In contrast, recent studies also suggest that alternative RNA processing also has a feedback regulation on epigenetic mechanisms. In this review, we discuss recent studies and summarize the current knowledge on the epigenetic regulation of alternative RNA processing. In addition, a feedback regulation of RNA processing on epigenetic regulators is also discussed.As a co-transcriptional process, RNA processing, including alternative splicing and alternative polyadenylation, is crucial for the generation of multiple mRNA isoforms. RNA processing mechanisms are widespread across all higher eukaryotes and play critical roles in cell differentiation, organ development and disease response. Recently, significant progresses have been made in understanding the mechanism of RNA processing. RNA processing is regulated by  Messenger RNA production is a fantastically complex process in eukaryotes, including transcription of mRNA precursors followed by capping, splicing, and polyadenylation. Alternative RNA processing, including splicing and polyadenylation (AS/APA), leads to the formation of distinct mRNA isoforms and explains how massive proteomic complexity can be accomplished with the relatively few genes in higher eukaryotes . AS/APA cis-regulatory sequences and trans-acting factors. In AS regulation, cis-regulatory sequences include splicing enhancers and silencers, typically 10 nt in length, the impact of which depends on their location and their preferential splice sites  site usage  in alternative RNA processing regulation. A feedback of alternative RNA processing on epigenetic regulation was also discussed.Despite the wide acceptance that these Arabidopsis revealed that two SU(VAR)3–9 homologs, SUVH1 and SUVH3, bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes , is a conserved and heritable DNA modification that affects gene expression in a genome-wide manner . The impar genes . Comparear genes . During ar genes .CD45 exon 5, which enables Pol II to transcribe more rapidly, giving rise to an exon 5 exclusion  binding protein 2 (MeCP2) binds to methylated regions to influence AS. MeCP2 is the earliest reported multifunctional protein that contains both methyl-CpG-binding domains and transcriptional repressor domains . Acting met1 mutant compared with those associated with the wild-type strain  mark is an active mark and is abundant in actively transcribed gene bodies . It has CD44 gene slows the elongation rate of Pol II, which in turn facilitates the recruitment of splicing factors such as U2AF65 and PRP8 to alternative exons, resulting in the inclusion of alternative exons , which has three isoforms in humans, HP1α, HP1β, and HP1γ, binds directly to histone H3 lysine 9 trimethylation H3K9me3; . A previve exons . Unsurprve exons .Arabidopsis encodes two homologs of human MRG15, MRG1 and MRG2, which bind to H3K4me3/H3K36m3-modifying histone marks and trigger temperature-induced flowering via the florigen gene FT -ASI1 immunoprecipitated protein 1 (AIPP1)-enhanced downy mildew 2 (EDM2) (AAE) complex, was identified targeting genic heterochromatic elements to regulate APA  have been identified . More reIn addition to histone variants, chromatin remodeling factors also affect chromosome segregation and transcription . During In addition to the identification of many alternative RNA processing events based on chromatin level, an interesting finding suggests that ncRNAs may play a key role in RNA processing regulation . General2CR by binding to a silencing element in exon Vb. PWS patients do not express HBII-52. They have different 5-HT2CR messenger RNA (mRNA) isoforms than healthy individuals  complex. Small Nucleolar RNA C.D Box 50A (SNORD50A), a U/A-rich C/D-box snoRNA, inhibits mRNA 3 processing by disturbing the Fip1-poly(A) site (PAS) interaction  binds to the highly conserved spliceosome component PRP8a, thereby impairing the recognition of specific flagellin-related transcripts by PRP8a  is the most widely studied lncRNA. Malat1 was first identified in human non-small cell lung cancer NSCLC; . A numbeby PRP8a . Actuallin vivo, resulting in the production of a non-transposase-encoding mature mRNA isoform in Drosophila germ cells (de novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway, and thereby inducing global AS/APA events  have been known as regulators of AS functioning in auxin-associated developmental processes such as lateral root formation , which is an evolutionarily conserved chromatin-remodeling factor . A recenIn addition, alternative RNA processing events can also lead to the formation of ncRNAs . More reEpigenetic modifications are dynamically regulated by different catalytic enzymes and reader proteins. This feature makes epigenetic mechanisms suitable for multiple biological processes, ranging from cell differentiation, development and environmental stress responses. RNA processing, a widespread mechanism of gene expression in eukaryotic cells, also play vital roles in multiple biological processes. During the last two decades, a great deal of efforts has been made in the crosstalk between epigenetic mechanisms and alternative RNA processing. As shown in Different from the chromatin-based alternative RNA processing, ncRNA impact AS/APA on RNA level, mainly by disturbing the binding of RNA processing factors . They caStudy on the interplay between epigenetic regulation and alternative RNA processing is a novel field which is still at an early stage. In addition to the important researches described above, there are still some outstanding questions regarding the underlying mechanism of alternative RNA processing due to the space constraints not discussed in this review, such as the identification of conserved factors involved in such regulation, a comparison of epigenetic regulation in RNA processing between animals and plants, and the precise epigenetic mechanisms of tissue- and environment-specific AS/APA events. Addressing the remaining questions will undoubtedly expand our understanding of the chromatin codes in the regulation alternative RNA processing.JZ and C-GD designed this review. JZ and Y-ZZ wrote the draft. JJ and C-GD edited it. All authors contributed to the article and approved the submitted version.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.


ColQ1 is active throughout the slow-twitch muscle, whereas pColQ1a is active at the motor endplate of fast-twitch muscle. We made a transgenic mouse line that expresses nuclear localization signal (NLS)-attached Cre recombinase under the control of pColQ1a (pColQ1a-Cre mouse). RiboTag mouse expresses an HA-tagged ribosomal subunit, RPL22, in cells expressing Cre recombinase. We generated pColQ1a-Cre:RiboTag mouse, and confirmed that HA-tagged RPL22 was enriched at the NMJ of tibialis anterior (TA) muscle. Next, we confirmed that Chrne and Musk that are specifically expressed at the NMJ were indeed enriched in HA-immunoprecipitated (IP) RNA, whereas Sox10 and S100b, markers for Schwann cells, and Icam1, a marker for vascular endothelial cells, and Pax3, a marker for muscle satellite cells, were scarcely detected. Gene set enrichment analysis (GSEA) of RNA-seq data showed that “phosphatidylinositol signaling system” and “extracellular matrix receptor interaction” were enriched at the motor endplate. Subsequent analysis revealed that genes encoding diacylglycerol kinases, phosphatidylinositol kinases, phospholipases, integrins, and laminins were enriched at the motor endplate. We first characterized the gene expression profile under translation at the motor endplate of TA muscle using the RiboTag technique. We expect that our gene expression profiling will help elucidate molecular mechanisms of the development, maintenance, and pathology of the NMJ.The neuromuscular junction (NMJ) is a prototypic chemical synapse between the spinal motor neuron and the motor endplate. Gene expression profiles of the motor endplate are not fully elucidated. Collagen Q (ColQ) is a collagenic tail subunit of asymmetric forms of acetylcholinesterase and is driven by two distinct promoters. p At the  pColQ1a . pColQ1 Massive parallel sequencing of RNA (RNA-seq) enabled the identification of gene expression profiles in specific tissues and cells at an unprecedented level of accuracy. Also, tissue-specific and cell-type-specific transcriptomic profiling is made available by fluorescence-activated cell sorting (FACS), laser capture microdissection, and single-cell RNA-seq  showed that mRNA immunoprecipitated with the HA tag from tibialis anterior (TA) muscle of pColQ1a-Cre:RiboTag mouse was enriched for motor endplate-specific genes, and mostly depleted for genes specific for Schwann cells and vascular endothelial cells. RNA-seq analysis of HA-immunoprecipitated mRNA revealed enrichment of genes encoding diacylglycerol kinases, phosphatidylinositol kinases, phospholipases, integrins, and laminins.In our study, we first generated a transgenic pColQ1a promoter  was amplified by PCR using genomic DNA of C57BL/6 mouse. We ligated pColQ1a, NLS, and Cre cDNA to generate pColQ1a-Cre . The nuclear localization signal (NLS) of the SV40 large T antigen sequence (5’-GAGCACCATGCCAAAAAAGAAGAGAAAGGTATCCAATTTAC-3’) was synthesized by Sigma–Aldrich. A 3519-bp mouse plQ1a-Cre . As a coTA and soleus muscles were harvested and fixed in 4% paraformaldehyde (PFA) for 1 h and were teased into muscle fibers in phosphate-buffered saline (PBS). The teased fibers were incubated in 100 mM glycine in PBS for 15 min at room temperature (RT). The fibers were washed in PBS three times for 15 min each and were incubated in a blocking buffer  for 1 h at RT. After blocking, the fibers were incubated with mouse monoclonal anti-Cre recombinase antibody  or anti-HA antibody  in a blocking buffer overnight at 4°C. After washed in 0.1% Triton X-100 in PBS several times, the fibers were incubated with FITC-conjugated goat anti-mouse IgG . AChR was stained with Alexa 594-conjugated α-bungarotoxin  for 2 h at RT. Finally, the fibers were covered by a coverslip with Vectashield with 4’,6-diamidino-2-phenylindole . Fluorescence signals were quantified by the IX71 microscope (Olympus) equipped with the MetaMorph imaging software (Molecular Devices).Frozen sections of TA muscle at 10 μm were incubated with a primary