In treating patients with castration resistant prostate cancer (CRPC), enzalutamide, the second-generation androgen receptor (AR) antagonist, is an accepted standard of care. However, clinical benefits are limited to a median time of 4.8 months because resistance inevitably emerges. To determine the mechanism of treatment resistance, we carried out a RNA sequence analysis and found increased expression levels of neuroendocrine markers in the enzalutamide-resistant LNCaP human prostate cancer (CaP) cell line when compared to the parental cell line. Subsequent studies demonstrated that Transcription Factor-4 (TCF4), a transcription factor implicated in WNT signaling, mediated neuroendocrine differentiation (NED) in response to enzalutamide treatment and was elevated in the enzalutamide-resistant LNCaP. In addition, we observed that PTHrP mediated enzalutamide resistance in tissue culture and inducible TCF4 overexpression resulted in enzalutamide-resistance in a mouse xenograft model. Finally, small molecule inhibitors of TCF4 or PTHrP partially reversed enzalutamide resistance in CaP cells. When tissues obtained from men who died of metastatic CaP were examined, a positive correlation was found between the expression levels of TCF4 and PTHrP. Taken together, the current results indicate that TCF4 induces enzalutamide resistance via NED in CaP. Prostate cancer (CaP) is the most common non-cutaneous cancer diagnosed among men and the second leading cause of male cancer deaths in the United States . In 2017Enzalutamide is a FDA-approved second-generation androgen receptor (AR) antagonist that blocks ligand binding, nuclear translocation, DNA binding, and coactivator recruitment of ARs . In multTCF4, also known as transcription factor 7-like 2 (TCF7L2), is an important effector of the canonical WNT signaling pathway . AlthougHuman prostate cancer cell lines, LNCaP, 22Rv1, and VCaP were obtained from the American Type Culture Collection  and maintained in the standard culture media: RPMI-1640 supplemented with 10% fetal bovine serum (FBS). There are several prostate cancer cell lines which are AR positive. Among them, C4-2B cell line is derivative of LNCaP. Therefore, we selected LNCaP, 22Rv1, and VCaP cell lines. TCF4 cDNA  was cloned into pLenti4/V5-Dest expression vector . After making pLenti4/V5-Dest/TCF4 viral supernatant with the Optimized Packaging Mix, the supernatant was added to the Virapower T-Rex LNCaP. After selection with Zeocin and Blasticidin, TCF4-inducible LNCaP (TCF4/LNCaP) were selected and the expression of TCF4 was screened using quantitative PCR (QPCR). Selected clones were cultured in RPMI-1640/10% FBS media containing 100 mg/ml of Blasticidin and Zeocin . To establish enzalutamide resistant CaP cell lines, cells were treated continuously with 10μM enzalutamide . After three months, stable cell lines were established . Unless specified, the standard culture media for these three enzalutamide-resistant cell lines included 10 μM enzalutamide. The β-catenin degradation activator was purchased from .2 /0.361. Doxycycline-inducible TCF4-expressing cells were subcutaneously injected into five mice per group. Mice were supplied with water containing 2 mg/ml of doxycycline  in 5% sucrose to induce TCF4.All mice and experimental procedures were conducted using protocols approved by and in accordance with the Rutgers Cancer Institute of New Jersey Institutional Animal Care and Use Committee approval (PROTO999900168) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For anesthesia, 3% isoflurane gas inhalation method was used. Rag2-/-, γc-/- mice were purchased from The Jackson Laboratory . The study was approved by the Institutional Animal Care and Use Committee at the Rutgers University . Where indicated, surgical castration was carried out via bilateral orchiectomy. Tumor size was measured using calipers and tumor volume was calculated using the formula: tumor volume = length x widthc-/- immunodeficient mice. When tumors reached an average size of 3 mm in diameter, all animals were surgically castrated and divided into four groups of ten mice each. TCF4/β-catenin interaction was disrupted with PKF118-310 while PTHrP was blocked with PTHrP(7–34). PKF118-310 was purchased from Millipore  and PTHrP(7–34) Bachem . Mice were injected daily with the PKF118-310  and/or PTHrP(7–34)  for four weeks. Where indicated, enzalutamide was administered daily via oral gavage at 10 mg/kg in 1% carboxymethyl cellulose, 0.1% Tween-80, and 5% DMSO [2 asphyxiation after PKC118-310 and/or PTHrP(7–34) treatment were completed.To explore the therapeutic implications of targeting TCF4/NED pathway, LNCaP-EnzR was injected into forty Rag2-/-, γ 5% DMSO –17. TumoRNA was purified using DirectZol RNA purification kit  from LNCaP, charcoal stripped FBS-resistant LNCaP (LNCaP-csFBSR), and LNCaP-EnzR. Then, RNA sequencing was performed by Macrogen, Inc.  using purified RNA.One μg of a plasmid containing TCF4 cDNA  was transfected into indicated prostate cancer cell lines such as LNCaP, 22Rv1, and VCaP on a 6-well plate. Three μl of lipofectamine 3000  was used for each transfection.GAAGAGGAGGAGGAGGAGGA, reverse: CACTCAGGCCCTTCTCTCTG); human β-actin ; human TCF4 ; human NSE ; human PTHrP .Total RNA was isolated with TRIzol LS reagent , and 1–2 μg of total RNA was used for synthesizing cDNA with High-Capacity cDNA Reverse Transcription Kit . cDNA was then used for QPCR in a StepOnePlusTM  with SYBR Green ROX QPCR Mastermix . Sequences of PCR primers used are as follows: human CHGA  containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were centrifuged and protein in the supernatant was measured. After separating of 25–50μg protein using SDS-PAGE, samples were incubated with TCF4 , CHGA , NSE , PTHrP , POU2F2  or β-actin  antibodies. For CHGA, NSE, PTHrP, and POU2F2 immunoblots, 1:1000 diluted antibody solutions in 5% skim milk was used. For the β-actin immunoblot, 1:10000 diluted antibody solution was used. All membranes were incubated overnight at 4°C. Following the incubation with appropriate secondary antibody, immunoblot was analyzed using SuperSignal West Femto Maximum Sensitivity Substrate .CaP cells were collected and lysed with lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM NaTCF4 MISSION shRNA was purchased from Sigma-Aldrich . TCF4 shRNA lentiviral supernatant was generated with ViraPower Lentiviral Packaging Mix  and used to infect LNCaP cells. The expression of TCF4 was analyzed using QPCR and immunoblot as described above.CRPC tissue microarray (TMA) was obtained from the University of Michigan’s rapid autopsy program. The rapid autopsy program was approved by the Institutional Review Board of University of Michigan and supported by Specialized Program of Research Excellence in Prostate Cancer  and the Prostate Cancer Foundation  (This prIn vitro experiments were repeated a minimum of three times. Continuous variables were expressed as mean ± standard deviation (SD).Statistical significance was calculated using the Student’s t-test for paired comparisons of experimental groups and, where appropriate, by Wilcoxon rank sum test, and by 2-way ANOVA. To investigate the mechanism of enzalutamide resistance in CaP cells, an enzalutamide-resistant human CaP cell line was initially generated by continuously treating LNCaP with 10 μM enzalutamide in RPMI-1640 supplemented with 10% charcoal stripped fetal bovine serum (csFBS). After three months, cells began to proliferate consistently and were designated as LNCaP-EnzR. Simultaneously, LNCaP cultured chronically under csFBS was also generated (LNCaP-csFBS). With these cells, RNA seq followed by an unsupervised data analysis was carried out. When compared to the LNCaP-csFBS, there was no obvious differences in AR signaling related gene expression levels. However, NED markers such as chromogranin A (CHGA), neuron-specific enolase (NSE), and PTHrP were significantly higher in LNCaP-EnzR . A similhttp://alggen.lsi.upc.es/). This effort identified consensus sequences for binding of five transcription factors within the promoters of NED markers: TCF4, POU2F2 (Oct2.1), MRF-2, LCR-F1, and MBF-1 (EDF-1). Quantitative PCR (QPCR) confirmed that mRNA levels of TCF4 and POU2F2 were significantly higher in LNCaP-EnzR when compared to the parental cell line  [(7–34) significantly decreased the cellular proliferation of all three cell lines in a concentration-dependent manner over a three-day period. Between the two inhibitors, PTHrP(7–34) had a more moderate effect. In the three enzalutamide-resistant cell lines , PKF118-310 again inhibited the cellular proliferation in a concentration-dependent manner up to 50 μM after 3 days ((7–34) with/without 10uM enzalutamide treatment. In the presence of 10 μM Enz, PKF118-310 or PTHrP(7–34) increased enzalutamide sensitivity in the enzalutamide resistant prostate cancer cell lines, LNCaP-EnzR, and parental cell line, LNCaP ((7–34) decreased cell growth compared with single treatment of PKF118-3130 or PTHrP(7–34) ((7–34) treatment also decreased cell count of LNCaP-EnzR, 22Rv1-EnzR, and VCaP-EnzR in a concentration-dependent manner up to 1 mM after 3 days. To assess whether PFK118-310 and PTHrP(7–34) affected enzalutamide sensitivity, we next cultured the three enzalutamide-resistant cell lines with a fixed concentration of PKF118-310 (5 μM) or PTHrP(7–34) (10 μM) and varying concentrations of enzalutamide (0–10 μM). The results demonstrated that both PKF118-310 and PTHrP(7–34) reversed enzalutamide resistance in LNCaP-EnzR, 22Rv1-EnzR, and VCaP-EnzR . Then, of the NED markers, we focused on PTHrP as a potential mediator of enzalutamide resistance because this peptide has been demonstrated to mediate castration resistance , 20. Con(7–34) , 25. Botr 3 days  Figs. Ine, LNCaP . FurtherrP(7–34) . SimilarCaP-EnzR . It shouTo clinically validate these observations, we next carried out immunofluorescence (IF) microscopy on the CRPC tissue