Limiting the Persistence of a Chromosome Break Diminishes Its Mutagenic Potential

To characterize the repair pathways of chromosome double-strand breaks (DSBs), one approach involves monitoring the repair of site-specific DSBs generated by rare-cutting endonucleases, such as I-SceI. Using this method, we first describe the roles of Ercc1, Msh2, Nbs1, Xrcc4, and Brca1 in a set of distinct repair events. Subsequently, we considered that the outcome of such assays could be influenced by the persistent nature of I-SceI-induced DSBs, in that end-joining (EJ) products that restore the I-SceI site are prone to repeated cutting. To address this aspect of repair, we modified I-SceI-induced DSBs by co-expressing I-SceI with a non-processive 3′ exonuclease, Trex2, which we predicted would cause partial degradation of I-SceI 3′ overhangs. We find that Trex2 expression facilitates the formation of I-SceI-resistant EJ products, which reduces the potential for repeated cutting by I-SceI and, hence, limits the persistence of I-SceI-induced DSBs. Using this approach, we find that Trex2 expression causes a significant reduction in the frequency of repair pathways that result in substantial deletion mutations: EJ between distal ends of two tandem DSBs, single-strand annealing, and alternative-NHEJ. In contrast, Trex2 expression does not inhibit homology-directed repair. These results indicate that limiting the persistence of a DSB causes a reduction in the frequency of repair pathways that lead to significant genetic loss. Furthermore, we find that individual genetic factors play distinct roles during repair of non-cohesive DSB ends that are generated via co-expression of I-SceI with Trex2.     

A Human Proteome Detection and Quantitation Project

The lack of sensitive, specific, multiplexable assays for most human proteins is the major technical barrier impeding development of candidate biomarkers into clinically useful tests. Recent progress in mass spectrometry-based assays for proteotypic peptides, particularly those with specific affinity peptide enrichment, offers a systematic and economical path to comprehensive quantitative coverage of the human proteome. A complete suite of assays, e.g. two peptides from the protein product of each of the ∼20,500 human genes (here termed the human Proteome Detection and Quantitation project), would enable rapid and systematic verification of candidate biomarkers and lay a quantitative foundation for subsequent efforts to define the larger universe of splice variants, post-translational modifications, protein-protein interactions, and tissue localization.There is growing interest in the idea of a comprehensive Human Proteome Project (1) to exploit and extend the successful effort to sequence the human genome. Major challenges in defining a comprehensive Human Proteome Project (and distinguishing it from the genome effort) are 1) the potentially very large number of proteins with modified forms; 2) the diversity of technology platforms involved in their study; 3) the variety of overlapping biological “units” into which the proteome might be divided for organized conquest; and 4) sensitivity limitations in detecting proteins present in trace amounts. The process of analyzing and discussing these issues may (and ought to) be lengthy, as it addresses core scientific unknowns as well as decisions about the organization and scale of biomedical research in the future. The benefits of taking time to involve the entire biological research community, and especially the medical research segment, in these discussions are substantial.Progress in systematically measuring proteins, however, need not wait for the conclusion of such discussions. We propose a near-term tactical approach, called the human Proteome Detection and Quantitation (hPDQ)¹ project that will enable measurement of the human proteome in a way that would yield immediately useful results while the strategy for a comprehensive Human Proteome Project is worked out. The hPDQ project is aimed at overcoming present difficulties in answering basic biological questions about the relationship between protein abundance (or concentration) and gene expression, phenotype, disease, and treatment response; i.e., the growing field of protein biomarkers. It is thus focused on the study of biological variation affecting protein expression rather than study of structure and mechanism and in this initial form does not directly address splice variants or most post-translational modifications. It is aimed at providing immediately useful capabilities to the human biology research community, in a way that does not adversely impact funding for individual investigators and does not generate administrative constraints on their ability to set and change courses in the conduct of research. Specifically, the goal of the hPDQ is to