A minimal cell can be thought of as comprising informational, compartment-forming and metabolic subsystems. Imagining the abiotic assembly of such an overall system, however, places great demands on hypothetical prebiotic chemistry. The perceived differences and incompatibilities between these subsystems have led to the widely held assumption that one or other subsystem must have preceded the others. Here, we have experimentally investigated the validity of this assumption by examining the assembly of various biomolecular building blocks from prebiotically plausible intermediates and one-carbon feedstock molecules. We show that precursors of ribonucleotides, amino acids and lipids can all be derived by reductive homologation of hydrogen cyanide and some of its derivatives and thus that all the cellular subsystems could have arisen simultaneously through common chemistry. The key reaction steps are driven by UV light, use hydrogen sulfide as reductant and can be accelerated by Cu(I)-Cu(II) photoredox cycling.Viewing the cell as an ensemble of subsystems 1 begs the question 'did the subsystems emerge together, or one after the other at the origin of life?' The consensus that sequential emergence is more likely 2 (though with opinions differing as to which subsystem came first [3][4][5] ) has been based on the notion that different, mutually incompatible chemistries are needed to make the various subsystems. We set out to explore this experimentally by evaluating the assembly chemistry of the various subsystems. Investigation of the assembly chemistry of an informational subsystem based on RNA led to our discovery of an efficient synthesis of activated pyrimidine ribonucleotides 6 . In this synthesis (Fig. 1a, bold, blue arrows), the C 2 sugar glycolaldehyde 1 undergoes phosphate-catalysed condensation with cyanamide 2 to give 2-aminooxazole 3. This heterocycle then participates in a C-C bond Reprints and permissions information is available online at www.nature.com/reprints. Additional informationSupplementary information and chemical compound information are available in the online version of the paper. Competing financial interestsThe authors declare no competing interests. forming reaction with the C 3 sugar glyceraldehyde 4 giving rise to a mixture of pentose aminooxazolines. Reaction of the arabino-configured aminooxazoline 5 with cyanoacetylene 6 then furnishes an anhydronucleoside 7 which on heating with phosphate in urea 8 -a by-product of the first step of the sequence -is transformed into ribo-cytidine-2′, 3′-cyclic phosphate 9. UV irradiation then partially converts this nucleotide into uridine-2′, 3′-cyclic phosphate 10 and destroys stereoisomeric impurities. Europe PMC Funders GroupWe subsequently showed that the C 2 and C 3 sugars, 1 and 4, can be sequentially provided by a Kiliani-Fischer-type homologation of hydrogen cyanide 11 using Cu(I)-Cu(II) photoredox chemistry (Fig. 1a, bold, green arrows) 8,9 . Using hydrogen sulfide 12 as the stoichiometric reductant -in which case the inclusion o...

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.

Histone modifications play an important role in the process of transcription. However, in contrast to lysine methylation, the role of arginine methylation in chromatin structure and transcription has been underexplored. The globin genes are regulated by a highly organized chromatin structure that juxtaposes the locus control region (LCR) with downstream globin genes. We report here that the targeted recruitment of asymmetric dimethyl H4R3 catalyzed by PRMT1 (protein arginine methyltransferase 1) facilitates histone H3 acetylation on Lys9/Lys14. Dimethyl H4R3 provides a binding surface for P300/ CBP-associated factor (PCAF) and directly enhances histone H3 acetylation in vitro. We show that these active modifications are essential for efficient interactions between the LCR and the ␤ maj -promoter as well as transcription of the ␤-globin gene. Furthermore, knockdown (KD) of PRMT1 by RNA interference in erythroid progenitor cells prevents histone acetylation, enhancer and promoter interaction, and recruitment of transcription complexes to the active ␤-globin promoter. Reintroducing rat PRMT1 into the PRMT1 KD MEL cells rescues PRMT1 binding, ␤-globin transcription, and erythroid differentiation. Taken together, our data suggest that PRMT1-mediated dimethyl H4R3 facilitates histone acetylation and enhancer/ promoter communications, which lead to the efficient recruitment of transcription preinitiation complexes to active promoters. (Blood. 2010;115:2028-2037 IntroductionCovalent modifications of N-terminal histone tails are critically involved in transcriptional activation and repression. 1 The interplay between individual modifications may exert distinct regulatory effects on different gene loci during development and cellular differentiation. For example, H3K9 and H3K27 methylations are generally linked to gene repression, whereas methylation of H3K4 correlates with transcriptionally active euchromatin. 2 However, in the ␤-globin locus H3K9 methylation was also detected in the active globin genes. 3 Arginine methylation of histones is associated with both transcriptional repression and activation. 4 PRMT6-mediated H3R2 dimethylation negatively regulates deposition of H3K4 trimethylation at active promoters, 5 whereas dimethyl H4R3 correlates with transcriptional activation. 6,7 Asymmetric dimethylation of H4R3 residues by protein arginine methyltransferase PRMT1 is essential in vitro and in vivo for the establishment or maintenance of active histone acetylation patterns. 7,8 The interdependence of these modifications appears to be important for the transcription of a p53-dependent reporter gene in a cell-free system with reconstituted chromatin templates. 9 Furthermore, PRMT1 was associated with the activation of HNF4 and HoxA9 genes during tissue development and oncogenesis, respectively. 10, 11 We showed recently that PRMT1 directly interacts with transcription factor USF1 (upstream regulatory factor 1), which has been implicated in chromatin barrier function and ␤-globin gene regulation. [12][13][14][15] The ...

Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are not currently available that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1 (that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not influence CDCP1 cleavage, but increased the association of E-cadherin with γ-catenin. This effect on E-cadherin/γ-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA) and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors, both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through novel, complementary mechanisms that converge on E-cadherin.

The central role of protein-protein interactions in a wide range of cellular processes makes them a target for research and drug discovery. A variety of methods, both experimental and theoretical, exist for probing protein interfaces for residues that affect activity and binding affinity. Using as an example a protein-protein complex between trypsin and a nine-residue synthetic peptide, we experimentally assay-binding affinities for a variety of mutants and determine their relative free energy of binding, ΔΔG, to rank the importance of interface residues to binding. We then compare how accurately, precisely and reliably computational methods for calculating ΔΔG can replicate these results. We find that a 'post-process alanine scanning' protocol of a single native complex trajectory gives results with better accuracy than running separate molecular dynamics (MD) trajectories for individual mutants. Compared across 10 independent simulations, we find that results from the post-process alanine scanning are also more precise and are obtained over five times faster than their equivalent with the 'full MD' protocol. These results suggest that, although not suitable in every case, post-process alanine scanning is a useful and reliable tool in predicting important residues at protein interfaces with potential for modulation.

Summary Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1+ mesoderm and then promotes hematopoietic differentiation through regulating hoxb gene pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated KD or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb gene expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1+ precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1+ precursors and differentiation of Flk1+ cells into hematopoietic lineages.

The main aim of origins of life research is to find a plausible sequence of transitions from prebiotic chemistry to nascent biology. In this context, understanding how and when phospholipid membranes appeared on early Earth is critical to elucidating the prebiotic pathways that led to the emergence of primitive cells. Here we show that exposing glycerol-2-phosphate to acylating agents leads to the formation of a library of acylglycerol-phosphates. Medium-chain acylglycerol-phosphates were found to self-assemble into vesicles stable across a wide range of conditions and capable of retaining mono- and oligonucleotides. Starting with a mixture of activated carboxylic acids of different lengths, iterative cycling of acylation and hydrolysis steps allowed for the selection of longer-chain acylglycerol-phosphates. Our results suggest that a selection pathway based on energy-dissipative cycling could have driven the selective synthesis of phospholipids on early Earth.

BackgroundSurgical procedures are now very common, with estimates ranging from 4% of the general population having an operation per annum in economically-developing countries; this rising to 8% in economically-developed countries. Whilst these surgical procedures typically result in considerable improvements to health outcomes, it is increasingly appreciated that surgery is a high risk industry. Tools developed in the aviation industry are beginning to be used to minimise the risk of errors in surgery. One such tool is the World Health Organization's (WHO) surgery checklist. The National Patient Safety Agency (NPSA) manages the largest database of patient safety incidents (PSIs) in the world, already having received over three million reports of episodes of care that could or did result in iatrogenic harm. The aim of this study was to estimate how many incidents of wrong site surgery in orthopaedics that have been reported to the NPSA could have been prevented by the WHO surgical checklist.MethodsThe National Reporting and Learning Service (NRLS) database was searched between 1st January 2008- 31st December 2008 to identify all incidents classified as wrong site surgery in orthopaedics. These incidents were broken down into the different types of wrong site surgery. A Likert-scale from 1-5 was used to assess the preventability of these cases if the checklist was used.Results133/316 (42%) incidents satisfied the inclusion criteria. A large proportion of cases, 183/316 were misclassified. Furthermore, there were fewer cases of actual harm [9% (12/133)] versus 'near-misses' [121/133 (91%)]. Subsequent analysis revealed a smaller proportion of 'near-misses' being prevented by the checklist than the proportion of incidents that resulted in actual harm; 18/121 [14.9% (95% CI 8.5 - 21.2%)] versus 10/12 [83.3% (95%CI 62.2 - 104.4%)] respectively. Summatively, the checklist could have been prevented 28/133 [21.1% (95%CI 14.1 - 28.0%)] patient safety incidents.DiscussionOrthopaedic surgery is a high volume specialty with major technical complexity in terms of equipment demands and staff training and familiarity. There is therefore an increased propensity for errors to occur. Wrong-site surgery still occurs in this specialty and is a potentially devastating situation for both the patient and surgeon. Despite the limitations of inclusion and reporting bias, our study highlights the need to match technical precision with patient safety. Tools such as the WHO surgical checklist can help us to achieve this.