microarray (TMA) obtained from the University of Michigan’s rapid autopsy program. Although this CRPC TMA was established prior to the formal approval of enzalutamide by the United States Food and Drug Administration, co-localization of PTHrP and TCF4 was frequently observed in CRPC tissues . PTHrP ain vivo, we established a doxycycline-inducible TCF4-expressing cell line, LNCaP-TCF4. After screening multiple clones, the one with the highest induction level of TCF4 on treatment with 1 μg/ml of tetracycline was selected and further characterized , or both in combination were delivered daily. At the end of seven weeks, the results demonstrated that PKF118-310 and PTHrP(7–34) dramatically when combined with enzalutamide decreased the tumor xenograft growth dramatically compared to that of enzalutamide monotherapy (control) over a seven-week period ((7–34). Again, H&E staining showed no significant changes in the histology of the treated xenografts ((7–34) treatment again had no effect on TCF4 expression. Also consistent with the mechanism of action of PKF118-310 in which the interaction between TCF4 and β-catenin is disrupted, PKF118-310 treatment did not alter the expression levels of TCF4. These observations collectively support our concept that TCF4 is an upstream signaling molecule of PTHrP. Supporting the immunofluorescence MFI result, immunoblot revealed a decrease in PTHrP protein levels following PKF118-310 but not PTHrP(7–34) treatment ((7–34) administration. Result of the mRNA quantitation for TCF4 and PTHrP was consistent with that of immunoblot  as a monotherapy  at the same dosage had a more moderate effect on the growth of LNCaP-EnzR xenografts over a six-week period , TCF4 and PO2F2 mRNA expression level were higher than the parental cell line (LNCaP and VCaP).(TIF)Click here for additional data file.S2 Fig(7–34) (0–1000 μM) for 72 hours and viable cells were counted. Both PKF118-310 and PTHrP(7–34) inhibited cellular proliferation in a concentration-dependent manner.Three androgen sensitive prostate cancer cell lines  were treated with indicated concentrations of PKF118-310 (0–50 μM) or PTHrP(TIF)Click here for additional data file.S3 FigA. Three enzalutamide-sensitive human prostate cancer cell lines  and B. three enzalutamide-resistant human prostate cancer cell lines  were treated with 10 μM enzalutamide and increasing concentrations of PKF118-310 and PTHrP(7–34) as indicated. After 48 hours, viable cells were counted. In the presence of 10 μM Enz, PKF118-310 or PTHrP(7–34) increased enzalutamide sensitivity in the enzalutamide resistant prostate cancer cell lines, LNCaP-EnzR, VCaP-EnzR, and 22Rv1-EnzR.(TIF)Click here for additional data file.S4 FigA. Selected TCF4-LNCaP cell clones were treated with tetracycline and TCF4 expressions were confirmed with QPCR. Clone 3, demonstrating the highest level of induction of TCF4 on treatment with tetracycline (1 μg/ml), was selected for further characterization. Con = vehicle. B. The effect of TCF4 on proliferation of clone #3 was assessed. Over a 4-day period, there was a slightly faster proliferation rate when TCF4 was induced via tetracycline but the difference was not statistically significant. Con = vehicle. C. The effect of TCF4 induction on enzalutamide resistance was assessed in clone #3. On induction of TCF4, there was a relative resistance to enzalutamide (0 to 10 μM) in a concentration-dependent manner over a 72-hour period. Con = vehicle. D. After inducing TCF4 expression with tetracycline for 72 hours, mRNA expression levels of neuroendocrine markers  were measured via QPCR. The results demonstrated an increase in neuroendocrine differentiation following TCF4 induction.(TIF)Click here for additional data file.S5 Figc-/- immunodeficient mice, all mice underwent a bilateral orchiectomy. Subsequently, all animals were divided into four groups of five each and treated with vehicle (con), enzalutamide , PKF118-310 , or PTHrP(7–34) (0.2 mg/kg daily subcutaneous) as indicated. A. PKF118-310 or PTHrP(7–34) moderately decreased tumor growth compare with vehicle treatment control group. Enzalutamide treatment demonstrated a slight inhibitory effect on tumor growth but this was not significant. B. H&E staining. There was no difference among all groups. C. Immunofluorescence staining for TCF4 (green), PTHrP (red) with DAPI (blue) staining. Again, only the PKF118-310 treatment decreased PTHrP protein levels. There was no significant effect on TCF4 protein levels with either PFK118-310 or PTHr(7–34). D. Immunoblot and E. QPCR demonstrated that treatment with PKF118-310 decreased PTHrP protein and mRNA expression. Error bars indicate average ± SE and * p-value<0.05.After injection of LNCaP-EnzR cell line into the flanks of twenty Rag2-/-, γ(TIF)Click here for additional data file.


Orbivirus , Dianthovirus  and Alphainfluenzavirus . Despite their distinct genomic features and diverse host ranges  each of these viruses uses trans-acting RNA–RNA interactions (tRRIs) to facilitate co-packaging of their segmented genome. The tRRIs occur between different viral genome segments and direct the selective packaging of a complete genome complement. Here we explore the current state of understanding of tRRI-mediated co-packaging in the abovementioned viruses and examine other known and potential functions for this class of RNA–RNA interaction.RNA viruses represent a large and important group of pathogens that infect a broad range of hosts. Segmented RNA viruses are a subclass of this group that encode their genomes in two or more molecules and package all of their RNA segments in a single virus particle. These divided genomes come in different forms, including double-stranded RNA, coding-sense single-stranded RNA, and noncoding single-stranded RNA. Genera that possess these genome types include, respectively,  RNA viruses are important pathogens that infect a wide variety of hosts. The RNA genomes of these viruses can exist in different forms . Those ttrans-acting RNA–RNA interactions (tRRIs), could also facilitate co-packaging. Indeed, the latter strategy appears to be involved in this process for a number of segmented RNA viruses . B. B37]. BCollectively, these results provide a solid foundation for identifying additional BTV interactions and exploring the assembly pathways involved. Future studies will need to integrate all of the existing validated tRRIs into a unified model. Currently, most of the larger segments have not been analyzed extensively, thus their proposed roles in later stages of tRRI network assembly warrant further investigation. Also, it remains to be determined whether accurate assembly requires a tRRI network that includes all ten segments and, if so, how is a complete ensemble of ten “sensed” by the virus.Rotavirus is in the same Sedoreovirinae subfamily as orbiviruses, but these viruses contain eleven dsRNA genome segments. Similar to BTV, in vitro studies on rotaviruses (RVs) support the involvement of mRNA tRRI networks in segment packaging [The genus ackaging ,40, howeackaging . In vitrackaging . It has ackaging . VP6 of ackaging . InsteadTombusviridae is a large plant virus family whose members contain (+) RNA genomes [Dianthovirus. Red clover necrotic mosaic virus (RCNMV) is the best characterized species in this genus [ genomes . All memis genus . Its genis genus . RNA2 coAlthough RNA1 is capable of genome replication on its own, the transcription of the sg mRNA during infections requires the presence of both RNA1 and RNA2, signifying a communication requirement between the viral genome segments . Furthertrans-activator (TA). Base pairing of this TA loop with its complementary partner sequence in RNA1, the trans-activator binding site (TABS), forms a higher-order bimolecular RNA structure that halts progression of the RdRp , which can affect both RNA-protein and RNA–RNA interactions . FinallyA variety of approaches including computational, biochemical, genetic, and cellular techniques have been used to identify and study the functions of viral tRRIs. The initial identification of putative interacting genome segments and corresponding complementary sequences involved computer-assisted RNA structural analyses , RNA–RNAThe tRRIs that form in different viruses are predicted to be disrupted at different stages after assortment. In BTV, inter-mRNA links are presumably lost when the messages are converted to dsRNA genomes during virion assembly, making them relatively transient. In contrast, tRRIs in IAV are maintained in virions and may even persist following fusion-mediated delivery into the cytosol of newly infected cells, possibly mediating codelivery of vRNPs to the nucleus . For RCNThe presence of tRRIs in coding regions is also relevant to experiments that assess virus protein function by modifying its corresponding coding sequence, as substitutions or deletions can concomitantly disrupt network connections. The same knowledge may also be pertinent to certain applications, such as optimizing the design of expression vectors by integrating appropriate partner tRRIs with introduced foreign genes. As one would expect, tRRIs also influence the efficiently of reassortment, as demonstrated experimentally for IAV . LikewisThe study of tRRIs in segmented RNA viruses is a new and growing field in virology. Understanding the roles of this class of interaction will provide important basic knowledge on how selective packaging occurs in segmented RNA viruses. Information on tRRIs will also be useful in assessing the likelihood of new viral strains emerging via reassortment, and could guide the development of antiviral treatments based on inhibition of these interactions. Moreover, as observed for RCNMV, the possibility exists that the functions for tRRIs extend beyond their established role in genome assortment. As the field expands, it will be interesting to see if novel functional tRRIs are active in other types of segmented RNA viruses.