enable individual biological researchers to measure defined collections of human proteins in biological samples with 1 ng/ml sensitivity and absolute specificity, at throughput and cost levels that permit the study of meaningfully large biological populations (∼500–5,000 samples).We clearly do not have this capability today. If an investigator defines a set of 20 proteins hypothesized to change in relation to some biological process or event, assays for only a minority (often none!) will typically be available. Further, these assays will lack absolute specificity and will not easily be multiplexed. Current proteomics research platforms are focused mainly on discovery; providing increasingly broad protein sampling surveys, generally at low throughput and high cost. Such approaches generally do not yield an economical or accurate measurement of a defined set of proteins in every sample. There is thus a fundamental barrier to hypothesis testing in quantitative proteomics, where relationships between protein abundance and biology are sought. A particularly important instance of this limitation occurs in the effort to establish useful biomarkers of disease, for diagnosis, for measuring efficacy of treatment, and for monitoring of disease recurrence. This limitation is largely responsible for the research community''s failure in recent years to bring forward significant numbers of new proteins as Food and Drug Administration approved diagnostic tests (2). However, if a robust, economical, and widely diffused capability to measure all human proteins existed, the research community would have the collective means to assess the utility of all human proteins as biomarkers in hundreds of diseases and other processes in the most efficient way.The need for new or improved biomarkers in many areas of healthcare has become critical. Early detection of cancer, coupled with surgical intervention, has the potential to radically improve survival (3), provided early markers exist and can be found. Without good biomarkers, degenerative diseases such as Alzheimer and chronic obstructive pulmonary disease (COPD) are difficult to detect early enough to benefit from the potential therapies. Clinical development of new drugs increasingly depends on identification of biomarkers for pharmacodynamic assessment of drug action to help guide dose and schedule, and predictive biomarkers for selection of patients who will benefit from therapy (4). Companion diagnostics are the currency of personalized medicine and represent those predictive or response biomarkers that are linked to specific therapeutics, substantially increasing their clinical value. Surrogate biomarkers (those biomarkers that substitute for a clinical outcome or response) are the most difficult to discover and to verify because of the long timeframe required but can radically shorten appropriate clinical trials. The impact of a vigorous increase in clinical biomarkers could thus be enormous, both in terms of patient well being and financial viability of healthcare systems worldwide.Protein measurements are also likely to play an important role in assessing the quality of material stored in large clinical sample collections (Biobanks). Much discussion has occurred recently regarding the value of banked samples because of unknown degrees of protein degradation occurring during acquisition, processing, and storage. This matter is of acute concern in the case of serum, where coagulation initiates a plethora of proteolytic cleavage events. The hPDQ may provide the opportunity to determine the value of each sample through the development of prototypic peptides tracking the stability of labile proteins.An attractive technology for achieving the objective of hPDQ is quantitative mass spectrometry, the sensitivity, and specificity of which are well established in the measurement of small molecules (5,6) and peptides (7,8). To achieve comprehensive quantitation of proteins, given the immense variability in their physical properties, these larger molecules are digested to component peptides using an enzyme such as trypsin, and protein amount is measured using proteotypic peptides (9,10) as specific stoichiometric surrogates. Multiple peptides from a target protein provide independent confirmation of this stoichiometry (equivalent to having multiple enzyme-linked immunosorbent assays with different antibody pairs), serving to control for the possibility of incomplete digestion or subsequent losses. Accurate calibration is achieved by spiking digested samples with known quantities of synthetic stable-isotope labeled peptides as internal standards (11,12). The sensitivity of this approach for multiplexed analysis of proteins in plasma has been extended from the microgram (13) to nanogram/ml levels by depletion of abundant proteins and limited peptide