antibody  overnight at 4°C. The sections were added with FITC-conjugated goat anti-mouse IgG  and Alexa 594-conjugated α-bungarotoxin  followed by incubation for 1 h at RT. Finally, the sections were covered by a coverslip with Vectashield with DAPI, and fluorescence signals were quantified as stated above.ColQ1a-Cre. Then, 50 μl of each plasmid  was injected into the TA or soleus muscle and applied electrical pulses at 50 volts. The electric pulses were delivered in parallel to the muscle fibers with forceps-shaped electrodes  connected to an electric pulse generator . The electrical pulses were comprised of three pulses of a rectangular wave for 25 ms at a rate of 3 Hz, followed by three pulses of the opposite polarity with the same protocol. TA or soleus muscle was harvested on day 5 after electroporation.All mouse experiments were approved by the Animal Care and Use Committee of the Nagoya University Graduate School of Medicine and were performed following the relevant guidelines. Under deep anesthesia of a 6-week-old C57BL/6 mouse, 36 U hyaluronidase  was injected into the TA muscle 2 h before injection of pCMV-Cre or pColQ1a-Cre . Then, anti-HA antibody  was added to 1,200 μl of cleared homogenates and incubated for 6 h at 4°C. Then protein G magnetic beads  in an IP buffer were added and incubated overnight at 4°C. After incubation, beads were washed four times for 5 min in a high-salt buffer  in the presence of 400 units/ml RNase Inhibitor (Toyobo) and the cOmplete™ protease inhibitor . RNA was eluted in an RLT buffer with β-mercaptoethanol (Qiagen) and was purified by RNeasy Micro Kit (Qiagen) with DNase treatment. Total RNA was quantified and its quality was evaluated by RNA integrity number  of pooled TA muscles of 6-week-old pGapdh), and also to the ratio of input RNA. For both IP RNA and input RNA, TA muscles of 10, 11, 12, and 16 pColQ1a-Cre:Ribotag mice were pooled into four independent samples to be analyzed in quadruplicate. qRT-PCR primers are indicated in After confirming that the RIN was higher than 7.0, total RNA was reverse-transcribed using random hexamer primers (Thermo Fisher Scientific) and ReverTra Ace (Toyobo). Quantitative RT-PCR (qRT-PCR) was performed with the LightCycler 480 (Roche Applied Science) using the TB Green Premix ExTaq II (Takara Bio). Gene expression levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase , concentration (>1 ng/μl), no contamination of DNA, no degradation of RNA confirmed by rRNA peaks , and RIN (>7.0). RNA-seq of Input RNA and IP RNA was performed at Macrogen and BGI, respectively. At both companies, a sequencing library was prepared using the TruSeq Stranded mRNA kit (Illumina), and the library was read on Illumina NovaSeq 6000 (150 bp paired-end reads). As the amount of IP RNA remained low even after pooling TA muscles, IP RNA was linearly amplified by the Smart-seq2 protocols  q-value <0.25 were used as the cut-off criteria  and Cre cDNA to generate a pColQ1a-Cre plasmid , representing the quality of RNA, of the IP and input RNA were more than 7.0 , vascular endothelial cells (Icam1), and muscle satellite cells (Pax3) were markedly attenuated in IP RNA  was 0.6225  of 22,383 protein-coding genes in IP RNA and input RNA showed that the coefficient of determination , phosphatidylinositol kinases (PIK*), and phospholipases (PL*). Similarly, the gene set “ECM receptor interaction” included genes encoding integrins (ITG*) and laminins (LAM*). We made our own gene sets for the five gene families and performed GSEA analysis, and found that these genes were indeed enriched at the motor endplate of TA muscle  database. We found that two gene sets of “phosphatidylinositol signaling system” and “ECM receptor interaction” were enriched at the motor endplate  in pColQ1a binds to the myogenic differentiation factors (MDFs) of the MyoD family and drives muscle-specific gene expression  in pColQ1 and a fast intronic regulatory element (FIRE) in pColQ1a  and DGK-θ (DGKQ) were 140-fold and 27-fold enriched at the motor endplate, respectively, and were more enriched than DGK-ζ .FASTQ files are available at the DNA Data Bank of Japan (DDBJ) under the accession number DRA010277 (The animal study was reviewed and approved by Animal Care and Use Committee of the Nagoya University Graduate School of Medicine.KO conceived the project. KH, MI, and KO designed experiments. KH and JL performed most of the experiments with the help of MI, BO, and AM. RNA-seq analysis was performed by J-IT and TO. KH, MI, and KO wrote the article.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.


Acanthaster spp.) are among the most extensively studied coral reef taxa, largely owing to their devastating impacts on live coral cover during population outbreaks. Much of this research has however, been conducted in the western Pacific, although it is now apparent that there are several distinct species of Acanthaster spp. across the Indo-Pacific. The purpose of this study was to test for biogeographical variation in behaviour, comparing between Acanthaster planci at Lankanfushi Island in the Maldives and Acanthaster cf. solaris at Rib Reef on Australia’s Great Barrier Reef. The extent to which CoTS were exposed  was substantially higher (63.14%) for A. planci at Lankanfushi Island, compared to 28.55% for A. cf. solaris at Rib Reef, regardless of time of day. More importantly, only 52% of individuals were exposed at night at Rib Reef compared to >97% at reefs around Lankanfushi Island. Biogeographic variation in the behaviour of Acanthaster spp. was independent of differences in the size structure of starfish and coral cover at specific study sites, but may be attributable to other environmental factors such as habitat complexity or prey availability. This is the first study to explicitly test for biogeographical differences in the biology and behaviour of Acanthaster spp., potentially linked to species-specific differences in the causes and explanations of population outbreaks. However, we did not find evidence at this stage of differences in behavior among regions, rather behavioural differences observed were most likely products of different environments.Crown-of-thorns starfish (CoTS;  Acanthaster spp.) have gained considerable notoriety over the last few decades following outbreaks throughout the Indo-Pacific [Crown-of-thorns starfishes  has -Pacific –21 and tA. planci, as distinct from Acanthaster brevispinus that occurs in deep-water non-reefal environments [Acanthaster planci [Acanthaster solaris [CoTS were initially described from several different geographical regions across the Indo-Pacific and assigned distinct species names . Howeverronments . Extensironments  consisteronments . Most imr planci , which i solaris  in both  solaris  and appe solaris . Species solaris .Acropora and Montipora corals , leadiniewed by , 31) areiewed by . Similariewed by . For exaiewed by  reportediewed by , especiaiewed by , 28 and iewed by . GeograpA. planci from the northern Indian Ocean and A. cf. solaris from the western Pacific. In particular, we documented the number of starfish that were out in the open  and whether they were observed feeding, resting or moving, at different times of day. This study is important because it represents the first explicit test of inter-specific differences in the biology and behaviour of CoTS, potentially accounting for biogeographical differences in the incidence and severity of CoTS outbreaks [A. planci from the northern Indian Ocean, which is particularly underrepresented in previous research on CoTS [The purpose of this study was to explicitly test for biogeographical differences in behaviour of CoTS potentially linked to inter-specific differences, comparing between utbreaks , while a on CoTS . The ext on CoTS , but may on CoTS .A. planci was studied in the Maldives, in the central Indian Ocean. The earliest reports of outbreaks of A. planci on Maldivian reefs are from the 1970s [A. planci were again recorded in the Maldives in 1999 to 2007 [A.planci have affected South Male and Ari Atolls [A.planci in comparison with nearby reefs. Despite localised culling between October 2015 and June 2016, outbreaks of A. planci had caused extensive coral loss in the study area, which was further compounded by severe coral bleaching in May 2016 [Behaviour of he 1970s , though he 1970s . Elevate to 2007 . Most rei Atolls , startinMay 2016 , 40, 41.th to March 29th, 2017, two divers carefully searched for CoTS within a 1m wide belt on either side of the transect line, along each of the six transects. Divers swam parallel to each other, whilst visually checking crevices and overhangs across the 2m belt to allow for different angles of visual perception, paying particular attention to areas where feeding scars were observed to ensure the likelihood of detection of both exposed and cryptic animals. In all, each of the six transects were surveyed at four different times of day. Times were grouped into categories including morning (surveys between 06:30–10:00), midday (11:30–13:00), afternoon (14:00–17:15) and night (18:00–22:00). Surveys were undertaken with permission from Gili Lankanfushi Resort, who manage the section of reef surveyed, negating the need for a permit.To test for diurnal changes in the behaviour of CoTS at Lankanfushi Island, three permanent replicate 100m transects were established on both the reef crest (8m) and the reef slope (15m) on the inner atoll rim reef between Lankanfinolhu Island and Himmafushi Island (4°17’53.0”N 73°33’13.7”E). During surveys, which were conducted randomly at various times of day and night from February 25A. cf. solaris was conducted at Rib Reef, in the central GBR, Australia . In deeper, less complex habitats, surveys were extended to 5m either side of the transect line to ensure enough CoTS were captured to allow for statistical analysis. As densities of CoTS were analysed, rather than abundance per site, this did not confound results. Each of the ten transects were surveyed at several different times of the day, including at least one survey during daylight hours and one survey after dark. Survey times were placed into the same time categories as the Maldives surveys for comparative analysis. Surveys at Rib Reef were undertaken under Marine Parks permit