Nidovirales stands out for including viruses with the largest RNA genomes currently known. Nidoviruses employ a complex RNA-synthesizing machinery comprising a variety of non-structural proteins (nsps). One of the postulated drivers of the expansion of nidovirus genomes is the presence of a proofreading 3′-to-5′ exoribonuclease (ExoN) belonging to the DEDDh family. ExoN may enhance the fidelity of RNA synthesis by correcting nucleotide incorporation errors made by the RNA-dependent RNA polymerase. Here, we review our current understanding of ExoN evolution, structure, and function. Most experimental data are derived from studies of the ExoN domain of coronaviruses (CoVs), which were triggered by the bioinformatics-based identification of ExoN in the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) and its relatives in 2003. Although convincing data supporting the proofreading hypothesis have been obtained, from biochemical assays and studies with CoV mutants lacking ExoN functionality, the features of ExoN from most other nidovirus families remain to be characterized. Remarkably, viable ExoN knockout mutants were obtained only for two CoVs, mouse hepatitis virus (MHV) and SARS-CoV, whose RNA synthesis and replication kinetics were mildly affected by the lack of ExoN function. In several other CoV species, ExoN inactivation was not tolerated, and knockout mutants could not be rescued when launched using a reverse genetics system. This suggests that ExoN is also critical for primary viral RNA synthesis, a property that poorly matches the profile of an enzyme that would merely boost long-term replication fidelity. In CoVs, ExoN resides in a bifunctional replicase subunit (nsp14) whose C-terminal part has (N7-guanine)-methyltransferase activity. The crystal structure of SARS-CoV nsp14 has shed light on the interplay between these two domains, and on nsp14’s interactions with nsp10, a co-factor that strongly enhances ExoN activity in vitro assays. Further elucidation of the structure-function relationships of ExoN and its interactions with other  members of the CoV replication machinery will be key to understanding the enzyme’s role in viral RNA synthesis and pathogenesis, and may contribute to the design of new approaches to combat emerging nidoviruses.Among RNA viruses, the order  RNA viruses typically exhibit a high mutation frequency. This intrinsic biological property facilitates rapid adaptation of the virus to changing circumstances, a major contributor to the frequent outbreaks of mutated or newly emerging RNA viruses in humans, livestock, and other host organisms. The poor fidelity of RNA virus genome replication is attributed primarily to the fact that errors made by the viral RNA-dependent RNA polymerase (RdRp) go uncorrected. This lack of proofreading results in “quasispecies” populations of closely related viral genomes that are subject to continuous natural selection. As the accumulation of an excessive number of deleterious mutations can result in “error catastrophe,” the low fidelity of their replication is thought to have restricted genome size, which for most RNA virus families is (well) below 15 kilobases . This evNidovirales, an order of positive-stranded RNA (+ RNA) viruses that includes the coronavirus (CoV) family as its best-studied taxon. Recent nidovirus additions  and helicase enzymes . These fThe advent of metagenomics has taken our understanding of nidovirus host and genome diversity to the next level , even thThe rapid expansion of the nidovirus order has highlighted the strict conservation of an array of five “core replicase” domains: (i) the main (or “3C-like”) protease, (ii) the nidovirus RdRp-associated nucleotidyl transferase (NiRAN), (iii) the RdRp, (iv) a Zn-binding domain (ZBD), and (v) the superfamily 1 helicase domain (HEL1), with which the ZBD is always associated . When prAs mentioned above, for most of the novel metagenomics-derived nidoviruses we only know genome sequences, and their replication properties and enzymes have remained biologically uncharacterized thus far. This is clearly different for CoVs, which have a track record as an important group of (zoonotic) human and veterinary pathogens. With some exceptions , also thnidus = nest), is a prominent CoV feature that drives the expression of the genes located downstream of ORF1b, encoding structural and accessory proteins  replicative enzyme functions. Increasingly supported by the availability of structural information, several key CoV enzymes were probed by site-directed mutagenesis, either using in vitro assays or by launching engineered mutant CoV genomes from cloned cDNA templates. The CoV replicase subunits nsp7 to nsp14 and nsp16 are most intimately associated with viral RNA synthesis, either as enzymatic entity or as important co-factor . Key plews, see . Severalews, see , processews, see . Severalews, see . ClearlyThe C-terminal two-thirds of the CoV nsp12 subunit are occupied by a canonical RdRp domain containing the commonly encountered motifs A to F . ConservA structural prediction of the nsp12-RdRp domain was described as early as 2003 , but a cde novo initiation were reported [reviewed in  nsp7 and nsp8, despite being structurally similar to their SARS-CoV equivalents, a higher-order complex quite different from the hexadecamer was described: a heterotrimer consisting of two copies of nsp7 and a single copy of nsp8  229E could not establish nsp8-associated primase or RdRp activities, but instead revealed a 3′-terminal adenylyl transferase activity that may serve to equip viral transcripts with their 3′-poly(A) tail (Early leotides . This actitution . Most re(A) tail . AlthougEscherichia coli DNA polymerase I  5′-terminal cap structure of CoV mRNAs, a modification that is critical for mRNA recognition by the cellular translation machinery -methyltransferase (N7-MTase) activity . Followiachinery . The bimachinery  and seveachinery . The latCrystal structures of SARS-CoV nsp14 in complex with its nsp10 co-factor (PDB entries 5C8U and 5NFY) revealed several unique structural and functional features . Below wThe CoV ExoN domain was originally identified on the basis of sequence similarities with distant cellular homologs  and clasSaccharomyces cerevisiae, which could rescue a mutant yeast strain lacking the native N7-MTase (S-adenosyl methionine (SAM) as methyl donor  activity (in vitro mismatch excision activity was reported recently for the ExoN domain of the bafinivirus white bream virus (WBV), using dsRNA substrates containing up to three mismatches  and single-stranded (ss) RNA substrates in the 3′-to-5′ direction, with a preference for dsRNA substrates . ExoN acactivity . Mutagenactivity , 2014. Tactivity . Furtheractivity . Importaactivity . A more activity . Howeveractivity . Comparasmatches . These ain vitro studies revealed that both the ExoN and N7-MTase domains of nsp14 are involved , may function as a proofreading enzyme that preferentially targets 3′-terminal single mismatches. Indeed, SARS-CoV nsp14 and the nsp7/nsp8/nsp12 complex were able to associate with each other, while retaining their RNA synthesis, N7-MTase, and exoribonuclease activities . Althouginvolved . Recent involved . Using ainvolved . Strikininvolved . Similarinvolved .in vitro single-nucleotide incorporation assays the SARS-CoV RdRp complex  displayed a lower fidelity than the RdRp of dengue virus, a flavivirus with a three-fold smaller genome (in vitro comparison of the properties of (distantly related) viral RdRps is not straightforward. Moreover, our perception is “fragmented” (at best…) when it comes to the biochemical evolution of RdRp and ExoN features following the postulated acquisition of the latter by an ancestral nidovirus , the above data strongly suggest that ExoN contributes to the fidelity of CoV RNA synthesis. It is striking, that r genome . Clearlyidovirus . ExoN acidovirus . This woidovirus  and  SARS-CoV backbone , but that the mutant genome accumulated to about 10-fold lower levels later in infection (24 h p.i.), possibly due to the rapid accumulation of unfavorable mutations  . SimilarE. coli polymerase I  play a more direct and basic role in CoV RNA synthesis than merely safeguarding the long-term fidelity of replication.in vitro produced full-length RNA into the cytosol of BHK-21 cells, thus providing an equal environment for the first, critical rounds of replication in a cell line that is known to be compromised in its innate immune response  to various degrees of impairment, with residual RNA production supporting the generation of infectious progeny only in the case of MHV and SARS-CoV. For both these betacoronaviruses, in spite of their “mutator phenotype,” the long-term consequences of ExoN inactivation seem limited during propagation in cell culture. Viral RNA synthesis might indeed be expected to tolerate, at least to a certain extent, the inactivation of a proofreading activity that was postulated to not be directly required for RdRp activity, but to merely boost the overall quality of the replication process. Clearly, when replicating in the absence of a functional ExoN, deleterious mutations would first have to accumulate before viral fitness would begin to decrease. This does not appear to be the case for a third betacoronavirus, MERS-CoV, and for two alphacoronaviruses, HCoV-229E and TGEV, for which the immediate (full to strong) disruption of viral RNA synthesis was observed when ExoN-knockout mutants were launched by transfection of full-length RNA or DNA. In our opinion, technical variations are unlikely to explain these viability differences: with the exception of TGEV, ExoN knockout mutants were commonly launched by electroporation of response . During Among the CoVs investigated thus far, MHV and SARS-CoV (in our experience) do stand out as the two viruses exhibiting the most robust RNA synthesis and overall replication in cultured cells. Possibly, the replication activity of ExoN-deficient mutants somehow needs to cross a certain “threshold” to result in infectious progeny, and for ExoN-deficient mutants this is only achieved with the CoVs that replicate most efficiently. However, when considering the phenotypic differences of knockout mutants, in terms of virus viability and sensitivity to mutagenic agents in cell culture, it remains difficult to reconcile the reported 1- to 2-log reduction of progeny titers for ExoN-knockout MHV and SARS-CoV with the complete loss of infectious progeny reported for the ExoN-knockout mutants of various other CoVs. It is also relevant to consider the fact that low levels of residual enzymatic activity of ExoN active site mutants may go unnoticed in biochemical assays, but could still support a certain level of replication when launching the RNA of an ExoN-knockout CoV mutant. Despite the conservation of ExoN among CoVs and most other nidoviruses, the extent to which particular mutations affect enzymatic activity can only be assessed when studying these specific viral proteins in a biochemical assay , 2018.Interactions with the host’s innate immune system have been suggested to co-determine the phenotype of ExoN-knockout CoV mutants . It has Several cellular interferon-stimulated gene products have been implicated in the hypermutation of viral genomes, so it remains to be established how directly the properties of ExoN mutants are determined by a lack  or surmiCurrently, there are no FDA-approved antiviral drugs for the treatment of CoVs, which is mainly due to limited interest from the side of the pharmaceutical industry, despite the loss of human lives during the short-lived SARS outbreak and the continuing MERS epidemic. Moreover, antiviral hits identified so far often suffered from poor selectivity indexes. Drug development efforts were further restricted by the limitations of available animal models and potency failure in clinical trials . Taking in vivo and in vitro  or may inhibit virus replication indirectly, for example by inducing lethal mutagenesis or perturbing intracellular nucleoside triphosphate pools. The ExoN proofreading function might counteract these compounds mode of action and, in order to circumvent this, a nucleoside would need to be incorporated more efficiently than it will be excised by ExoN, or should be resistant to ExoN-mediated removal . Recentlin vitro . Comparein vitro .in vitro: adenosylhomocysteine, aurintricarboxylic acid (ATA), and sinefugin  to the largest RNA virus genomes currently known . Upon its discovery, promoted by the relationship with other proofreading exonucleases, the ExoN domain was postulated to have played an important role in nidoviral evolution and genome expansion  is a remarkable issue. In this case, it appears to be particularly challenging to integrate the results from biochemistry, structural biology, (reverse) genetics, and the analysis of CoV-infected cells into a coherent model of ExoN function. It might also be instructive to reassess the increased mutation frequency and evolution of ExoN-knockout mutants using more advanced deep-sequencing methods that have been developed during recent years .in vitro, it is still quite unclear how these findings can be extrapolated to the viral enzyme complex in the infected cell  and their co-factors  now have been characterized ted cell . The impted cell  nicely iFurther elucidation of the structure-function interplay between ExoN and other  members of the CoV replication machinery will be key to understanding their role in viral RNA synthesis, immune evasion and pathogenesis. Such information will contribute to the design of new antiviral approaches, or the improvement of existing ones, including those relying on inducing “lethal mutagenesis” . LikewisNO, CP, and ES conceived, wrote, and edited this review. FF, ED, and BC revised and extended the parts of the text – specifically in the areas of the biochemistry and structural biology, and provided various other useful suggestions and contributions. FF prepared The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We use an interactive story design in which participants read short stories and make two consecutive plot choices about whether protagonists commit low- or high-violence actions. Our study has four main findings. 