple aliases, their disambiguation an arcane and thankless task. On the other hand, recent diagnostic advances have allowed several classic cell lines to be reclassified, e.g. HL-60 (from AML-M3 to AML-M2), SK-N-MC (from neuroblastoma to Ewing-family tumor). The most complete coverage, that of human leukemia lymphoma cell lines offered by the DSMZ repository barely reaches 50% of 609 examples hitherto characterized, the latter only ∼40% of the published total.10 Repository holdings are mainly limited by the generosity of donors. While publication to death by retentive originators is only human, less understandable is hoarding to trade against co-authorships or pecuniary advantage. Avoiding reposition means evading authentication, leaving authors free to publish findings unencumbered by worries concerning outside replication.11 As cited by Lacroix,12 the problems occasioned by CLCC are illustrated by the sad though instructive case of ECV-304, derived from the classic bladder cancer cell line T-24,13 inadvertently republished as a ‘spontaneously immortalized human endothelial cell line’.14 Although there are several human cell lines purporting to be endothelial in both origin and character, only one, EA.hy926, produced by fusing human umbilical vein endothelial cells with the lung cancer cell line A549 is widely used at present.15 Despite being hybrid and therefore potentially unstable, the genomic composition of EA.hy926 awaits full characterization. Miraculous birth inspires faith, and the equally prodigious ECV-304 quickly became a popular in vitro endothelial cell model in its heyday (Fig. 1), not least because, unlike EA.hy926, its originators granted distribution by major cell repositories, prior to the advent of STR-profiling which might have revealed its true identity. Notwithstanding doubts concerning, inter alia, its lack of von Willebrand factor secretion, ECV-304 became widely regarded as the leading endothelial cell model. Citation history of ECV-304. Main figure shows yearly (March-February) citations of ECV-304 (http://highwire.stanford.edu/). Plot shows percentage citation as ‘endothelial’ 1999–2006. Hand indicates 1999 when ECV-304 was first unmasked as T-24.8, 15 Note stable citation 2000–2006. In 1999, just as its popularity took off, the journal where ECV-304 was originally described published our letter announcing its derivation from T-24, based on DNA profiles of samples obtained from their respective originators.16 This finding was rapidly confirmed by other ECV-304 recipient cell banks, pointing inexorably to originator CLCC. Simultaneously, this journal published our general survey of CLCC affecting cell lines sourced from their originators, including ECV-304.8 Although it might be argued that denunciation halted ECV-304 in its tracks, the level of citation reached in 1999 has scarcely abated. Disappointingly, the percentage of articles describing ECV-304 as ‘endothelial’ remains stable, and may even be increasing (Fig. 1) notably in China—by now a major scientific hub. Although ECV-304 is widely used there, its former status remains unchallenged like several other false cell lines. For various reasons, including linguistic barriers, back door distribution, and the inaccessibility of Chinese cell repository websites, the CLCC message has yet to come across. We are drafting an updated list of false leukemia-lymphoma cell lines which will be submitted to the Chinese Journal of Hematology. Most ECV-304 citations seem blissfully unaware of its true origin, while a significant remainder seem coy (Fig. 1). The latter are divided between the frank—admitting identity to T-24, and opportunists who have simply replaced ‘endothelial’ by ‘epithelial’. In most cases the ‘guiding hand’ of referees is clear to see. Just how many papers suffered the ‘clunking fist’ of rejection is unknown, of course. Although a notably egregious example, the case of ECV-304 is by no means untypical17 and points to chronic structural weaknesses in the response of the scientific community to a specific form of misrepresentation, however inadvertant. Although various solutions have been proposed to combat CLCC,18 including ‘passports’ detailing provenances and movements of cell lines,19 there is little evidence of takeup, e.g. by inclusion within editorial notes for referees. Non-interventionists argue that science inevitably generates misleading data condemned to oblivion given their inherent unrepeatability. Consideration of ECV-304 suggests that rather than cumulating burdensome discrepancies, most users reported endothelial features (Fig. 1), 1 group claiming that endostatin promotes apoptosis in ECV-304 unlike bladder cancer cell lines.20 In the light of CLCC, conclusions of a significant proportion of the 1361 articles