G15/37363.1 issued by the Great Barrier Reef Marine Park Authority.Reef access was much more limited at Rib Reef, compared to Lankanfushi Island, in the Maldives. As such, two-four permanent replicate 50m transects were established during each of three distinct reef visits, with sampling conducted in a different location on each visit to account for prevailing conditions and changes in the distribution and abundance of CoTS. In all, five transects were surveyed on reef crest habitats (3m) and five on slope habitats (6m). CoTS were surveyed within 2.5m either side of transects on the reef crest, where there was high cover of tabular in situ to the nearest cm. The extent to which individuals were visible from above (or at 90° perpendicular to the substrate on particularly steep sections of reef) was then recorded to the nearest 10%. There were however, two distinct modes in these data, where the majority (85%) of starfish were either completely (100%) exposed or entirely concealed (0% exposed) beneath the reef substrate and only visible from an acute angle. As such, we categorised starfish as either cryptic, where ≤50% of the starfish was exposed, versus exposed where >50% of the starfish was visible from above. In addition to exposure, we also recorded whether each starfish was moving versus stationary, based on sustained movement of the body of the starfish. For starfish that were not moving, one or more arms were lifted away until it was possible to see whether the stomach was everted. Starfish with stomachs partially or fully everted were recorded as feeding, whereas individuals with stomachs completely retracted were considered to be resting.All CoTS recorded within the area of belt transects at both Lankanfushi Island, Maldives and Rib Reef, central GBR, were measured (diameter from arm tip to opposite arm tip) Coral cover was determined along each of the transects, which were demarcated by a fibreglass tape, at both locations before CoTS surveys began. Benthic cover was determined to the lowest possible taxonomic level, with scleractinian corals identified to genus and growth form, using the point intercept method every 50cm.To assess the variation in CoTS density between reef locations and habitats, a linear model with gaussian error distribution was constructed, to which the time of observation  and interaction terms for location:Habitat and location:Time was also included. Similarly, a linear model was constructed to determine variations in the size of CoTS individuals between reef locations, habitats and time of day, using the same interaction terms as above. Analysis of variance and post-hoc comparisons (using R package ‘lsmeans’) of factor levels was also conducted to determine the significance of these variations in density and size. Potential interactions were tested and retained if they improved model fit on the basis of the corrected Akaike information criterion . To test2). Exposure was recorded as a binary variable (either cryptic or exposed) and thus modelled using a Bernoulli distribution using the R package ‘brms’ [To test for variation in the cryptic behaviour of CoTS, we constructed a Bayesian generalised linear model to investigate the effects of 7 different predictor variables; location (Lankanfushi Island versus Rib Reef), habitat (crest and slope), body size, coral cover, behaviour (moving or stationary), time of day  and CoTS density (per 100me ‘brms’ . All pote ‘brms’ . Effects2 ±1.08 SE) were significantly higher than recorded at Lankanfushi Island, Maldives  however, there was no difference detected among habitats  or time of day  , however there were also significant interactions between reef and observation time  and reef and habitat ). At Rib Reef, slightly larger starfish were found on the slope (32.12cm ± 0.39SE) compared to the crest  and starfish observed in the morning were significantly larger compared to all other time periods  however, these differences were very low in magnitude . In contrast to Rib Reef, slightly smaller starfish were observed in the morning compared to the night and afternoon , however these differences were also low in magnitude (~3cm) (Average densities of CoTS recorded at Rib Reef (6.29 starfish per 100m = 0.44) . The sizectively . The avee 1-2cm, . At Lanke (~3cm) .Porites being the most abundant coral genus at Lankanfushi Island (15.44% ± 6.38SE), while coral cover at Rib Reef was dominated by the preferred prey Acropora (31.75% ± 4.88SE) , with  4.88SE) .Overall, CoTS were exposed  in only 37.14% (410/1104) of records. The proportion of CoTS exposed was however, substantially higher at Lankanfushi Island (63.14%) compared to Rib Reef (28.55%), regardless of time of day. The proportion of exposed CoTS increased through the day at both locations, with lowest levels recorded in the morning (33.97% at Lankanfushi Island and 13.78% at Rib Reef) and highest levels of exposure recorded at night (97.47% at Lankanfushi Island and 52.09% at Rib Reef).Acropora at each site, as well as the size , and the behaviour of starfish    however, this model had poorer performance overall according to widely applicable information criterion.The best model  to account for variation in cryptic behaviour of CoTS across both locations included an interaction between reef (Lankanfushi Island versus Rib Reef) and time of day , percent  moving) . At both moving) . The pro moving) . Unsurpr moving) . The pro = 0.09) . HoweverA. planci that were observed feeding (as opposed to moving or resting) was higher at Lankanfushi Island (44.03%) compared to A. cf. solaris at Rib Reef . Conversely, a significantly higher proportion of CoTS were recorded resting at Rib Reef (56.99%) compared to Lankanfushi Island , whereas the proportion of starfish actively moving was similar between locations . It was apparent that the proportion of individuals observed feeding at Lankanfushi Island was more consistent throughout the day (ranging from 38–64%) and was significantly higher during the midday surveys (64.5%) compared to night time , whereas the incidence of feeding by A. cf. solaris at Rib Reef increased throughout the day. The proportion of A. cf. solaris recorded feeding at night (65.4%) was more than twice that recorded at any time during the day . To test whether this result was attributable to the large number of smaller  starfish at Rib Reef, whereby smaller starfish may be more cryptic and tend to feed nocturnally [A. cf. solaris ≥25 cm total diameter, the proportion of individuals feeding increased from 13.02% in the morning to 62.92% at night.The proportion of turnally , 3, 28, Acanthaster spp. over the last three decades , wiiewed by ). Despitiewed by , there hiewed by , 35). Itiewed by ) from diA. planci from the Maldives and A. cf. solaris on the GBR were higher levels of cryptic behaviour for CoTS on the GBR, especially at night. The proportion of starfish exposed at night was higher than during the day at both locations, but for A. cf. solaris on the GBR only 52% of individuals were found out in the open during the night, while a similar proportion remained largely concealed within and/ or beneath live or dead corals. In contrast, at Lankanfushi Island in the Maldives, virtually all individuals of A. planci (>97%) were fully exposed at night. Similarly, Ormond and Campbell [Acanthaster spp. from the Red Sea, where CoTS were reported to feed almost exclusively at night. Increased nocturnal exposure in the Maldives did not translate into higher feeding incidence, though movement was greatest at night. At Rib Reef on the GBR however, greater exposure at night was accompanied by pronounced nocturnal feeding. In some locations, Acanthaster spp. are reported to switch from feeding mainly at night to feeding day and night with the onset of population outbreaks . These Acropora) at this location. Reef habitats with high cover of complex corals are likely to offer more opportunities for Acanthaster spp. to remain concealed, even while continuing to feed. While coral composition and habitat (slope vs crest) had no effect on the probability of exposure for CoTS that were recorded, the effect of percent Acropora was likely masked in our model by the inclusion of reef location. Indeed, our results showed percent Acropora performing the same function as reef location, when the latter was removed from the model. Despite this, it is also likely that detectability itself varies with coral cover and habitat complexity [A. planci since 2015, have caused extensive depletion of acroporid and pocilloporid corals, which are generally preferred prey of Acanthaster spp. [Porites) [Acropora) at Rib Reef likely explains higher densities and wider size ranges of CoTS recorded at this location. Despite being subject to moderate bleaching in 2016 and 2017 [A. cf. solaris, there was still moderate cover of Acropora spp. at Rib Reef, allowing prey to not only sustain large densities of adult starfish, but also facilitate secondary waves of recruitment and replenishment.The differences in behaviour of CoTS between the two locations, particularly the increased levels of exposure at Lankanfushi Island, is most likely linked to the lower levels of complex branching corals  , combineand 2017  and sustAcanthaster. Predation risk will vary with size, abundance and composition of potential predators which will, in turn, vary due to inherent biogeographical patterns, as well as overarching influences of anthropogenic extraction and reef degradation [A. cf. solaris with sublethal injuries, presumed to be caused by sublethal predation, was higher inside a marine reserve compared to nearby areas that were open to fishing [Acanthaster spp. respond to increased predation risk inside marine reserves by being more cryptic, this might lead to differences in detectability, which will need to be considered when comparing density estimates inside versus outside of marine reserves . There s (e.g., , 55.A. planci at Lankanfushi Island in the Maldives and A. cf. solaris at Rib Reef on Australia’s GBR. These preliminary results from just two locations suggest these differences are a product of variation in extrinsic factors, in particular coral prey availability and structural complexity afforded by differences in coral composition, along with possible differences in predation risk. It is important, however, given recognition of species boundaries for Acanthaster spp. across the Indo-Pacific [A.planci in the Maldives, which is useful for the management and control of CoTS throughout this region, but more work is required to establish whether any behavioural differences are reflective of intrinsic biological differences between species of Acanthaster.This study has revealed differences in behaviour between -Pacific , 25, tha