1) People who choose high violence report greater satisfaction with the story, while those switching to or staying with no violence show lower satisfaction. 2) However, when participants encounter these stories without choices, they reliably rate higher-violence stories as less satisfying than lower-violence stories. 3) Regret seems to account for the low satisfaction of those who choose or switch to low violence. 4) There is a large segment of people (up to 66%) who can be persuaded by different story contexts  to choose extreme violence in interactive fiction and as a consequence of their choice feel satisfaction. We hypothesize that people who opt for high violence enjoy the story as a result of their choice. Overall, we suggest that choosing violence serves as a gateway for enjoyment by creating an aesthetic zone of control detached from morality. Depictions of violence are highly present in many interactive media, and a matter of great controversy. Some media users show an interest in violence and derive high enjoyment from it. At the same time, other media users do not enjoy but rather detest and avoid violence. However, there also seem to be some people in the middle who sometimes, but not always, enjoy and choose violence in media. We are interested to know more about this middle group, such as how large it is and under what circumstances they will opt for and enjoy violence in media. Moreover, we are curious about the connection between choosing violence and enjoying it.There have been mixed findings as to whether violence in media increases or decreases enjoyment. Some studies report increased enjoyment –2, some Black Mirror Bandernsnatch, video games like Quantic Dreams’ Heavy Rain, Beyond: Two Souls or Dontnod Entertainment’s Life is Strange, online role playing games (RPGs), and hypertext fiction attracting increasing numbers of participants.While many previous studies have focused on visual media, we focus on interactive fiction where readers choose how the plot should continue. Participatory or interactive fiction , a mediuInteractive fiction reduces the complexity of other media, such as video games, by eliminating the visual aspects and demands of immediate reaction, and allows focus on plot choices. By concentrating on interactive fiction, we want to draw more attention to this genre. Studies of interactive fiction might also provide insights that could apply to video games and narrative fiction in general. While the choice making process in interactive fiction is different from video games and narrative fiction, some similarities remain. This is obvious in video games where users also make choices, though they may be under time pressure to react and perceive the choices paired with visual stimuli. But it additionally applies to narrative fiction where readers and listeners often identify with the protagonist and thus co-experience the decision-making with the protagonist –8.The design of our studies uses interactive fiction by offering readers two consecutive plot decisions that contain varying degrees of violence. We ask our participants at different points of the study to rate their enjoyment with the narrative. Our studies use an understanding of violence as physical aggression in line with Anderson and Bushman: “violence refers to extreme forms of aggression, such as physical assault and murder” and aggression to “a behavior intended to harm another individual who is motivated to avoid that harm” . For thiOverall, we ask:1What are the genre and perspective conditions under which people are more likely to opt for highly violent plot developments?While a large part of the population may not enjoy violence in media, it has been established that some of the population has a predisposition to opt for and enjoy violence in media , particu2How large is the group that can be influenced to choose violence under these different genre and perspective conditions?We predict that a large portion of the population can be persuaded to opt for and enjoy violence under the conditions listed above.3How do different options for no, low, and high levels of violence affect the overall satisfaction with the story?when people opt for high violence in stories, they are more likely to enjoy the story. This hypothesis is especially relevant in context of Hypotheses 1 and 2. We conjecture that when those people from the middle group opt for high violence they will as a result of their choice enjoy the story more than if they had opted for no or low violence. By opting for violence, people opt for enjoyment.We reason that it is not predisposition alone that leads people to enjoy highly violent stories; it is also not control by itself (having a choice versus lacking a choice) that leads to higher satisfaction, even though both predisposition  and cont4Do people show signs of regret after violent choices?In the context of our studies, we define regret as the wish to correct a previous aggressive choice by opting for an apology in future choices. Our study design of two consecutive choices of low and high violence is well suited to record patterns of regret as a first choice for high violence followed by an apology. Our hypothesis is that this choice pattern of regret is linked to low enjoyment. It is not well understood when the rules of morality are fully suspended in game playing and fiction and when they are merely overshadowed. We suggest that regret choice patterns indicate that moral judgment is not fully suspended, but rather only momentarily overshadowed in a previous choice. Regret also raises the question of whether the suspension of morality is a general effect of games and fiction or an individual choice of players and readers .5What is the relation of choice to satisfaction in violent interactive fiction?We hypothesize that it is specifically the choice aspect of interactive fiction that drives satisfaction. Consequently, we hypothesize that when we make our narratives non-interactive, regardless of violence level satisfaction will be much lower than when participants are given the opportunity to make choices about how the story will precede. This would indicate that it is the choice for violence, not the violence alone, that accounts for enjoyment of violence in interactive narrative.A key factor for the enjoyment of violence is control and agency. In this study, we distinguish between control (having a choice) and agency (the feeling of control). Choice in media selection has been shown to influence reader reaction, with lack of choice increasing participant reactance to even preferred media when it is forced; having choices leads to higher satisfaction, though the effect is not always strong .While our studies also concern the difference between choice and forced choice , our focus is on the difference between specific choices for and against high violence. In a two-choice scenario between high and low violence, making a choice for high violence, rather than the choice against it, seems to indicate a higher degree of agency; put differently, the choice for high violence may feel more like agency. Our studies allow further insights how control and agency (feeling of control) interact.Within media context, it has been shown that violent video games provide people, particularly adolescent boys, with a context to voluntarily control the emotional situations they confront, meaning that it is partially control (agency) that makes violent video games pleasurable –22. In tMorality is a key inhibitor in enjoyment of violence, and Flesch  has suggAesthetic distance can overshadow morality, creating a path to moral disengagement that allows for enjoyment and pleasurable narrative control. Oatley  suggestsIn the context of our study, factors that are likely to increase enjoyment of violence thereby include:breaking out of the ordinary world/taboo breaking;making a story more interesting or surprising;agency that comes from opting for violence ;completion of narrative ;accomplishment ;eudaimonia ;meaningfulness to the narrative –32;fictionality;eventfulness ;specific genres and perspective (see our studies below).However, it is not clear whether particular choices or the very fact that one has a choice drives enjoyment. We also suggest that regret, as a moral emotion that follows an earlier transgression, needs to be considered in the context of these factors (see Hypothesis 4). In general, desensitization over prolonged exposure to violence may increase individual enjoyment of violent media through moral disengagement regardless of initial traits , 34. TheOur studies cannot explain whether violent media contributes to aggressive behavior. Active participation in mediated violence as in game playing may produce different responses than passively viewing violent behavior. The General Affective Aggression Model  has beenIn this set of studies, we focus on interactive fiction in order to examine the question of choice. We offer our participants two consecutive choices in a short story between high, low, and no violence and we ask them questions concerning their satisfaction, involvement, and perception of morality. Our particular design of choices allows us to track how many people switch from one path to another. We suggest that people who switch from a highly violent first choice to an unviolent second choice (apology) show signs of regret. Specifically, we measure how many people make which choices and how they rate their satisfaction after making their choices under different genre and perspective conditions.We created basic stories with variations in different genres and perspective conditions (second and third person). We reason that different genres create distinctive sets of expectations and levels of detachment that will have an influence on reader choice (Hypothesis 1), satisfaction (Hypothesis 3 and 5), likeliness for regret (Hypothesis 4), as well as the overall number of those for opt for violence (Hypothesis 2). We offer these stories with two consecutive plot choices , no choice (Study 4), and with a single choice without showing outcomes of the choice to test anticipation (Study 5).In this study, participants received a short story and were told to “read the following story” and then twice during the story were asked to “choose what comes next.” Each story introduced two main characters in conflict with one another. One of these characters is given a motive for being annoyed with the other character, such as a noise disturbance. After 3–4 sentences describing an encounter between these characters, participants were given their first choice of three options about how the story would continue. One choice was not violent, usually starting a conversation about topics that were not related to the subject of disturbance, the second involves a low level of violence, such as a slap, and the last one was highly violent, such as hitting the other character with a baseball bat. After making the first choice, participants were directed to a sentence depicting the consequences of the action, describing either the follow-up of the mundane conversation, or the confusion of the other character from the low violence action, or the injury of the other character from high violence. After that, participants made a second choice for the aggressive character between a highly violent or non-violent action. The non-violent choice could be ending the conversation peacefully (if following a first non-violent choice) or an apology if it followed a prior act of violence. This means that there are six patterns of choices by participants resulting from no violence (= N) low violence (= L) and high violence (= V): first no violent choice and end of the conversation (= NN); first no violent choice and then performance of a violent action (= NV); first slightly violent and then apology (LN); first slightly violent and then escalation (LV); first highly violent and then apology (VN); and first