citing ECV-304 must be deemed questionable, as with a legion of additional false cell lines. While CLCC affecting prototypes like ECV-304 is most harmful, more commonly established tumors may come to be represented by single exemplars thrust into prominence by expert recommendation, such as membership of the NCI-60 reference panel. Alas, NCI-60, originally used for anticancer drug evaluation and more recently for transcriptosome analysis, no longer comprises 60 cell lines following expulsion of several duplicated or false examples, most recently the purported drug-resistant breast cancer MCF-7/AdrR cell line, alias NCI/ADR-RES, recently shown to be subclones of OVCAR-8 ovarian adenocarcinoma.21 Woes concerning ECV-304 seem to have plagued certain journals more than others, implying that editorial policy can limit CLCC data dissemination. Although high throughput methods simplify detection, there is no simple nostrum to banish CLCC. Hence, multiple avenues of attack seem advisable, but to be effective consensus is prerequisite. To continue the debate recently started in an open letter to the USA Secretary of Health from Prof. Roland Nardone and 19 prominent co-signatories calling for zero tolerance for CLC—‘no grants; no publications’,22 we have identified distinct stages of the scientific process where intervention might be effective. Document their derivation from primary material (either by DNA profiling, or, where relevant, by cytogenetics). Offer unrestricted availability or public reposition. Test for and, if necessary, eliminate mycoplasma contamination—a problem which often accompanies CLCC.23 Infractions would be penalized by time-limited embargo by the affected journals. List false cell lines, including those thereby excluded from accessions; Collaborate on comparative STR profiling of all holdings to detect duplicates; Offer training courses, including cell line authentication. The DSMZ publishes a comprehensive list of known false leukemia and lymphoma cell lines (http://www.dsmz.de/human_and_animal_cell_lines/main.php?content_id=97). A similar scheme for solid tumor cell lines is planned. Encourage reports and reviews such as that of Lacroix; Furnish referees with lists of false cell lines; Insist that authors give provenances of cell lines used; Require indirectly provenanced material (i.e. obtained from secondary sources) to be authenticated by comparison with reference STR profile provided online by cell banks, or by cytogenetic analysis. Insist that definitive first reports of cell lines subsequently shown to false be retracted. Mandate use of cell lines provenanced directly from originator/repositories, or authenticated with reference to published STR profiles/karyotypes; Insist that cell lines established by funds provided be submitted to a public repository. To end on a more optimistic note, measures to avoid CLCC have been successfully implemented by the USA biopharma industry where only 2 cases of misidentified cells were discovered in over 900 samples tested from 1996 to 2006 (cited in Ref.22). The authors welcome all comments and suggestions from users of cell lines how best to combat CLCC. According to „Genome News”︁ (December 2007), The National Institutes of Health issued a notice that it expects researchers working with cultured cells to use authentication procedures in order to avoid misidentified or contaminated cultures that can significantly affect research outcomes. The NIH cautioned that grant applications that fail to employ authentication methods “would not be considered of the highest quality and such manuscripts would not fare well in the journal review process.” The notification is a response to an open letter from Roland Nardone, a professor at the Discovery Center for Cell and Molecular Biology at the Catholic University of America, and by a number of other biologists, to US Department of Health and Human Services Secretary Michael Leavitt, which said that misidentification of cell lines is a serious and potentially growing problem that can have the potential for “dire consequences.” The authors of the letter advised that “all researchers using cell cultures incorporate a specific cell line authentication protocol into their experimental framework” and that authentication be mandatory in order for researchers to receive grants. We thank Dr Hilmar Quentmeier for critically reading the manuscript. We also wish to thank Mr Peyton Hughes for drawing our attention to the article in “genome News” cited in the Note Added in Proof. Yours sincerely, Roderick A.F. MacLeod, Wilhelm G. Dirks, Hans G. Drexler Roderick A.F. MacLeod*, Wilhelm G. Dirks*, Hans G. Drexler*, * DSMZ, German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Inhoffenstr. 7b, 38124, Braunschweig, Germany.