fractionation prior to analysis (14) or by capture of the subset of glycopeptides (15). Sensitivity and throughput of peptide MS measurements can be further increased to levels required in hPDQ by specific enrichment of the target peptides using anti-peptide antibodies. This method, called SISCAPA (for “stable isotope standards and capture by anti-peptide antibodies”) (16) or iMALDI (for immuno-MALDI) (17), combines the enhanced sensitivity of immunoassays with the specificity of mass spectrometry, while maintaining multiplexing capability. For these reasons we emphasize SISCAPA and iMALDI in this hPDQ proposal, although proteins in the 100 ng/ml or higher concentration are readily accessible by targeted MS in plasma without antibody enrichment. Combining these elements results in a measurement system, with the potential to measure 10–100 selected proteins at ng/ml levels in small (∼10 μl) samples of human plasma in a single short analytical run. Sensitivity can be further increased through the use of larger samples and/or advances in MS sensitivity. In comparison to the conventional ELISA approach, MS-based SISCAPA assays are less expensive to develop (one antibody instead of a carefully matched pair), easier to multiplex (off-target interactions being less likely with peptides than proteins), and provide absolute structural specificity (by reading the masses of multiple specific peptide fragments). This improved specificity solves a major problem plaguing clinical immunoassays for proteins such as thyroglobulin (18) and has led to the development of first clinical SISCAPA assay (19). In addition, since the mass spectrometer functions as a “second antibody” that identifies the captured peptides, the anti-peptide antibody used for peptide enrichment need not have perfect specificity. This greatly reduces the cost of affinity reagents, currently a limiting factor in developing ELISA assays for large numbers of protein analytes.Achieving the hPDQ goal by this approach would require that four resources be generally available. 1) A comprehensive database of proteotypic (protein-unique) peptides for each of the 21,500 human proteins (20), coupled with experimental or computational data identifying the best peptides for MS measurement and associated optimized MS instrument parameters. 2) At least two synthetic proteotypic peptides, labeled with stable isotope(s) and available in accurately quantitated aliquots, for use as internal measurement standards for quantitation of each protein. Such peptides are readily available today through custom order, at rapidly declining prices. 3) Anti-peptide antibodies specific for the same two proteotypic peptides per target protein, capable of binding the peptides with dissociation constants < 1e-9 (the level required in theory and practice to enrich low-abundance peptides from complex sample digests). Such antibodies are now being made for a variety of targets, and a robust production pipeline is being developed. Monoclonal antibodies would be preferred, despite their higher development cost, to establish a stable reagent supply, especially for those targets that prove useful as biomarkers. 4) Robust and affordable instrument platforms for quantitative analysis of small (amol to fmol) amounts of tryptic peptides and for sample preparation. Existing triple-quadrupole mass spectrometers (with a current worldwide installed base of more than 6,000 instruments) coupled with nanoflow (∼300–600 nl/min) liquid chromatography systems can meet this requirement and are undergoing rapid improvement with declining cost. MALDI platforms may provide similar capabilities at even higher throughput.We estimate that an initial pilot phase targeting 2,000 proteins selected for biomarker potential could be completed in two years at a cost of less than $50 million through funding of existing academic and commercial resources in a distributed network. In the following five years, the remaining 18,500 proteins could be targeted for $250 million, making use of anticipated technical improvements, particularly in the strategies for generating suitable high affinity monoclonal antibodies (21) in large numbers at low cost (22).Although the natural mechanism for providing the hPDQ database (resource 1 above) is through an academic collaboration, perhaps modeled on the successful Global Protein Machine (23) and Peptide Atlas (24) databases, the other resources would benefit from commercial distribution by experienced providers of instruments and reagents. The required instrument platforms (4 above) serve existing markets, and their further development is unlikely to require additional funding for hPDQ applications. However, business economics does not presently justify the expense of developing well characterized antibodies and peptides for quantitation of proteins that are not already