highly violent and then escalation (VV), see After the story, the participants were asked “how satisfied do you feel with the events of the story?” They were given a scale from 0 to 10 with 0 being not satisfied and 10 very satisfied. We collected only whole numbers. For each story we created four versions that each fell into one of the following genres: realistic-contemporary, fantasy, Nazi-historical, and general-historical. We selected these genres since we reasoned that they would direct participants to either more positive (fantasy), expectation-congruent , negative , or expectation-incongruent and negative  attitudes about violence. In each version, we preserved the basic plot with the reason for the original annoyance (i.e. noise disturbance); the relationship between the characters, such as being neighbors; and the choices of violent actions, such as stabbing. The violent actions were identical or highly similar, (i.e. stabbing with a pocket knife versus a dagger) and the different genres featured appropriate settings. For each version, we also created second and third-person variations. All in all, we created three different stories (with different motivations and forms of violence), each having four different genre versions and two different perspective variations, resulting in a total of 24 different texts , violating the assumptions of the standard one-way parametric ANOVA.Additionally, we were interested in the potential influence of story genre and story perspective on participants’ story path outcomes, which is a classification problem with two nominal predictors. To answer these questions, we used multinomial logistic regression, which can be thought of as an extension of logistic regression featuring a nominal dependent variable with more than two levels. NN, fantasy, and second-person were used as the reference categories for story path, genre and perspective. The analyses described here were reused in Study 2 and 3. All analyses were performed in R: the multinomial regression made use of the mlogit package, and the Dunn post-hoc test used the PMCMRplus package, while the rest of the analyses were carried out with R’s base package.Out of 507 total participants, 283 (56%) made a non-violent choice then continued with no violence (NN), 56 (11%) made two high-violence choices (VV), while 168 (33%) made at least one violent option (L or V), but did not opt twice for high violence, see M = 7.09, SD = 2.9) were significantly more satisfied than participants in the LN story path , p < .01. And participants in the NN  story path were also significantly more satisfied than participants in the LN story path, p < .05. The tests revealed no other significant differences in satisfaction between the conditions.A Kruskal–Wallis test yielded a significant effect of story path on participant satisfaction, χ2(5) = 17.74, p < .01. A Dunn post-hoc test using the Bonferroni correction for p-values revealed two significant contrasts. Participants in the VV story path . 37 of 256 participants (14%) opted for VV in third-person compared to 19 of 252 in second-person (8%); and 24 of 256 participants (9%) opted for VN in third-person compared to 14 of 252 in second-person (6%).In other words, the fantasy genre influenced participants to choose VV and VN more frequently and NN less frequently when compared with all other genres. And the third-person perspective likewise influenced participants to choose VV and VN more frequently and NN less frequently than the second-person perspective.Given the choice patterns in this study, we can distinguish three populations of participants:People with a strong inclination for morality and/or against violence in all story settings that our conditions cannot persuade to opt against their disposition for the least violent pattern offered (NN in Study 1).A middle group who might opt for a choice of high violence in some story condition, but for no violence in others.People with a strong inclination for violence that our conditions cannot persuade to opt against their disposition and preference and who will opt for the most violent path (VV) Our data allows us to estimate the size of this middle group. We assume that the participants who opt for either extreme (NN and VV) most represent the portion of the population that is unaffected by story genre and perspective. By finding the smallest proportion of participants who follow these extreme paths, we can estimate the size of the population that has strong predispositions for or against violence. By subtracting this percentage from 100, we can then estimate the size of the population that can be influenced by story condition. The conditions with the fewest people opting consistently for high violence (VV) are second-person general-historical and second-person Nazi-historical, both of which had 1/63 people opting for the VV path (2%). The condition with fewest people making a non-violence choice (NN) is in third-person fantasy, with 20/62 opting for the NN path (32%). Consequently, we can estimate that 66% of our participants can be influenced by genre and perspective.There was no clear difference between males and females in choice and satisfaction. 146 females opted for NN and expressed a satisfaction of 6.86 compared to 135 males who rated their satisfaction as 6.67 in average. 28 females and 28 males chose VV, the females rated their satisfaction as 7.04 and the males as 7.14 in average. (Note again that 3 people preferred not to state gender).The astonishing finding of Study 1 is the link between high violence and high satisfaction. Overall, the study has five main findings:People were statistically significantly more likely to make highly violent choices in the fantasy genre than in any of the other genres.People were also significantly more likely to opt for VV in third-person perspective stories and more likely to opt for NN in second-person perspective.Participants who made only violent choices expressed notably higher levels of satisfaction with the story.Participants were more likely to show regret by moving from violence to no violence than to move from lower to higher violence. If they switched to no violence they showed notably lower satisfaction.There was a large middle group of 66% of the participants who could be persuaded to opt for violence.The unequal distribution of choice paths by genre and perspective indicates that it is not predisposition or specific traits alone that influence choice and satisfaction. Within the study design, it is likely that participants without a strong disposition for or against high violence reacted to both inhibitors and enablers for making violent choices. Second-person conditions, realistic and general-historical settings had an inhibiting effect, perhaps a reminder of personal responsibility and morality. Contrastingly, third-person and fantasy conditions, but not Nazi-historical stories, had an enabling effect, emphasizing fictionality and consequently allowing taboo breaking. Even though violence is expectation congruent for the Nazi-historical genre, unlike the fantasy genre, the Nazi-historical genre did not increase the number of highly violent choices made. Perhaps participants wanted to take the opportunity to have a Nazi character behave more morally than what would be expected in a story; or they may have wanted to distance themselves from the Nazi characters, and thus make choices that align with their own moral behavior. Note again that the stories were quite graphic in the descriptions of violence, including in fantasy, with conclusions such as you “take a piece of glass from the broken jar and cut his throat,” see Given that highly violent choice paths are linked to higher satisfaction, there is an intriguing possibility that choice for violence is a choice for satisfaction. Our next studies aim to clarify this possibility. In our next studies, we aim to investigate under what conditions and to what extent this middle group can be persuaded to choose highly violent choices in narrative. If we can indeed persuade more people to opt for high violence, we want to know whether these people display the same high satisfaction after making the choice.We wanted to know if more people opt for high violence when the option against violence is not available and the choice is only between low and high violence. We also wanted to know how people who can be persuaded to opt for high violence in certain conditions, such as limited choice, rate their enjoyment. Do these people still rate high violence as highly satisfying or do they show low satisfaction?We repeated Study 1 without the no-violence option in the first choice, using the same basic 24 stories see . For eacOut of 480 total participants, 315 (66%) made a low-violence choice then apologized (LN), and 78 (16%) made two high-violence choices (VV). 65 (14%) switched from a high violence choice to a second choice of no violence (VN). In comparison, only 22 (5%) increased violence after a first choice of low violence to high violence (LV), see Overall, participants reported the highest average satisfaction in the VV story path (6.5). The lowest averages occurred in second choice low violence , see M = 6.53, SD = 2.75) and LN , p < .001. And second, VV and VN , p < .001.A Kruskal-Wallis test yielded a highly significant effect of story path on participant satisfaction, χ2(3) = 35.77, p < .001. A Dunn post-hoc test using the Bonferroni correction for p-values revealed two highly significant contrasts. First, between participants who chose VV  opted for VV in third-person compared to 32 of 243 in second-person (13%); and 13 of 237 participants (5%) opted for VN in third-person compared to 9 of 243 in second-person (4%).In summary, a general similarity was established with Study 1’s findings. The fantasy genre and third-person perspective influenced participants to choose VV more frequently and LN less frequently.We calculated the middle group as in Study 1. The middle group was 47%.Females were less likely to opt for high violence than males and on average expressed lower satisfaction in all conditions. 181 women (satisfaction: 4.13) and 135 males (satisfaction: 4.95) opted for LL; while only 22 women (satisfaction: 5.35) but 55 men (satisfaction: 6.96) opted for VV. More women (39) switched from high violence to apology (VL) than men (26).The main finding of Study 2 is the fact that we can indeed make the middle group shift towards a choice for high violence. Overall, the study confirms four of study 1’s main findings:People were again statistically significantly more likely to make highly violent choices in the fantasy genre than in any of the other genres.People were also again significantly more likely to opt for VV in third-person perspective stories and more likely to opt for LN in second-person perspective.Participants who made only violent choices again expressed notably higher levels of satisfaction with the story, especially if their last option was for high violence.Few people changed their choice path, in line with theories of cognitive dissonance . Those wThe large middle group seems to have been persuaded to opt for high violence. The size of the middle group within the conditions of Study 2 was again high at 47%, but lower than in Study 1 that offered more choice options. Specifically, whereas in Study 1 11% (56 of 507) of participants chose the VV option, 16% (78 of 480) chose this option in Study 2. Consequently, by eliminating the non-violent option in the first choice we were able to persuade more participants to choose high violent options in Study 2. People seem to be swayed not only by narrative conditions but by how their choice is framed as well. To emphasize again, the satisfaction ratings for this VV group remain generally consistent across studies despite these differences in choice path distribution. However, unlike in Study 1, the participants in Study 2 who made more violent choices expressed significantly higher levels of satisfaction with the story than any other group, especially if their last option was for high violence. A likely explanation for this increased difference is that since we offered participants a forced choice with no non-violent option in Study 2, it is likely that many participants felt dissatisfied. These differences in choice path distribution along with the consistencies in satisfaction ratings seem to indicate that it is the choice for violence and not predisposition alone that drives satisfaction.There is a difference in gender between Study 1 and Study 2. While Study 1 records no clear differences, females in Study 2 were notably less satisfied. This might suggest that females had a stronger preference and reacted strongly to more limited choices and forced choices for some violence (L or V), while males were more flexible to adjust without being dissatisfied.We created a follow-up study with no participant choice for plot development. Participants were randomly given a completed version of the story with the choices already made. We created 32 versions of one of the stories, namely