In late December 2019, a cluster of patients with pneumonia caused by an unknown pathogen was reported from Wuhan, Hubei Province, China. The pathogen has been identified as a novel coronavirus, severe acute respiratory syndrome 2 (SARS-CoV-2) and the disease has been named as coronavirus disease 2019 (COVID-19). The objective of this study was to perform the first holistic scientometric evaluation of coronavirus publications.Our main source for this study was Web of Science Collection database. All items published between 1980 and 2019 were included. A distribution map of global production in coronavirus literature and scientometric networks were generated.The United States, China, Germany, the United Kingdom, and Netherlands were the most productive countries. Publications in coronavirus literature have been produced from almost every country in the world, except for some countries in Asia and Africa.While in the 1980s, the United States and developed countries from Europe were major source countries and the virus was identified only as an animal disease in the literature and its biological and genetic structure was investigated, in the 2000s, China became a major contributor of coronavirus literature because the SARS outbreak originated from southern China. Almost all most-cited publications in this period are related to SARS and the ACE2 protein. The disease was coined as coronavirus disease 2019 (COVID-19). Recently, the official name of the virus that causes COVID-19 has been announced by World Health Organization (WHO) as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).2In late December 2019, a cluster of cases with pneumonia caused by an unknown etiology was reported from Wuhan, Hubei Province, China.3 The academic literature lacks an in-depth scientometric study evaluating coronaviruses and disease caused by them. Herein, we aimed to perform a holistic scientometric assessment of the coronavirus literature.Scientometrics, also known as “Science of science,” is a popular statistical method analyzing scientific literature thoroughly in a certain field.4 We used keywords of “coronavirus,” “SARS”, “SARS-CoV”, “MERS”, “MERS-CoV”, and “COVID-19” for our analysis. All items published between 1980 and 2019 were included, and documents produced in 2020 were excluded for our major analysis. A distribution map of global production of coronavirus literature was generated by a free Web source named GunnMap.5 VOSviewer freeware was used to create scientometric networks.6Our main source of this study was Web of Science  Core Collection database. We preferred WoS as our main data source because it provides data analysis for publications and citations and allows the results to be sorted according to the number of citations. In addition, WoS attribution data are considered more reproducible and reliable than other databases, and WoS is used as the standard by certain official organizations.A total of 13,833 documents indexed in WoS Core Collection between 1980 and 2019 were found in coronavirus literature, 7339 of which were open access. The peak year for publication was 2016 with 837 papers and 106 articles have been produced in 2020 so far. English was the major language of coronavirus literature (96.957%) followed by French, German, Spanish, and Chinese . Original articles covered 80.518% with 11,138 documents of all coronavirus literature followed by reviews, proceeding papers and editorials  ranked first in coronavirus literature with 4894 articles (35.379%) followed by China, Germany, the United Kingdom (UK) and the Netherlands . Publications in coronavirus literature have been produced from almost every country in the world, except for some countries in Asia and Africa . Yuen frJournal of General Virology and University of Würzburg (Germany) stood out in their categories in this period. H-index of coronavirus literature for this period was calculated as 79 and total number of citations was 21,682 . Average citations per item were 33.83. The most cited document in this period was an original article titled “Characterization of An Efficient Coronavirus Ribosomal Frameshifting Signal - Requirement for An RNA Pseudoknot” written by Brierley et al. . The most prolific author was Termeulen V with 37 items. The y et al. .Journal of Virology was the prominent source title in this decade with 226 articles (13.501%), and University of Southern California (USA) was the most contributor institution with 96 papers (5.735%). H-index was measured as 105 and published documents were cited 63,445 times in this period . The most cited document was an original article titled “Community Study of Role of Viral-Infections in Exacerbations of Asthma In 9-11-Year-Old Children” by Johnston et al. . Lai from University of Southern California (USA) was the most prolific author with 70 documents. The n et al. . As we and “cat” .N = 1679, 1202, 324, 322, and 278 papers, relatively). University of Hong Kong (China), Chinese Academy of Sciences (China), and Chinese University of Hong Kong (China) were the most contributor institutions in this period . Yuen form the University of Hong Kong (China) was the most prolific author with 110 papers (2.287%) and the most productive source titles were detected to be Journal of Virology, Virology and Advances in Experimental Medicine and Biology . H-index for this decade was 165 and total number of citations was 189,424 . Average citations per item were 39.38. The most cited article in this period was an original article written by SARS Working Group, Ksiazek et al., titled “A novel coronavirus associated with severe acute respiratory syndrome” published in The New England Journal of Medicine in 2003  were open access and 80.7% of all items were original articles. The United States, producing 33.586% of all coronavirus literature, ranked first as ever, followed by China, Germany, Saudi Arabia, and The United Kingdom . University of Hong Kong (China), National Institutes of Health (USA), Chinese Academy of Sciences (China), and University of California System (USA) stood out from the rest  and Drosten from Charité – Universitätsmedizin (Germany) was the most prolific author of this period with 113 articles. A total of 6111 articles were analyzed for the citation analysis. H-index of this period was measured as 106. Indexed documents were cited 109,418 times . An original article titled “Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia” written by Zaki indexed in The New England Journal of Medicine in 2012 .By the date of the writing of this study , 6679 documents were indexed relevant to CoVid-19 in PubMed and 4605 items were included in WoS databases. All included documents were published and indexed in 2020. Total number of open-access coronavirus articles produced in 2020 was 4605. China ranked first with 1184 documents (25.711%) followed by the United States, the United Kingdom, Italy, Germany, India, Canada, Australia, France, Iran, Switzerland, Brazil, and South Korea . The most contributor institutions were University of London (UK), Huazhong University of Science Technology (China), Harvard University (USA), University of California System (USA), Wuhan University (China), University of Hong Kong (China), Chinese Academy of Sciences (China), Zhejiang University (China), University of Oxford (UK) and Fudan University (China) . Major source titles for 2020 were It is important to analyze the coronavirus literature based on periods, because it helps us understand the progression of the disease. In the 1980s, the United States and developed countries from Europe were major source countries. During this period, the virus was identified only as an animal disease in the literature, and its biological and genetic structure was investigated. In the second decade we investigated (1990s), the United States and European countries stood out again and the coronaviruses started to be examined in terms of whether it affected the health of animals and people with asthma . In fact7 In the literature of the 2000s, China became a major contributor of coronavirus literature because the SARS outbreak originated from Guangdong province of southern China. Almost all most-cited publications in this period are related to SARS and ACE2 protein. SARS-CoV is “thought to be an animal virus from an as-yet-uncertain animal reservoir, perhaps bats, that spread to other animals (civet cats)” according to WHO. We want to draw attention to one of the most cited articles in this period titled “Bats are natural reservoirs of SARS-like coronaviruses” written by Li et al. published in Science in 2005  were reported from 27 countries. Outbreaks caused by human-human transition in Saudi Arabia in 2014 and South Korea in 2015 occurred.10We found only one scientometric document relevant to coronavirus literature. It was a letter with limited data and no tables or images revealing scientometric networks. The authors reported that the United States and China had primary roles in the literature.2 By the date of writing this study, a total of 167,682 confirmed patients and 6456 deaths were reported from 157 countries and territories, and the COVID-19 outbreak has been officially declared as pandemic. We hypothesized that the COVID-19 pandemic could be prevented if the suggestions of the articles previously published, such as that by Lie et al., were taken into consideration and the wet-markets were completely closed to cut off the human-animal interaction. Scientometric studies should be performed in certain diseases that previously caused epidemics to prevent global spread in the future and to take urgent measures.In late 2019, a novel coronavirus causing SARS-like pneumonia was identified. This novel virus was temporarily named as SARS-CoV-2 and the disease has been coined as COVID-19 by the WHO.