recognized as pivotal in biological research (i.e. precisely those in need of the attention of the research community). Hence a substantial portion of the required funding for the proposed approach for such antibody and peptide reagents will be needed from government and philanthropic sources. A significant advantage of such diversified support would be the leverage it would provide in retaining in the public domain the identities of the selected peptides, their parameters and basic measurement protocols.The value of a general protein measurement capability for research is very substantial, but the proposed effort would not solve several larger issues that must await definition of a broader human proteome program. For example, the hPDQ project does not address the basic process of de novo proteome-wide discovery; the comprehensive exploration of splice forms, post-translational modifications, active fragments of preproteins or genetic variants (although once known, most of these can be targeted by the methods used here); interactions among proteins or with other molecules; or spatial arrangement of proteins in organs and tissues. Each of these areas would benefit from the resources proposed in hPDQ, but will likely require separate, coordinated large-scale efforts that are likely to identify additional sets of biomarkers. Thus although a complete suite of targeted assays is only a first step toward the complete human proteome, we feel that its fundamental importance for progress in biomarker research and its value as a foundation for protein quantitation justifies consideration as an initial step.In the beginning of the study of protein diagnostics, investigators at the Behring Institute discovered many of the well known plasma proteins and made associated specific antibodies and antibody-based quantitative tests available to the research community worldwide, spurring the initial round of plasma biomarker research. The application of monoclonal antibodies sparked additional discoveries through close coupling of protein “discovery” with simple quantitative monoclonal antibody-based assays - this “shortcut” to clinical measurement allowed investigators to publish more than 1,000 papers referring to the ovarian cancer marker CA125 (measured by ELISA) before the sequence of the protein was finally identified in 2001 (25). The broader proteomics technologies (beginning with the two-dimensional electrophoresis technology that formed the basis of the Human Protein Index Project (26) formulated by two of us almost 30 years ago, and extending to modern shotgun-style MS-based approaches) have radically expanded the universe of observable proteins. However, quantitative specific assay capabilities have not kept pace with this expansion, leading to the current gap between biomarker proteomics and clinical biomarker output. It is now time to address this gap and realize the benefits of a clinically accessible human proteome. Effective translation of basic research into tangible medical benefit requires it.     

Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA⁺/VacA⁺ H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.     

Biphasic ethylene production during the hypersensitive response in Arabidopsis: A window into defense priming mechanisms?

The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H₂O₂, salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the “priming” of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.Key words: hypersensitive response, biphasic patterns, potentiation, defense priming, ethylene, ArabidopsisThe Hypersensitive Response (HR) is a cell death process which occurs at the site of attempted pathogen attack and which has been associated with host resistance.¹ Much work on the regulation of the HR has indicated the importance of H₂O₂,² and NO.³ A feature of H₂O₂ generation during the HR is its biphasic pattern (Fig. 1A). The first rise reflects elicitation by pathogen-associated molecular patterns (PAMPs)⁴ and the second reflects the interaction between a pathogen-encoded avirulence (avr) gene product with a plant resistance (R) gene. A key aspect of the first rise is the initiation of salicylic acid (SA) synthesis which potentiates the second rise and hence the potency of plant defense and the HR.⁵Open in a separate windowFigure 1Patterns of defense signal generation during the Pseudomonas syringae pv. phaseolicola elicited-hypersensitive response in tobacco (Nicotiana tabacum). Generation of (A) H₂O₂ (●, Mur¹⁸); (B) nitric oxide (◇; Mur¹² (C) salicylic acid (SA, ■¹⁹) and (D) ethylene (○ Mur⁹) during a HR elicited by Pseudomonas syringae pv. phaseolicola (Psph) in tobacco cv. Samsun NN. In (A) a phase where SA acts to augment the second rise in H₂O₂—the potentiation phase—is highlighted. The potentiation phase is likely to be similar to defense “priming”.