all four paths for the realistic, fantasy, Nazi-centered historical, and general-historical genre with either second- or third person-perspective, as in Study 2, but wrote each of the possible outcomes as part of that story. For example, a participant would receive a second-person, realistic story with the slightly violent action followed by an apology (LN) already woven into the story. Participants read one of these narratives and immediately afterwards rated their satisfaction with the story on a scale from 0 to 10. Participants were randomly assigned a story version. Each of the 32 story conditions (4 story paths x 4 genre x 2 perspectives) was rated by 8–11 participants, for a total of 295 different raters. All three base stories behaved similarly in Study 2, and we selected the story that correlated most closely to the overall pattern of satisfaction in the genre categories and perspective conditions  compared to the average satisfaction for all paths).We combined the data from Study 2 (choice) and Study 3 (no choice) for our analyses of the influence of participant choice on satisfaction. We chose an ordinal-logistic-regression model where the nominal variables of choice presence, participants’ story choices, and their interaction were used to predict ordinal satisfaction ratings. The choice being present was used as the reference for the choice variable. LN was used as the reference category for story choice. The ordinal logistic regression was performed in R with the MASS package.M = 2.27, SD = 2.58) than with the less violent stories LN , see Participants who had no choice in the story progression consistently reported lower average satisfaction with the highly violent stories VV  still expressed higher satisfaction with the high violence condition in Study 3, representing a group that enjoys violence regardless of condition or presence of agency.Females showed notably lower satisfaction in Study 3 with no choices in all story paths, while there was no clear difference in gender in Study 1. Choice seems thus to play a larger role for satisfaction for females than males.We wanted to know at which point participants decide on their ratings of satisfaction—before or after seeing the outcomes of their choices—and how they explain their choice making.We created a survey based on Study 2 with one addition: right after making their initial choice and before seeing the outcome of that first choice, we inserted a question concerning anticipated satisfaction and several questions about the reasons why participants made their choice between the high or low violence option. We could not include these questions in Study 1 or 2 since such a question would have likely resulted in priming effects for the later tasks. We first asked participants to “predict how satisfied you will be with the story based on your choice for how the story should continue.” Afterwards, participants were asked: “To which degree did each factor influence your choice about how the story should continue?” They ranked the following on a sliding scale from 0 to 10 as in the other studies with the quoted explanations: realism , fun (“the story would be fun”), morality , curiosity (“I am curious to see what comes next”), violence (“I am interested in violence”), and distaste . As with Study 3, we used the eight versions of Story 2 because it correlated most closely to the overall pattern of satisfaction in the genre categories and perspective conditions. 241 participants rated one of the eight story conditions, with 33–57 for each genre condition.To analyze the influence of participants’ first story choice (L or V) on satisfaction we employed a Wilcoxon rank-sum test. As with the non-parametric tests used in Studies 1 and 2, the Wilcoxon test best fit our ordinal and non-normally distributed data.Additionally, we wanted to determine whether participants’ ratings of their motivations could predict whether they made a L or V first choice. And this question is well suited for a logistic regression model where the binary first choice is predicted by the ordinal motivation ratings (treated as continuous). All analyses were performed with R’s base package.M = 5.97, SD = 2.68) than the 178 participants who chose the less violent option  (Participants were asked to rate their satisfaction with the story after making their first choice and before seeing the outcome of their choice. 63 participants chose the highly violent first option and reported a higher level of anticipated satisfaction ( = 2.92) .W = 2429, p < .001. Consequently, we have strong evidence that overall satisfaction was a product of anticipation and largely determined prior to seeing the outcome of the story choices.A Wilcoxon rank-sum test revealed that this difference was significant, fun ratings reliably predicted the highly violent choice (p < .01), while higher distaste for blood and brutality ratings reliably predicted the less violent choice (p < .001). However, realism, moral motivation, curiosity, liking violence did not reliably predict story choice. Of particular note: the fact that moral motivation did not predict story choice suggests that participants did not see the story choice as a moral situation; and that liking violence was not a reliable predictor indicates that participants who chose the highly violent option do not possess or were not motivated by an inherent preference for violence.In addition, we asked participants to rate potential motivations for making the choice that they did, and we inputted this data into a logistic regression as continuous measures to predict their binary first choice, see Females anticipated notably lower satisfaction than males. In L, 76 females predicted a satisfaction of 3.27 and 99 males 5.12 in average. In VV, 26 females predicted a satisfaction of 5.08 and 37 males 6.59.The ratings show that as soon as they make their first choice people predict a significantly higher satisfaction. This indicates that satisfaction does not arise solely from seeing consequences of their decisions. Consequently, it suggests that the satisfaction cannot alone be explained by prediction-reward theory since participants did not see the outcomes.People who opted for the low violence option reported being guided by morality and distaste for violence and anticipated low satisfaction. People who opted for high violence reported being guided by fun and not morality and anticipated high satisfaction.The ratings for realism are close to equal in both conditions. Hence, there is no evidence of a detachment from reality in making the decision for high violence or difference in satisfaction. In this respect our finding differs from suggestions of Waddell  who showdecide to have fun, decide to discount morality, and make it happen in their decision. Those who choose the most violent option indicate that morality is not as high of a concern, supporting the disengagement theory [That means the first decision is already a decision about having fun and to discount morality, or vice-versa. This is important to emphasize since it provides a motivation for people who choose high violence. People who choose high violence t theory , 39, 36.People who chose highly violent plot developments generally enjoyed the stories more than people who made less violent choices. A large percentage of our participants (up to 66%) could be persuaded to opt for highly violent plot choices and as a result rated their satisfaction higher than people who did not make highly violent choices. However, people who did not have choices and read completed stories generally strongly disliked high violence. It is when participants are given control of a situation and execute it by opting for high violence that they find greater satisfaction. In short, choosing violence increases enjoyment.Specifically, our studies have five findings:Fantasy and third-person perspectives enabled people to choose high violence, while realistic and historical genres and second-person perspectives acted as deterrents of highly violent choices. Hypothesis 1 was partly supported. We did not, however, predict that the Nazi-historical genre would not enable highly violent choices.What are the conditions under which people are more likely to opt for highly violent plot developments? In our studies, the middle group was the largest of all groups. Depending on the study conditions, up to 66% of participants could be influenced to make different choices according to story condition (Study 1).How large is the group that can be influenced to choose violence under these different genre and perspective conditions? Generally, people who opt for highly violent plot choices were more satisfied than people who opt for low violent options . Moreover, satisfaction for the VV group remains high even when more people opt for high violence (VV) under different story or choice conditions. Hypothesis 3 was supported.How do different options for no, low and high levels of violence affect the overall satisfaction with the story? Regret plays a substantial role in decreasing satisfaction. It is much more common for people to switch from high violence to low violence than the other way around. And when people switched from high violence to low or no violence, they showed low satisfaction. Similar to our findings, the reaction model [Do people show signs of regret after violent choices? on model  would prWhen there is no choice, most people and especially females are highly dissatisfied with highly violent narratives, see What is the relation of choice to satisfaction in violent-interactive fiction? The enjoyment effect of high-violence choices disappears and reverses when there is no choice (Study 3). We had an unpredicted side finding. When choices are forced (Study 3) or appear forced (no N in Study 2 and 4), females show lower satisfaction. When all choices are available (Study 1), including no violence (N), females and males show the same satisfaction for different choice paths. This seems to suggest that women are more set on their preference, and that choice seems to play a larger role for satisfaction for females than males.Our data suggest that while disposition may play a significant role in determining choice paths and satisfaction, there is a large middle group in the population that can be swayed to choose high violence and enjoy doing so because of this choice. That means the people in this middle group might choose either path, and the actual choice determines satisfaction. As reported, this middle group consisted of up to 66% of our participants. This number is especially surprising considering the graphic nature of violence in our stories, including in the fantasy genre, see It is important to note that within Studies 1 and 2 respectively, the satisfaction ratings for different choice paths remained similar across different genre and person conditions, even though the distribution of choices varied significantly. Moreover, satisfaction ratings for the VV group in particular remained similarly high between Studies 1 and 2, even when more participants chose the VV path in Study 2. This distribution suggests that the actual choices people make, and not just personal disposition, matter for the satisfaction ratings. Specifically, participants in our studies mentally connected their initial choice for high violence with an opting-out of morality and opting-in for fun (Study 4). Apparently, the very act of opting for high violence simultaneously disconnects people from constraints of morality and responsibility and thereby opens an aesthetic realm of fun and satisfaction.In our proposed framework, it is not that the large middle group enjoys violence but that they accept it in media as a route to a sense of control (agency) that is satisfying. Our studies support prior research that has found that choice itself is a driver of pleasure by allowing for a greater sense of control , 37, 39 We suggest that by choosing high violence, people claim specific forms of agency over the media content, which leads to greater enjoyment. The appeal might not be the satisfaction of a disposition, but rather an act of choosing stories that break out of the ordinary and thus open up an aesthetic zone of enjoyment. Choosing violence is enjoyable, not violence itself. Reversely, people who, when given the choice, do not opt for high violence, do not claim agency, do not enter a zone of aesthetic enjoyment and do not enjoy the stories. Consequently, they stay in the realm of expected choices, feel confined by morality, experience feelings of guilt, and, despite having a choice, may not have the feeling of choice and control (agency). Our findings, if confirmed, are significant for understanding the appeal violence has for media users to satisfy a desire for aesthetic distance and heightened agency.S1 Interactive Stories(DOCX)Click here for additional data file.S1 Table(CSV)Click here for additional data file.S2 Table(CSV)Click here for additional data file.S3 Table(CSV)Click here for additional data file.S4 Table(CSV)Click here for additional data file.