Nucleic acids, aside from being best known as the carrier of genetic information, are versatile biomaterials for constructing nanoscopic devices for biointerfacing, owing to their unique properties such as specific base pairing and predictable structure. For live-cell analysis of native RNA transcripts, the most widely used nucleic acid-based nanodevice has been the molecular beacon (MB), a class of stem-loop-forming probes that is activated to fluoresce upon hybridization with target RNA. Here, we overview efforts that have been made in developing MB-based bioassays for sensitive intracellular analysis, particularly at the single-molecule level. We also describe challenges that are currently limiting the widespread use of MBs and provide possible solutions. With continued refinement of MBs in terms of labeling specificity and detection accuracy, accompanied by new development in imaging platforms with unprecedented sensitivity, the application of MBs is envisioned to expand in various biological research fields.  Subject Areas: Molecular Genetics; Biodevices; Nanotechnology Nucleic acids are biological polymers with unique properties such as specific base pairing, well-defined composition, and predictable structure and size, making them ideal materials for engineering artificial nanodevices that can interface with the biological world . For livAlthough MBs have shown great promise for intracellular RNA analysis, their potential has not yet been fully realized, as the majority of existing MB-based assays rely on bulk fluorescence measurements, which are suitable for visualizing highly abundant RNA but cannot reveal the spatiotemporal heterogeneity among individual RNA transcripts, information that is crucial for better characterization of the molecular basis of human health and disease . Given thttp://www.molecular-beacons.org/MB_publications.html#cap1. More recently, reviews that overview the field of single-molecule imaging methods have briefly outlined the capacity of MBs for illuminating RNA activities at the single-molecule level , a bacterial exotoxin that interacts with cholesterol, to form pores of 30 nm in size at the plasma membrane . It has O-methyl RNA (2Me MBs) nucleotides, it is now fairly clear that both types of MBs are highly prone to nonspecific opening caused by nuclease digestion or nonspecific protein binding inside cells, leading to the generation of false-positives. To circumvent this problem, one widely used approach has been to synthesize the MB backbone with unnatural nucleotide analogs that have been developed for protecting antisense agents from nuclease digestion or nonspecific protein binding (LOOP MB) can exhibit reduced nonspecific binding while maintaining nuclease resistance in various cell types  or 2′- binding   approach , in whicoskar mRNAs in Drosophila melanogaster oocytes  to label native  oocytes . By follTo illuminate RNA transcripts at the single-molecule level, a general approach has been to employ multiple fluorescent probes to label a single RNA transcript, such that each labeled transcript appears as a bright spot under fluorescence microscopy. For MB-based single RNA imaging, one highly adoptable approach involves genetic modification of a target RNA with an MB-tag containing multiple copies of the same sequence that can hybridize to a single type of MB  A. AlternLOOP MB  mRNA transcript to harbor 96 tandem repeats of a 50-nucleotide unique MB target sequence . Using 2LOOP MB . Specifioskar mRNAs in Drosophila oocytes by using the MS2 system  in a single guide RNA (sgRNA)-mediated fashion   , our gro system) . Hybridi system) . Additio system)   . Only th system)  with comDespite the great progress made in the MB technology, several challenges still remain to be overcome before MBs can be truly reliable in furthering our understanding of the molecular basis underlying normal physiology and pathophysiology. Below, some of these challenges are discussed and their potential solutions are provided.One crucial design advantage of MBs over conventional single-stranded unstructured (linear) probes is the self-quenchability in the absence of target hybridization, enabling sensitive live-cell imaging without the need to wash away unbound probes. However, due to general imperfect quenching of the fluorophores by currently available quenchers, MBs can still emit fluorescence in the closed configuration even if they are completely resistant to nonspecific opening. Therefore, when the use of MBs at a high concentration is required , as is the case when intracellular RNA is imaged, unbound MBs may accumulate within a detection region and the resulting background signal may mask the signal arising from specific MB-target hybridization within the region. We should mention that the detectable single β-actin mRNA dynamics with only two MBs in the growing axon reported by in situ hybridization (Based on studies in the test-tube environment, MBs have been reported to be capable of differentiating perfect targets from targets with a single base mismatch compared with single-stranded linear probes, as the stem domain can impose an energy barrier on loop-target hybridization . More redization .Besides being unique, the target RNA region for an MB should be free of secondary structure. Moreover, although it is thought that MBs exhibit improved binding capacity for their target RNAs when compared with RNA-binding proteins and thus may displace the bound proteins from target sites to result in successful labeling , displacWith the development of efficient probe delivery strategies, methods to minimize nonspecific signals, as well as high-resolution fluorescence microscopy, we have witnessed the transition of MBs from being only useful for reporting RNA activity based on bulk fluorescence to a highly sensitive tool capable of illuminating the spatiotemporal dynamics of specific RNAs and even genomic loci at the single-molecule level. With further possible approaches for enhancing the detection sensitivity and accuracy, accompanied by the use of more sophisticated imaging techniques such as multispectral imaging  and supe


Nearly 7.5% of all human protein-coding genes have been assigned to the class of RNA-binding proteins (RBPs), and over the past decade, RBPs have been increasingly recognized as important regulators of molecular and cellular homeostasis. RBPs regulate the post-transcriptional processing of their target RNAs, i.e., alternative splicing, polyadenylation, stability and turnover, localization, or translation as well as editing and chemical modification, thereby tuning gene expression programs of diverse cellular processes such as cell survival and malignant spread. Importantly, metastases are the major cause of cancer-associated deaths in general, and particularly in oral cancers, which account for 2% of the global cancer mortality. However, the roles and architecture of RBPs and RBP-controlled expression networks during the diverse steps of the metastatic cascade are only incompletely understood. In this review, we will offer a brief overview about RBPs and their general contribution to post-transcriptional regulation of gene expression. Subsequently, we will highlight selected examples of RBPs that have been shown to play a role in oral cancer cell migration, invasion, and metastasis. Last but not least, we will present targeting strategies that have been developed to interfere with the function of some of these RBPs. TP53, CDKN2A, RB1, RBL1/2) as well as activation of oncogenic signaling pathways including epithelial growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), and Janus kinase/signal transducers and activators of transcription (JAK/STAT) that contribute to oral cancer progression [TP53 loss-of-function mutations, which occur in early stages often combined with mutations in the Rb pathway [NOTCH1 have been identified suggesting a tumor-suppressive role of Notch signaling in head and neck squamous cell carcinomas (HNSCC) in general and OSCC in particular [Cancers of the lip, tongue and oral cavity were estimated to collectively account for approximately 355,000 newly diagnosed neoplasia and over 177,000 cancer deaths in 2018 ranking oral cancer on position 16 and 15 in terms of incidence and mortality, respectively ,5. Knowngression ,10. In H pathway ,12. In Hrticular . Howeverrticular ,14.https://seer.cancer.gov/csr/1975_2006/) [Identification of novel and high-confidence therapeutic targets in OSCC is an urgent need and despite significant advances in the diagnosis and treatment, the 5-year survival rate is ~60%, which decreases to ~30% for patients with advanced disease 5_2006/) ,16. One 5_2006/) . Moreove5_2006/) ,19,20. L06/ ,16. 5_2006/) ,22. The 5_2006/) . In cont5_2006/) ,24,25. T5_2006/) ,24. Once5_2006/) . Thus, uThe development of metastasis is a multifactorial phenomenon, which, in the case of head and neck cancers, includes the following factors that significantly increase distant metastasis: extracapsular extension, location of the primary tumor in the hypopharynx, higher stage grouping, N classification, locoregional failure, including relapse and persistent disease . HPV posOf note, cellular plasticity conferred by EMT and other mechanisms that lead to morphological and functional changes requires robust reprogramming of gene expression programs. For example, several thousand genes change their expression in a context-dependent manner during EMT . These pIn this review, we will focus on RNA-binding proteins and we will offer a brief overview about their general contribution to post-transcriptional regulation of gene expression. Subsequently, we will highlight selected examples of RBPs that have been shown to play a role in oral cancer cell migration, invasion, and metastasis. Last but not least, we will present targeting strategies that have been developed to interfere with the function of some of these RBPs.Over the past decade, RBPs have been increasingly recognized as important regulators of molecular and cellular homeostasis. RBPs regulate diverse cellular processes such as cell survival, pluripotency of embryonic stem cells, and immune cell function, as well as aid in the transition between cellular states in response to stimuli, e.g., during differentiation, cellular stress, or viral infection ,56,57,58In order to fulfill their cellular tasks and to influence the fate of hundreds to thousands of transcripts RBPs interact with specific sequences or RNA secondary structure elements within their respective target RNA to regulate its post-transcriptional processing, i.e., its alternative splicing, polyadenylation, stability and turnover, localization, or translation as well as editing and chemical modification. Therefore, RBPs typically contain discrete domains for the purpose of binding RNA and more than 40 RNA-binding domains (RBDs) have been described . ImportaWe have summarized the classical as well as emerging hallmark functions of RBPs in As mentioned earlier metastases are the major cause of cancer-associated death. However, the role and architecture of RBPs and RBP-controlled expression networks in the diverse steps of the metastatic cascade is only incompletely understood. Here, we introduce some examples of RBPs whose association with the malignant spread of oral cancer cells has been recently established.The adenosine deaminase acting on RNA 1 (ADAR1) protein is involved in adenosine-to-inosine (A-to-I) editing in double-stranded RNA and has been implicated in several human cancers ,98. BecaThe ATP-dependent DEAD-box RNA helicase DDX3 is involved in multiple steps of RNA metabolism from transcription to translation control, and DDX3 participates in