⁶ Methodological details are contained within the appropriate references. (E) A possible model for biphasic defense signal regulation during the Psph-elicited HR in tobacco. During an initial phase NO and H₂O₂ act to initiate SA biosynthesis, where SA and NO act to initiate a “H₂O₂ biphasic switch”. This could initially suppress both SA and the H₂O₂ generation but subsequently acts to potentiate a second phase of H₂O₂ generation. This in turn increases SA biosynthesis which could act with NO to initiate the “C₂H₄ biphasic switch” to potentiate ethylene production. These (and other) signals contribute to initiation of the HR and SAR.This potentiation mechanism appears to be similar to defense priming; when whole plants display systemic resistance to disease as opposed to a localized resistance against pathogens. Priming can be initiated (the “primary stimulus”) following attack with a necrotizing pathogen (leading to “systemic acquired resistance”, SAR) or non-pathogenic rhizosphere bacteria (to confer “induced systemic resistance”, ISR). In the primed state a plant stimulates a range of plant defense genes, produces anti-microbial phytoalexins and deposits cell wall strengthening molecules, but only on imposition of a “secondary stimulus”.⁶ Such secondary stimuli include SA³ or PAMPs⁷ and is likely to be mechanistically similar to the potentiation step in the biphasic pattern of H₂O₂ generation (shaded in Fig. 1A). Accordingly, the two phases in the biphasic wave represent primary and secondary stimuli in priming.Highlighting a similarity between local HR-based events and priming, adds further impetus to efforts aiming to describe the underlying mechanism(s), however both phenomena remain poorly understood. Besides SA, both jasmonates and abscisic acid (ABA) have been shown to prime defenses as have a range of non-plant chemicals, with β-aminobutyric acid (BABA) being perhaps most widely used.^6,8 Mutants which fail to exhibit BABA-mediated potentiation were defective in either a cyclin-dependent kinase-like protein, a polyphosphoinositide phosphatase or an ABA biosynthetic enzyme.⁸We have recently investigated biphasic ethylene production during the HR in tobacco elicited by the nonhost HR-eliciting bacterial pathogen Pseudomonas syringae pv. phaseolicola.⁹ As with H₂O₂ generation, this pattern reflected PAMP-and AVR-dependent elicitation events and included a SA-mediated potentiation stage. Crucially, we also showed that NO was a vital component in the SA-potentiation mechanism. When this finding is integrated with our other measurements of defense signal generation in the same host-pathogen system the complexity in the signaling network is revealed (Fig. 1). NO generation (Fig. 1B) appeared to be coincident with the first rise in H₂O₂ (Fig. 1A) which initiated SA biosynthesis^10,11 and together would contribute to the first small, but transient, rise in that hormone (Fig. 1C). In line with established models⁵ this momentary rise in SA coincides with the potentiation phase (shaded in Fig. 1A) required to augment the second rise in ROS. However, ethylene production seems to be correlated poorly with the patterns of NO, H₂O₂ and SA (Fig. 1D). Nevertheless, biphasic ethylene production was found to reflect PAMP and AVR-dependent recognition and included a SA-mediated potentiation step.⁹ Hence, ethylene production could be used as a post-hoc indicator of the potentiation mechanism. Therefore, our discovery that the second wave of ethylene production—a “biphasic switch”—is influenced by NO acting with SA could also be relevant to the H₂O₂ generation. Significantly, the second phases in both H₂O₂ and ethylene production occur exactly where SA and NO production coincides; in the case of H₂O₂ generation 2–4 h post challenge and with ethylene 6 h onwards (Fig. 1E).Thus, ethylene production represents a readily assayable marker to indicate perturbations in the underlying biphasic and possible priming mechanisms. As we have demonstrated, laser photoacoustic detection (LAPD) is a powerful on-line approach to determine in planta ethylene production in tobacco^9,12 but any mechanistic investigations would be greatly facilitated if the genetic resources offered by the model species Arabidopsis could be exploited.To address this, Arabidopsis Col-0 rosettes were vacuum infiltrated with either Pseudomonas syringae pv. tomato (Pst) avrRpm1 (HR-eliciting), the virulent Pst strain and the non-HR eliciting and non-virulent Pst hrpA strain. Ethylene production was monitored by LAPD (Fig. 2A). Significantly, Pst avrRpm1 initiated a biphasic pattern of ethylene production whose kinetics were very similar to that seen in tobacco (compare Figs. 2A with ​with1D).1D). Inoculations with Pst and Pst hrpA only displayed the first PAMP-dependent rise in ethylene production. Thus, these data establish that Arabidopsis can be used to investigate biphasic switch mechanism(s) in ethylene production during the HR and possibly defense priming. When considering such mechanisms, it is relevant to highlight the work of Foschi et al.