E. coli. Compared with rBMP2, rBMP2-E8 displayed markedly enhanced binding to HA disks and was better retained on the disks following exposure to vigorous wash steps. Furthermore, rBMP2-E8 was purified using a heparin column, and evaluated for its capacity to stimulate osteoblastic cell signaling. Treatment of SAOS2 cells with rBMP2-E8 induced SMAD 1/5 activation, confirming that the protein retains activity. Collectively these results suggest that the E8 domain serves as an effective tool for improving rBMP2 coupling to graft materials. The increased retention of rBMP2-E8 on the graft surface is expected to prolong BMP2’s osteoinductive activity within the graft site, while simultaneously reducing off-target effects.Nonautologous bone grafts have limited osteoinductive potential and thus there is substantial interest in reconstituting these graft materials with osteogenic factors such as bone morphogenic protein 2 (BMP2). However, one limitation of this approach is that BMP2 is typically weakly bound to the graft, which can lead to side effects associated with BMP2 dissemination. In the current study we added a hydroxyapatite (HA)-binding domain onto BMP2 to increase coupling to the graft surface. A sequence consisting of eight glutamate residues (E8) was inserted into the C-terminus of BMP2, and the recombinant protein (rBMP2-E8) was expressed in  Bone grafting is an increasingly common procedure with over 500,000 grafts performed annually in the US and over 2 million worldwide , 2. BoneNonautologous graft materials derive from a variety of sources including allograft and xenograft, however these materials must be sterilized and/or chemically treated to make them safe for human use , 7. ThesRecombinant Bone Morphogenic Protein 2 (rBMP2) has been utilized as an osteoinductive molecule for bone regeneration since it was first FDA-approved for lumbar spinal fusions in 2004 . rBMP2 iIn the present study, we utilized a molecular domain found within native bone proteins to improve the binding of rBMP2 to HA materials. This domain is comprised of repeating glutamate residues which bind to the calcium within HA via the carboxylate groups within glutamate . There iin vivo. HA or ABB grafts coated with oligoglutamate-modified mimetic peptides were found to retain the peptides for at least 2 months following graft implantation into rat subcutaneous pouches [In view of the strong interaction between oligoglutamate and HA, we and others have added this domain to a variety of biomimetic peptides with the goal of enhancing the anchoring of such peptides to the surface of calcium phosphate graft materials. Oligoglutamate domains have been added to DGEA (a collagen-derived peptide), RGD (integrin-binding peptide), and KRSR and FHRRIKA (proteoglycan-binding peptides), in addition to peptides derived from BMP2 and VEGF –31. In e pouches .Importantly, better peptide/graft coupling has been shown to improve osteoregeneration. Our group synthesized a BMP2-derived mimetic peptide with a heptaglutamate (E7) domain and examined the peptide’s osteoinductive activity in a rat mandibular implant model. ABB particles were coated with either unmodified BMP2-derived peptide (BMP2pep) or E7-modified BMP2 peptide (E7-BMP2pep). As compared with BMP2pep-coated ABB, E7-BMP2pep-coated grafts stimulated significantly more new bone formation, confirming that better anchoring of the peptide to ABB was effective in enhancing tissue regeneration . FurtherPfu Polymerase and the following primers:A clone of the human BMP2 gene encoding the pre-pro-protein was obtained from SINO Biological. All PCR and cloning enzymes were from New England Biolabs. The mature coding sequence, encoding Gln283 to the terminal Arg396, was amplified by PCR using 5’-CATATGGGTCAAGCCAAACACAAACAGCGGForward (N-term): 5’-CTCGAGTTATTCCTCTTCTTCTTCCTCTTCTTCGCCACCAGCTGCAC CAGCGCGACACCCACAACCCTCCACReverse (C-term): NdeI site, as well as a Gly-encoding codon (GGT) between the ATG start and the codon for Gln283. The 3’ primer included sequence for an Ala-Gly-Ala-Ala-Gly-Gly spacer, followed by eight glutamic acids (E8 domain), the stop codon and a terminal XhoI site. The PCR product encoding mature BMP2-E8 was cloned into the pCR-Blunt-II (Life Technologies) cloning vector, and verified by DNA sequencing  and ampicillin to 100 μg/mL. Expression of rBMP2-E8 was induced by the addition of IPTG to 0.5 mM. Cells were harvested at three hours post-induction, and the cell pellet was frozen. The cell pellet was thawed and suspended in sonication buffer  and lysed by sonic disruption. Insoluble inclusion bodies containing the IPTG-induced rBMP2-E8 were isolated by low-speed centrifugation, and inclusion body pellets were washed by re-suspension in sonication buffer and re-centrifugation three more times. The pellets were then suspended and solubilized in denaturing buffer (6 M guanidine-HCl (GnHCl), 100 mM Tris-HCl pH 8.5, 1 mM EDTA, 100 mM DTT). The solubilized protein was dialyzed against the same buffer with succeeding decreases in the concentration of GnHCl to 3, 2, and 1 M. The protein was then dialyzed into refold buffer , 1 mM oxidized glutathione (GSSG)) and finally into storage buffer . Hereafter, the refolded inclusion body preparation is referred to as IBP. At the end of this process, we obtained a yield of 10 ml of IBP solution containing 1.35 mg/ml of rBMP2-E8, as measured by ELISA, from a starting liter culture of E. coli. Aliquots were collected at each step of the expression and isolation procedures and the various samples were resolved by SDS-PAGE using Nu-PAGE Bis-Tris 4–12% gradient gels in MES buffer (Life Technologies), followed by Coomassie Blue staining of the gels.The pET-BMP2-E8 plasmid was transformed into BL21(DE3) 1 ml heparin columns  were used to purify rBMP2-E8 from the IBP. In brief, the column was equilibrated with binding buffer  before adding the inclusion body lysate. The lysate was allowed to bind the column for 15 minutes, and then the column was washed extensively with binding buffer to remove unbound proteins. The rBMP2-E8 protein was then eluted from the column with elution buffer  and the eluate concentrated using Amicon Ultra-2 mL Ultracel-10K centrifugal filters. The affinity-purified protein was then diluted 1:20 into a refolding buffer  as described in . BuffersSAOS2 cells were obtained from ATCC and maintained in Hyclone DMEM  supplemented with 10% fetal bovine serum  and 1% antibiotic/antimycotics (Invitrogen). Prior to signaling experiments, cells were serum-starved for 24 hours. BMP2 activity was assessed by monitoring SMAD 1/5 phosphorylation in cells treated for 15 minutes with either 100 ng/ml of total protein from the inclusion body preparation  or 100 ng/ml of pure rBMP2 . Following this treatment, cells were lysed using radioimmune precipitation assay (RIPA) buffer (Thermo Fisher) containing protease and phosphatase inhibitors (Sigma). Samples were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were incubated in 5% nonfat dry milk dissolved in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST). The blots were probed with antibodies for pSMAD 1/5 , SMAD 1 , or rBMP2 . Subsequently, blots were incubated with the appropriate HRP-conjugated secondary antibodies . Blots were also probed with anti-β-tubulin or anti-β actin (both from Abcam) to control for protein loading. Proteins were detected by enhanced chemiluminescence using Clarity Western ECL substrate .Relative levels of BMP2 in the inclusion body preparation vs. commercial rBMP2 solution were evaluated using an enzyme-linked immunosorbent assay (ELISA). An ELISA plate was coated overnight with a rBMP2 capture antibody  and then washed and blocked with 0.1% BSA. Afterwards, varying concentrations of total protein from the inclusion body preparation (measured by BCA), as well as a known reference amount (5 ng/ml) of commercial rBMP2 (Peprotech), were added to the plate and allowed to bind for 2 hours. Plates were subsequently washed and incubated for 2 hours with a distinct rBMP2 antibody used for detection . A biotinylated secondary antibody was added, followed by streptavidin-HRP. The BMP2 signal was monitored by adding a TMB colorimetric substrate (Thermo Fisher), and then quantifying absorbance  using a plate reader.A) to be 0.921 μm using a Federal Surfanalyzer 4000 [HA disks were prepared from hydroxyapatite fast flow powder  using a 15.875 mm die under 3000 psi as in our prior publications . Afterwazer 4000 . Disks wThe native BMP2 molecule contains a pro-domain that must be enzymatically cleaved before the protein becomes active. We therefore cloned the E8 domain into the DNA sequence encoding the mature BMP2 protein . E8 was added to the C-terminus of BMP2, along with an intervening spacer sequence composed of alanine and glycine residues . This spE. coli were transfected with the pET21c/rBMP2-E8 vector, and protein expression was induced by IPTG. Cells were lysed, and then whole cell lysates resolved by SDS-PAGE, followed by Coomassie staining of the gels . The protein concentration of the IBP was quantified by BCA assay. Disks were also coated with BSA as a negative control. After a 1-hour coating with BSA, rBMP2, or IBP, disks were washed briefly to remove unbound proteins, and a modified ELISA assay for BMP2 was used to detect proteins bound to the disks. Specifically, coated disks were incubated with an anti-BMP2 antibody, followed by a fluorescent secondary antibody. Disks were washed in TBS, and then imaged by fluorescence microscopy . As shown in To test the strength of the interaction between rBMP2-E8 and HA, we coated HA disks overnight with IBP or rBMP2, and then washed the disks at 4˚C with agitation for 5 days. At the end of this interval, the difference in fluorescence signal between disks coated with rBMP2 or IBP was even more striking . The morNotably, the amount of rBMP2 in the initial coating solutions was actually in excess of that of rBMP2-E8. For the modified ELISA, 100 ng/ml of either total IBP protein (measured by BCA) or commercial rBMP2 (pure rBMP2) was used to coat disks, however the IBP clearly contains many proteins other than rBMP2-E8. To estimate the amount of rBMP2-E8 within the IBP, we used an ELISA for BMP2. Total IBP protein was added to the ELISA in either 12.5 or 25 ng/ml concentrations. As a reference, 5 ng/ml of commercial rBMP2 was also included. These studies indicated that ~20 ng/ml of total IBP protein had the equivalent of 5 ng/ml of rBMP2, suggesting that ~25% of the total protein within IBP was comprised of rBMP2-E8. In view of these data, the binding differences shown in E. coli-derived proteins may interfere with cell signaling. Secondly, data shown in E. coli-derived proteins, on cell signaling, we proceeded to purify rBMP2-E8 from the IBP.Having confirmed that the E8 domain confers binding for HA, we next evaluated whether the rBMP2-E8-containing IBP was capable of activating BMP2-dependent signaling. SAOS2 osteoblastic cells were treated for 15 minutes with IBP, rBMP2, or left untreated, and then cells were lysed to probe for pSMAD 1/5, a marker of BMP2 signaling. As shown in E. coli, we therefore adopted this approach. Using standard protocols [Heparin affinity columns are commonly used to purify rBMP2 from rotocols , heparinrotocols . In contE. coli proteins and enrich for the rBMP2-E8 dimer, we next tested the activity of the purified rBMP2-E8. SAOS2 cells were either left untreated or incubated with purified rBMP2-E8, the refolded rBMP2-E8 sample, IBP, or commercial rBMP2. As before, cells were lysed and probed for pSMAD 1/5. Significantly, purified rBMP2-E8 induced a marked increase in SMAD 1/5 phosphorylation relative to the untreated control, confirming activity Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.