several signaling processes, e.g., innate immune response and Wnt signaling ,105. MecELAVL1. HuR, in contrast to its family members HuB (ELAVL2), HuC (ELAVL3) and HuD (ELAVL4), is ubiquitously expressed in human tissues and is a well-established regulator of post-transcriptional gene expression whose activity is not only controlled by abundance, but also by its subcellular localization [p < 0.025), grade (p < 0.014), lymph node (p < 0.050), and distant metastases (p < 0.025) as well as reduced survival (p < 0.023) [One of the best characterized RBPs in human cancer cells is HuR, the protein product of the embryonic lethal and abnormal vision gene lization ,114,115.lization ,117,118.lization . RNA binlization . Importalization ,122,123.lization . In orallization . Moreovelization . Analysi< 0.023) . Thus, c< 0.023) .in situ compared to normal epithelium. Moreover, higher expression of ESRP1/2 was also detected in advanced OSCC and cancer nests in metastatic lymph nodes. In contrast, careful analysis revealed a loss of ESRP1 and ESRP2 expression in cancer cells that penetrated through the basement membrane into the stroma as well as in those cells that invaded from cancer nests into stromal tissues. Hence, down-regulation of ESRP1 and ESRP2 in OSCC might be restricted to cells that acquire a motile phenotype during cancer invasion [Over the past decade RNA-sequencing technologies and sophisticated analysis pipelines have led to a wave of discoveries regarding the causes and functional relevance of alternative splicing, which has increased the awareness of its potential role in the development and progression of cancer and other diseases . These ainvasion . These finvasion . Anotherinvasion . Of noteThe mammalian Insulin-like growth factor 2 mRNA-binding protein (IGF2BP) family comprises of three RBPs. Two members of the family, IGF2BP1 and IGF2BP3, are de novo synthesized in various human cancers and function as bona fide oncofetal proteins . All famLIN28B gene belongs to the lin-28 family that was initially discovered in Caenorhabditis elegans where it controls developmental timing [The l timing . In humal timing . The difl timing . A huge l timing . Intrigul timing . In the l timing . Neverthl timing . Notablyl timing . Anotherl timing . Again, l timing . In ordel timing . Moreovel timing . Taken tIn the last couple of years, chemical modifications in RNA—also referred to as ‘RNA epigenetics’ or the ‘epitranscriptome’—have been extensively studied and their dynamic distribution as well as the impact of modified bases on the metabolism and fate of both coding and non-coding transcripts has been revealed ,166,167.In summary, several individual RBPs have been identified as important regulators of oral cancer cell phenotypes as well as prognostic markers in OSCC. The herein presented examples highlight the broad variety of RBP functions and mechanisms of activation and establish these RBPs as clinically relevant targets in OSCC. Additional RBPs with a role in oral cancer cell migration, invasion, and metastasis are summarized in In this final chapter we will focus on small molecules and chemistry-based strategies to target OSCC-associated RBPs. We will not discuss other genetic or non-genetic option, e.g., gene therapy, small interfering RNA (siRNA), circular RNAs, aptamers, or RNA-targeted therapies, which were recently discussed elsewhere ,189.Small molecules have been developed to target disease-related proteins like ion channels, G-protein-coupled receptors, nuclear receptors, and kinases and these molecules are commonly and successfully used as therapeutics in clinical applications . HoweverA widely applied strategy to interfere with the function of an RBP is to modulate the interaction with its key coding or non-coding target RNA that is thought to be essential for the disease-related function of the RBP. This can be achieved either via targeting the RNA or the RBP directly. RNA-targeting strategies, which include nucleotide-based agents that target unstructured regions of RNA as well as small molecules that bind directly to structured RNAs are showing promising clinical success and have been discussed elsewhere ,224,225.First, small molecule inhibitors of RBPs are commonly developed from hits initially identified in high-throughput screen and the chemical space covered in the screening libraries will therefore determine the biological activity and specificity of the hit compound, and ultimately the success of the screen. Thus, more complex and diverse libraries should be used in order to retrieve compounds with new biological activities against RBPs. To this end, libraries should include both protein-targeting and RNA-targeting molecules to ensure that RBP-RNA interactions are comprehensively interrogated. Second, the structures of RBPs, both in the presence and absence of their RNA substrate(s) should be systematically solved and catalogued. This will allow subsequent hit optimization by structure-activity relationship analysis and will ultimately shift the discovery of RBP inhibitors from screening-based approaches towards rational drug design-based approaches.Second, the screening goals should be re-evaluated. For example, instead of aiming to identify small molecules that compete with RNA for binding to RBPs, it might be possible, e.g., by leveraging structural information, to identify allosteric binding pocket in RBPs that could be targeted by small molecules to block functional relevant conformational changes in RBPs. Additionally, unbiased screening to identify high-affinity binders irrespective of their binding sites on the RBP might enable the development of novel, chimeric molecules, so called proteolysis targeting chimeras (PROTACs) that could be used to induce proteasomal degradation of the target RBP .Another aspect that should be considered in the design of future screening and targeting strategies is the dual functioning of some RBPs. For example, RBPs might not only act by modulating RNA metabolism, but might also bind to DNA and other proteins, or contain (pseudo)kinase and RING domains, which suggest non-canonical, potentially RNA-independent functions and novel options to modulate the activity of RBPs ,233,234.Last but not least, functional genetic screens could be used to identify synthetic vulnerabilities that could be exploited to target cancer cells that show an altered expression or mutations in RBP genes ,239,240.RNA-binding proteins comprise a huge and diverse class of important regulators of cellular plasticity and homeostasis. Their significant contribution to disease phenotypes has been broadly established. However, the therapeutic targeting of RBPs remains challenging. Thus, future research endeavors should try to identify disease-specific RBP-dependencies, explore indirect (synthetic) vulnerabilities, and develop innovative targeting approaches or leverage existing concepts to make this class of proteins accessible for RBP-directed clinical interventions.


Chinese employees may experience and respond to guanxi human resource management (HRM) practice . Little has been done to examine the linkage between guanxi HRM practice and employees’ occupational well-being. This study investigates the psychological process of how guanxi HRM practice affects employees’ occupational well-being. The theoretical model of this study proposes that employee psychological safety mediates the relationship between guanxi HRM practice and occupational well-being, while collectivistic team culture moderates the relationship between guanxi HRM practice and psychological safety. Multi-level data from 297 employees nested within 42 teams support all hypotheses. This study reveals the cross-level effects of guanxi HRM practice and providing practical suggestions for future research on psychologically safe and healthy work environments. Over the past 40 years, occupational well-being has been gaining momentum as it has been realized to be a central concern not only for individual health but also for managers and organizations in terms of performance and overall productivity ,2. Many Recent research that has focused on guanxi HRM practice has regarded it as the stressor of employee outcomes, which is defined as the extent to which HRM decisions are made based on personal relations . Guanxi Furthermore, we still have much to learn about the “black box” of how guanxi HRM practice affects employees’ occupational well-being. Of the studies examining the influence of guanxi HRM practice on employee emotional exhaustion , few haThirdly, it remains largely unanswered whether the negative effects of guanxi HRM practice emerge in all circumstances. Wu and Chaturvedi  providedThe present study makes several contributions to the literature. First, to the best of our knowledge, few empirical studies have examined the effects of HRM on employees’ occupational well-being. We address this research gap by investigating one pervasive HRM style in the workplace of China: guanxi HRM practice, which may play an important role in connecting occupational well-being to that perceived by employees. Secondly, we identify employees’ perceptions of psychological safety as a key mediating mechanism that links guanxi HRM practice to occupational well-being. Finally, since there are unclear boundary conditions for the effects of guanxi HRM practice, we draw from the cultural value perspective to examine when guanxi HRM practice is related to employees’ perception, which, as a result, decreases employees’ psychological safety. The overall theoretical model is presented in Guanxi, a Chinese term referring to mutually and reciprocal interpersonal connections, has been pervasive and extensively examined in China and the Chinese-language areas of Taiwan, Singapore, and other overseas Chinese communities for the last few centuries ,18,19. GGuanxi HRM practice refers to the degree to which HRM decisions, such as recruitment, task allocation, performance appraisal, promotion, and compensation, are influenced by personal relationships . In othePsychological safety is defined as “feeling able to show and employ one’s self without fear of negative consequences to self-image, status, or career”  (p. 708)Collectivism is typically conceptualized as the opposite of individualism, especially when contrasting East Asian and Western cultures. Within a workgroup, collectivistic culture presents a pattern of shared values, beliefs, and norms that emphasize interdependence, cooperation, and harmony between teams of employees . CollectWell-being is a multidimensional construct containing different types of well-being  . In the As a resource-based theory of stress, conservation of resources (COR) theory provides a theoretical explanation of whether and how HRM practice impacts employee well-being. COR theory acknowledges the importance of resources for sustaining employee well-being. When these resources are threatened or lost, their loss may evoke distress, eventually resulting in decreased well-being . In the According to the COR theory, when employees realize they do not have many resources, they will lose their enthusiasm and report more burnout and pressure, resulting in lower occupational well-being. Guanxi HRM practice as a work demand may threaten or deplete the individual’s valued resources. There is evidence that guanxi HRM practice is related to employee psychological conditions. For instance, Chen and O’Leary  found thAccording to the COR theory, guanxi HRM practice can be viewed as a job demand as it exerts an energy-draining effect on employees through a stressful process that may undermine employees’ occupational well-being. On the one hand, guanxi HRM practice may