¹³ who observed that biphasic activation of a monomeric G protein to cause phase-specific activation of different kinase cascades. Interestingly, ethylene has been noted to initiate biphasic activation of G proteins and kinases in Arabidopsis, although differing in kinetics to the phases seen during the HR.¹⁴ Further, plant defense priming has been associated with the increased accumulation of MAP kinase protein.⁶Open in a separate windowFigure 2Ethylene in the Pseudomonas syringae pv. tomato elicited-hypersensitive response in Arabidopsis thaliana. (A) Ethylene production from 5 week old short day (8 h light 100 µmol.m².sec⁻¹) grown Arabidopsis rosette leaves which were vacuum infiltrated with bacterial suspensions (2 × 10⁶ colony forming units.ml⁻¹) of Pseudomonas syringae pv. tomato (Pst) strains detected using laser photoacoustic detection (LAPD). Experimental details of the ethylene detection by LAPD are detailed in Mur et al.⁹ The intercellular spaces in leaves were infiltrated with the HR-eliciting strain Pst avrRpm1, (■), the virulent strain Pst (△) or the non-virulent and non-HR eliciting derivative, Pst hrpA (◇). (B) The appearance of Arabidopsis Col-0 and etr1-1 leaves at various h following injection with 2 × 10⁶ c.f.u.mL⁻¹ with of Pst avrRpm1. (C) Explants (1 cm diameter discs) from Arabidopsis leaf areas infiltrated with suspensions of Pst avrRpm1 were placed in a 1.5 cm diameter well, bathed in 1 mL de-ionized H₂O. Changes in the conductivity of the bathing solution, as an indicator of electrolyte leakage from either wild type Col-0 (◆), mutants which were compromised in ethylene signaling; etr1-1 (□), ein2-2 (▲) or which overproduced ethylene; eto2-1 (●) were measured using a conductivity meter. Methodological details are set out in Mur et al.⁹A further point requires consideration; the role of ethylene as a direct contributor to plant defense.¹⁵ The contribution of ethylene to the HR has been disputed,¹⁶ but in tobacco we have observed that altered ethylene production influenced the formation of a P. syringae pv. phaseolicola elicited HR.⁹ In Arabidopsis, cell death in the ethylene receptor mutant etr1-1 following inoculation with Pst avrRpm1 is delayed compared to wild type (Fig. 2B). When electrolyte leakage was used to quantify Pst avrRpm1 cell death, both etr1-1 and the ethylene insensitive signaling mutant ein2-1 exhibited slower death than wild-type but in the ethylene overproducing mutant eto2, cell death was augmented (Fig. 2C). These data indicate that ethylene influences the kinetics of the HR.Taking these data together we suggest that the complexity of signal interaction during the HR or in SAR/ISR could be further dissected by combining the genetic resources of Arabidopsis with measurements of ethylene production using such sensitive approaches as LAPD.     

Development of Functional Human NK Cells in an Immunodeficient Mouse Model with the Ability to Provide Protection against Tumor Challenge

Studies of human NK cells and their role in tumor suppression have largely been restricted to in vitro experiments which lack the complexity of whole organisms, or mouse models which differ significantly from humans. In this study we showed that, in contrast to C57BL/6 Rag2^−/−/γ_c ^−/− and NOD/Scid mice, newborn BALB/c Rag2^−/−/γ_c ^−/− mice can support the development of human NK cells and CD56+ T cells after intrahepatic injection with hematopoietic stem cells. The human CD56⁺ cells in BALB/c Rag2^−/−/γ_c ^−/− mice were able to produce IFN-γ in response to human IL-15 and polyI:C. NK cells from reconstituted Rag2^−/−/γ_c ^−/− mice were also able to kill and inhibit the growth of K562 cells in vitro and were able to produce IFN-γ in response to stimulation with K562 cells. In vivo, reconstituted Rag2^−/−/γ_c ^−/− mice had higher survival rates after K562 challenge compared to non-reconstituted Rag2^−/−/γ_c ^−/− mice and were able to control tumor burden in various organs. Reconstituted Rag2^−/−/γ_c ^−/− mice represent a model in which functional human NK and CD56+ T cells can develop from stem cells and can thus be used to study human disease in a more clinically relevant environment.     

Fresh powder on Waddington's slopes

The Keystone Symposium on Stem Cell Differentiation and Dedifferentiation attracted nearly 500 participants affiliated with academia, the biotechnology industry, scientific publishing and clinical practice. This broad group examined current advances in stem‐cell biology, with a particular focus on induced pluripotent stem cells, and discussed both their potential and limitations in therapeutic applications.