The origins of life require the emergence of informational polymers capable of reproduction. In the RNA world on the primordial Earth, reproducible RNA molecules would have arisen from a mixture of compositionally biased, poorly available, short RNA sequences in prebiotic environments. However, it remains unclear what level of sequence diversity within a small subset of population is required to initiate RNA reproduction by prebiotic mechanisms. Here, using a simulation for template-directed recombination and ligation, we explore the effect of sequence diversity in a given population for the onset of RNA reproduction. We show that RNA reproduction is improbable in low and high diversity of finite populations; however, it could robustly occur in an intermediate sequence diversity. The intermediate range broadens toward higher diversity as population size increases. We also found that emergent reproducible RNAs likely form autocatalytic networks and collectively reproduce by catalyzing the formation of each other, allowing the expansion of information capacity. These results highlight the potential of abiotic RNAs, neither abundant nor diverse, to kick-start autocatalytic reproduction through spontaneous network formation. Reproduction of information-carrying polymers is integral to the origins of life. In prebiotic environments on the early Earth, reproducing molecules must have originated from limited resources because their supply solely relied on geological factors and inherent chemistry, without the help of complex biological catalysts . LimitedAs envisioned in the RNA world theory, a promising candidate for a prebiotic informational polymer on the early Earth is ribonucleic acid, because it can possess catalytic function, potentially even reproducing itself ,4. AlthoPertaining to such abiotic RNA oligomers, one possible reproduction mechanism is template-directed recombination ,16: A phA plausible organization of prebiotic reproduction is an autocatalytic network, in which each molecule multiplies one of the others, allowing collective reproduction , as suppHere, using computer simulations, we investigated the required diversity of finite populations of short (8-mer) RNAs for the onset of autocatalytic reproduction under prebiotically possible catalytic reactions. We mainly focused on template-directed recombination, but we also compared it to template-directed ligation. We found that in both cases, a high diversity of an RNA population was not required to initiate reproduction; in fact, it rather diminished the chance of reproduction in finite populations. The reproduction at high diversity was improved to some extent with increased population size. Conversely, extremely limited variation of RNA sequences also made reproduction unlikely. We discovered that the critical sequence diversity within a population for the initiation of reproduction is in an intermediate range, and in that range, autocatalytic networks were favorably formed with a high probability.X as distinct 8-mer sequences , and prepared a fixed number of each X distinct sequence  as an initial population. Each distinct 8-mer sequence was randomly sampled from all of the possible combination of 4 nucleotide variants, assuming adenine (A), guanine (G), cytosine (C), and uracil (U), as seen in RNA. Assumed from physiological RNA chemistry, A can form a base-pair with U, and G with C. During one reaction step, two sequences were randomly picked up as potential substrates from the population, and another sequence was also picked up as a potential template, and then template-directed recombination or template-directed ligation occurred if they satisfied criteria described below. In a simulation, performed in the Python 3.6 environment on a PC, supplied RNA sequences randomly reacted with each other according to a template-directed recombination or a template-directed ligation mechanism as described below. We defined Template-directed recombination occurs as a combination of cleavage of one substrate RNA and subsequent ligation of it to the other substrate RNA, supported by a template RNA that forms base-pairs with the two substrates ,18. We aWe fixed the concentration of reactable RNAs in a population over iterative reactions so that the time of a reaction could be assumed the same, and that the population size did not change. To do this, we added one molecule chosen randomly from an initial population if ligation occurred, or substituted the molecule for an unreactable short RNA (<3-mer) generated by recombination. For simplicity, degradation of RNAs by hydrolysis was not considered because we did not have sufficient empirical data of relative hydrolysis rate to the rate of the template-directed reactions. We also did not consider template-inhibition for the subsequent reaction, a potential problem for prebiotic reactions, although they could be overcome in fluctuating environments . The seqIn a simulation, every pair of catalysts (template) and products (ligated oligomers) for every reaction was recorded to create a directed graph. Only the products that remained in the final population of a simulation was considered. To remove randomness, only the reactions that occurred more than once were considered. The directed graph was created by the Python software package NetworkX. Closed catalytic reaction sets, including the self-reproduction reaction, was obtained using the same package. To estimate the maximum size of a closed autocatalytic network, the sizes of as many as possible closed networks were calculated, also using the same package. Because the calculation time exponentially increases as the number of edges involved in a network increases, we set 2.0 s for the maximum calculation time, which was long enough for a calculated maximum size to be saturated in our simulation, although calculation time was less than 0.2 s in most cases.To understand whether reproducing RNAs emerge by iterative template-directed recombination over the simulation , we intrX in a starting population of 300X 8-mer RNA sequences. X represents the number of distinct sequences in the population, and the multiplicity of each distinct sequence was fixed (300). We increased the number of reaction steps as the population size increased, and performed 10,000X, 30,000X, or 100,000X steps for each simulation. We repeated each simulation with a fixed parameter 100 times. Not surprisingly, if X was too low (X = 2 or 5), in most cases, recombination did not occur at all in the first place, hence reproduction could not be initiated (X (X was too high (X = 80 or 160), for simulations with the three different number of reaction steps, although recombination occurred many times, autocatalytic networks or self-reproducers rarely emerged (X = 10–20). At these diversities, reproduction occurred even after a small number of reaction steps . In most of these cases, RNAs formed an autocatalytic network, and reproduced collectively. We also analyzed the number of distinct catalysts in the largest network as an indication of complexity. This number increased over reactions in the investigated parameters, reaching at most 23 on average for X = 10  during the simulations. The average variation of reactions increased as the starting materials became more diverse because the number of possible combinations of recombinable RNAs also increased a. As a riversity b. Therefncreased c, confirX with three different population sizes: 100X, 300X, and 1,000X. We set 10,000X, 30,000X, and 100,000X reaction steps for each population size, respectively, so that the number of recombinations per sequence is comparable among different population sizes (X = 5–20) did not significantly change depending on the population sizes, the probability of reproduction at higher sequence diversity (X = 40–160) was improved as the population size increased (X) tested, a complex autocatalytic network emerged even at a relatively higher diversity X = 40   e, suggesThe origin of reproducing information-carrying polymers is one of the long-standing (but heretofore unknown) phenomena important for the emergence of life. Here, we constructed and analyzed an RNA-based catalytic reaction model under prebiotically possible template-directed recombination and ligation. These modes of reproduction would have been more accessible to emergent polymeric pre-life than template-directed replication ,18. UsinEmergent reproducing RNAs at the intermediate sequence diversity tended to form autocatalytic networks and cooperatively catalyzed each other’s reproduction c. FurtheX = 2–160). Although it was difficult to examine a much higher diversity on our simulation as a consequence of finite computational resources, the expansion of the optimal diversity depending on the population size  suggests that reproduction could have also started from a highly diverse concentrated RNA population. It should be noted, however, that we focused on only one type of reaction (a template-directed recombination or ligation) in a given simulation, and did not consider other reactions . If multiple different reactions happened simultaneously, it would generate more unrelated or potentially interfering RNAs, making reproduction increasingly difficult. In such a chaotic milieu, the incorporation of new factors such as sequence specificity of template-directed recombination and ligation, if any, may be required to achieve reproduction. One may argue that the actual diversity of RNA sequences on the early Earth could have been higher than the range investigated in the study  of autocatalytic networks through continuous supply of resources and degradation or dilution, valuable aspects for the propagation of genetic information ,24. ThisIn this study, we demonstrated that a subset of short RNAs with limited diversity had potential to kick-start autocatalytic reproduction thorough successive primordial recombination and ligation. The dominant mode of reproduction was an autocatalytic network, supporting the plausibility of cooperation among abiotic RNAs. These results indicate that the limited resources possibly available in prebiotic environments may have been sufficient for the foundation of reproduction, a key living process for the origins of life, both on the Earth and elsewhere in the universe. Although our simulation presented here only focused on the initiation of reproduction, further simulations could contribute in future to characterizing sustainability and evolvability of reproducing RNA sets, and if combined, key parameters for gradual steps toward life-like reproduction during abiogenesis would be unlocked.