decrease employees’ job resources, such as fairness perception. Guanxi HRM practice creates a work environment in which opaque promotion rules, informal exchange of opinions and sharing of valuable information, and lack of performance orientation in compensation and appraisal are common characteristics in workgroups . When guOn the other hand, guanxi HRM practice may deplete employees’ personal resources, such as certainty and perceived control. Employees may experience high uncertainty when they perceive that recruiting processes, task allocations, and performance appraisal are often decided based on guanxi rather than rules and regulations . SuperviHypothesis 1.Guanxi HRM practice is negatively related to employees’ occupational well-being.From a COR theory perspective, guanxi HRM practice may trigger a spiral loss of psychological safety which may, in turn, harm employees’ occupational well-being at follow-up. Psychological safety is perceived as the freedom to show and employ oneself . The CORHypothesis 2.The negative relationship between guanxi HRM practice and occupational well-being is mediated by psychological safety.Due to the blurring of organizational boundaries and the proliferation of self-managing teams, team culture is increasingly important to HR practice . People Hypothesis 3.Team-level collectivistic culture moderates the relationship between guanxi HRM practice and psychological safety; the negative relationship is strengthened when collectivistic culture is higher and weakened when collectivistic culture is lower.Data were collected from two private firms and two state-owned companies in central China. We contacted the senior directors of these firms and introduced the research project. They showed interest in this project and agreed to take part in anticipation of receiving an overall report regarding the results of the study. We explained how this study would be carried out so that they could instruct their employees as to the details of this investigation. All respondents were assured that all information will be kept confidential and gave their informed consent electronically when the survey was carried out.To minimize the potential for common method bias, the data were collected from multiple sources. Five hundred and eighty-eight employee participants were asked to respond to demographic questions and scales related to guanxi practice, psychological safety, and occupational well-being. A separate survey was employed to collect supervisors’ evaluations of the team’s collectivistic culture. After deleting missing data and matching employee data with team leader data, complete survey responses were available for 42 of the 55 teams invited (76.4 percent), with a total of 297 employees (50.5 percent). The employees’ average age was 32 years, and 57.5 percent were women. In total, 99 percent had completed a high school degree, and 69 percent held bachelor’s degrees or above.We used well-established scales to measure the constructs . Following the translation and back-translation procedure, we created a Chinese version of the scales for measuring these variables. All items in the study were rated on a 5-point Likert scale (1 = Strongly disagree to 5 = Strongly agree).p < 0.000), and high ICC (intraclass correlation coefficient) values  [Guanxi HRM practice was measured with a 5-item scale of Chen et al. . The res = 0.47) . Thus, sPsychological safety was measured using the Edmondson  seven-itOccupational well-being was measured using the Yvonne, Rodney and Kate  four-iteCollectivistic culture was measured with a ten-item scale from Wagner and Moch . The meaFollowing advice from previous studies . Afterward, hierarchical multiple regression and Sobel test were applied to test hypotheses.2/df = 5.91, RMSEA (root mean square error of approximation) = 0.13, SRMR  = 0.16, CFI (comparative fit index) = 0.40, TLI (Tucker–Lewis index) = 0.34), thereby suggesting that CMB is minimized in the current study. Based on the analyses mentioned above, the dataset was appropriate for further analysis.As the variables in the current study were self-reported, common method bias (CMB) might be a concern, and confirmatory factor analysis with Mplus 7.0 was conducted to check the problem of CMB. The results indicate that one-factor measurement model with Guanxi HRM, psychological safety, occupational well-being and collectivistic culture loaded on one general factor did not fit the data well  and individual-level variables . We tested our study hypotheses in three interlinked steps. First, we examined a model of guanxi HRM practice and occupational well-being (Hypothesis 1). Second, we examined a mediation model (Hypothesis 2). Third, we integrated the proposed moderator variable into the model and empirically tested the moderation model (Hypothesis 3).γ = −0.427, SE = 0.089, p < 0.001), supporting Hypothesis 1.Hypothesis 1 posited that guanxi HRM practice was negatively related to employees’ occupational well-being. In Model 1 of p < 0.001), while the direct path from guanxi HRM practice to occupational well-being became less significant , as shown in Model 2. Meanwhile, guanxi HRM practice was negatively associated with psychological safety, as shown in Model 3 , indicating a partial mediation. Hence, Hypothesis 2 was preliminarily supported. To further test Hypothesis 2, Sobel’s [p < 0.001), suggesting that psychological safety partially mediates the main effects of guanxi HRM practice on employees’ occupational well-being, thus providing support for Hypothesis 2.Hypothesis 2 stated employee psychological safety mediated the relationship between guanxi practice and employees’ occupational well-being. Model 2 showed that when psychological safety was included, the link between psychological safety and occupational well-being was significantly positive . This finding suggests that collectivistic culture positively moderates the relationship between guanxi HRM practice and psychological safety and supports Hypothesis 3. Following Aiken and West’s [Hypothesis 3 assumed that team collectivistic culture positively moderates the relationship between guanxi HRM practice and psychological safety. As indicated by Model 4 of d West’s  methods,There has been growing interest in understanding the impact of guanxi HRM practice on employee outcomes . The prThis study contributes to the literature in several ways. First, drawing on the COR theory, we investigated whether and how guanxi HRM practice affects employees’ occupational well-being. The present study is meaningful because most research regarding HRM has focused on the financial return ,82. FurtSecond, our study further addresses the question of why guanxi HRM practice relates to employees’ occupational well-being by outlining a plausible mediating mechanism, such as psychological safety. Gu, Nolan, and Rowley’s  study ofThird, by isolating a potential boundary condition for guanxi HRM practice, we further offer an elaborate understanding of when guanxi HRM practice impedes psychological safety. Recently, COR theorists have begun to search for potential contextual factors that may constrain or facilitate the conservation of resources process . AlthougFrom a practical perspective, the present study provides meaningful messages for HR managers. Occupational health psychology aims to create a psychologically healthy and safe workplace to promote workers’ well-being and prevent harm to their health . ConsideSecond, our results suggest that the effect of guanxi HRM practice on employees’ occupational well-being is mediated by psychological safety. Thus, when guanxi HRM practice has previously occurred and the members have become highly psychologically unsafe, it might be necessary to build a psychologically safe climate to enhance occupational well-being. For example, leaders can work to create practices such as shared goals, shared knowledge, job autonomy, and mutual respect to foster psychological safety and attempt to maintain fair and socially favorable interactions, as well as creating a supportive work environment in which employees may discharge any insecurities and anxieties.Finally, our findings regarding the exacerbating impact of guanxi HRM practice under highly collectivistic culture suggest that organizations should not build collectivistic cultures to deal with the ill effects of guanxi HRM practice; in fact, employees place a premium on maintaining relationships within a context in which guanxi HRM practice interacts with collectivistic culture. Indeed, managers should create a supportive diversity climate conveying a positive message to all employees, in which employees place more emphasis on uniqueness and norms , which iDespite these important contributions, several limitations warrant further discussion and future research. First, the present finding may be contaminated by common method variance, as the data related to guanxi HRM practice, psychological safety, and occupational well-being came from the same source. However, the assessment of collectivistic culture was performed by immediate supervisors, which helps to reduce potential issues with common method bias . Second,This study explored the negative effects of guanxi HRM practice on employees’ occupational well-being and tested the mediating role of psychological safety and the moderating role of collectivistic culture. Our results provide important theoretical and practical implications for employee well-being and guanxi HRM practice. Future studies on employee well-being should expand the existing COR theory, which focuses on the relationship between HRM and employee outcomes. Moreover, future implementation efforts must consider improving the occupational well-being of employees and enhance their psychological safety to reduce the negative impact of guanxi HRM practice on health-related productivity.


E. coli, followed by purification and kinetic analysis of an untagged NSP12/7/8 RdRp complex. Pre-steady-state kinetic analysis shows that our reconstituted RdRp catalyzes fast (kcat = 240–680 s−1) and processive (koff = 0.013 s−1) RNA polymerization. The specificity constant (kcat/Km) for Remdesivir triphosphate (RTP) incorporation (1.29 μM−1s−1) is higher than that for the competing ATP (0.74 μM−1 s−1). This work provides the first robust analysis of RNA polymerization and RTP incorporation by the SARS-CoV-2 RdRp and sets the standard for development of informative enzyme assays to screen for new inhibitors.COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently being treated using Remdesivir, a nucleoside analog that inhibits the RNA-dependent-RNA polymerase (RdRp). However, the enzymatic mechanism and efficiency of Remdesivir have not been determined, and reliable screens for new inhibitors are urgently needed. Here we present our work to optimize expression in   •E. coli gives soluble SARS CoV2 RdRpCo-expression of NSP12/7/8 with chaperones in •Tag-free RdRp complex catalyzes fast and processive RNA polymerization•Polymerization rates are sufficient to replicate the 30 kb genome in 2 min•Remdesivir is incorporated with a specificity constant twice that observed for ATP Biological Sciences; Biochemistry; Molecular Biology; Molecular Biology Experimental Approach A small outbreak of SARS-CoV-2 originated in Wuhan, China in December 2019  and swifin vivo but Remdesivir seems to show promise despite this hurdle. Therefore, accurate data are required to understand the mechanistic and kinetic basis for the effectiveness of Remdesivir and to use that knowledge to screen for and develop additional antiviral compounds. Although drug screening based on propagation of virus in cell culture has been effective in developing new therapies, this method is expensive and time consuming, making it difficult to screen millions of compounds in the search for new inhibitors. Assays based on analysis of well-defined kinetics of polymerization catalyzed by the viral RdRp in vitro could provide a critical component to find new drug candidates. However, preliminary studies attempting to address the kinetics and mechanism of Remdesivir incorporation were flawed because they were conducted using substandard enzyme preparations requiring 30 min of incubation to see a product band on a gel . A recent paper  was constructed so that it contains codon-optimized genes for NSP12, NSP7, and NSP8 in a single expression plasmid  was created (E. coli/pG-Tf2 harboring either pQE-(NSP12)-pcIts,ind+-(NSP7-NSP8) or pQE-(NSP12-TEV-8xHis)-pcIts,ind+-(NSP7L8) to test solubility of the complexes when co-expressed with chaperones from pG-Tf2. The chaperone/co-expression strategy yielded virtually all soluble protein for the untagged construct , then was mixed with 250 μM ATP, CTP, and UTP to start the reaction. Adding three of the four nucleotides afforded extension of the primer by 12 residues before a C in the template was encountered, terminating synthesis. Following the addition of the three nucleotides, rapid extension of the RNA primer to form the 32 nt product was observed in 0.2 s . This indicates that under our experimental conditions, NSP8 does not have measurable primer extension activity and that there is no detectable RNase activity in our tag-free preparation of NSP8.As a control experiment, we also tested NSP8 alone for primer extension activity as a paper on the SARS-CoV  suggeste2+ was mixed with varying UTP concentrations (2.5–150 μM) to start the reaction. Reactions were quenched and analyzed as in kcat = 220 ± 23 s−1 and Km = 74 ± 11 μM for incorporation. To achieve more accurate results, the parameters reported in this paper were obtained by global data fitting based on numerical integration of the rate equations using KinTek Explorer to derive estimates for kcat and Km directly (kcat (k2 and k4), and Km (((k-1+k2)/k-1), and ((k-3+k4)/k3))) for the two UTP incorporations. The value of kcat/Km was then calculated from the estimates for kcat and Km. The parameters kcat/Km, kcat, and Km for each UTP incorporation derived from the simulation-based data fitting are summarized in kcat/Km.The next experiment was designed to measure the UTP concentration dependence on the rate of polymerization. A pre-equilibrated mixture containing 2 μM NSP12/7/8 complex, 6 μM NSP8, 100 nM FAM-20/40 RNA, and 5 mM Mgdirectly . Data fidirectly , and condirectly . The sch2+ was mixed with 150 μM UTP to start the reaction. Reactions were quenched with EDTA, and the amount of total extended primer was determined by capillary electrophoresis. The concentration of product formed versus time at different enzyme concentrations is shown in Kd = 1.9 ± 0.2 μM), signifying that the binding of the enzyme to the RNA is weak compared with the fixed RNA concentration (200 nM) in the titration. This experiment suggests that the amplitude of 60 nM in Kd estimated from the global fit by simulation was 1.8 ± 0.2 μM for the enzyme binding to RNA, assuming 80% of the enzyme is active, as described below. This information aids in designing additional experiments in this paper, as the amount of product expected to form in a single turnover can be estimated based on the measured Kd for RNA binding.Because only 60% of the RNA primer was extended in the previous experiments in spite of a large excess of enzyme over the RNA substrate, the following experiment was performed to differentiate low active enzyme concentration from weak binding to the RNA. A mixture of varying concentrations of the NSP12/7/8 complex (0.2–10 μM), 20 μM NSP8, 200 nM FAM-20/40 RNA, and 5 mM Mg2+ was allowed to equilibrate for 30 min, then mixed with 2 mg/mL heparin to start the reaction. The use of 2 mg/mL heparin has been shown to be an effective concentration (−1 (koff in the Scheme in Success of the experiment described in ntration  as a trantration E. This sntration E. The di−1 and fit the enzyme titration experiment in −5 s−1) and allowed to reach equilibrium  before adding RNA in a separate step and then nucleotide in a final step, both of which occur on timescales fast enough to not perturb the E to X equilibrium.To obtain a minimal estimate of the active fraction of enzyme, we locked the RNA dissociation rate at 0.013 sKactive, and the resulting kon are shown in Kd (from varying kon) changes based on the active fraction. At 60% active enzyme, the resulting Kd for RNA binding is 1.3 μM, whereas at 100% active enzyme the resulting Kd would be 2.2 μM, giving the range of possible Kd values from the data. The RNA dissociation rate does not vary significantly over this range because it is well defined by the data in kon[E]. The apparent RNA binding rate constants are far below diffusion limits , which could be attributed to a two-step binding mechanism; however, the experiments presented here cannot differentiate multiple binding steps, so the simple one-step binding model was used.Confidence contours for kcat and Km values for the first and second UTP incorporations are summarized in Globally fitting the data shown in kcat/Km) for ATP provides a benchmark for evaluating the relative efficiency of Remdesivir incorporation. A solution of 1.25 μM NSP12/7/8 complex, 6 μM NSP8, 100 nM FAM-20/40 RNA, and 5 mM Mg2+ was mixed with 200 μM UTP and varying concentrations of ATP (5–400 μM) to start the reaction. The RNA substrate used in these studies encodes for two UTPs followed by four ATPs and then another UTP to form the 27 nt product. The data for each product band at 225 μM ATP are shown in Because Remdesivir D is an AK5 and k6). The data for the total product ≥23 nt at each ATP concentration are shown in Km = (k-5+k6)/k5 and kcat = k6  to start the reaction. Data at 315 μM RTP are shown in k1 through k4 in the scheme in kcat/Km = 1.29 ± 0.06 μM−1s−1, kcat = k6 = 68 ± 2 s−1, and Km = 53 ± 2 μM  and slower kcat (3.6–3.8 s−1) leading to lower Km values  incorporation by the SARS-CoV-2 NSP12/7/8 complex, the concentration dependence of Remdesivir on the kinetics of its incorporation were measured by rapid quench methods, as described for ATP. A solution of 1.5 μM NSP12/7/8 complex, 6 μM NSP8, 100 nM FAM-20/40 RNA, and 5 mM Mg3 ± 2 μM . Fitting3 ± 2 μM E affordem values . The sloConfidence contours in As of October 25, 2020, the World Health Organization reports over 43 million confirmed cases and over 1.15 million deaths due to SARS-CoV-2 globally with new infections increasing by 33% in a last week . Althougkcat/Km values for RTP versus ATP (1.29 versus 0.74 μM−1s−1). Although RTP is incorporated at least three-fold slower than ATP (68 s−1 versus 240 s−1), it has a six-fold higher kcat/Km value due to a six-fold lower Km relative to ATP (53 μM versus 320 μM). We define a discrimination index as the ratio of specificity constants for the normal nucleotide divided by that for the analog—this term quantifies the ratio by which the enzyme discriminates against the nucleotide analog (AnTP) relative to the normal nucleotide (NTP).We found that Remdesivir is incorporated by the RdRp complex more efficiently than ATP, defined by the relative kcat/Km = 0.0007 μM−1s−1 compared with 0.1 μM−1s−1 for UTP, giving a discrimination index = 140 ± 45 , which indicates that the analog is a better substrate than the cognate nucleotide. Of the nine nucleoside analogs approved by the FDA to treat HIV infections, only Stavudine  has a discrimination index less than one, but it has a higher toxicity index due to incorporation by the human mitochondrial DNA polymerase . Certain140 ± 45 . The phy140 ± 45 , so givekcat/Km values lower than that for the first RTP mostly because the rate constants for incorporation (kcat) were 18-fold slower .Questions about or requests for materials should be directed to and will be fulfilled by the Lead Contact, Kenneth A. Johnson  – 160656 pQE-(NSP12)-pcIts,ind+-(NSP7-NSP8) – 160540.Plasmids used in this study have been deposited to Addgene: pcIThe published article includes all datasets generated or analyzed during this study.All methods can be found in the accompanying


Tetranychus urticae Koch (two‐spotted spider mite) according to three distinct host‐choice selection treatments for ten generations. Additionally, we tested the presence of induced habitat choice mechanisms and quantified the adaptive value of a choice before and after ten generations of artificial selection in order to gather insight on the habitat choice mechanisms at play. Unexpectedly, we observed no evolution of habitat choice in our experimental system: the initial choice of cucumber over tomato remained. However, this choice became maladaptive as tomato ensured a higher fitness at the end of the experiment. Furthermore, a noteworthy proportion of induced habitat choice can modify this ecological trap depending on past environments. Despite abundant theory and applied relevance, we provide the first experimental evidence of an emerging trap. The maladaptive choice also illustrates the constraints habitat choice has in rescuing populations endangered by environmental challenges or in pest control.Individuals moving in heterogeneous environments can improve their fitness considerably by habitat choice. Induction by past exposure, genetic preference alleles and comparison of local performances can all drive this decision‐making process. Despite the importance of habitat choice mechanisms for eco‐evolutionary dynamics in metapopulations, we lack insights on the connection of their cue with its effect on fitness optimization. We selected a laboratory population of  DispersFast environmental change can disconnect a habitat preference cue from its former fitness advantage, resulting in a maladaptive choice. This transforms habitat choice from an ecological opportunity to an ecological trap  population. We report on an artificial selection experiment where we select populations making a habitat choice between tomato and cucumber plants. We selected each generation for ten generations according to three habitat choice selection treatments. If evolvable, we expect our particular selection regime to evolve habitat preferences, which results in an evolved choice, reflecting the selection treatment independent of the habitat choice mechanisms involved in T. urticae. Additionally, we test the contribution of different habitat choice mechanisms involved in host choice. While the initial choice for cucumber did not evolve over ten generations of artificial selection, quantifying life‐history traits before and after the experiment revealed that the mites adapted to tomato relative to cucumber. This now maladaptive habitat choice for cucumber is one of the first accounts of an ecological trap supported with information on preference and fitness before and after the onset of the trap and with insights on the choice mechanisms involved. Our experiment can add perspective to maladaptive choices in similar agricultural pest systems and broader ecology‐driven systems such as conservation, agriculture and evolutionary medicine.Here, we study the evolvability of habitat choice and the mechanisms involved in a 22.1Tetranychus urticae (two‐spotted spider mite) population. T. urticae is a cosmopolitan phytophagous mite species, a generalist best known as a pest in greenhouses. The mites have a sexual mating system with unfertilized females still able to produce males: arrhenotoky. Furthermore, it is also frequently used as model species in evolution and ecology  over Cucumis sativus L. tanja (cucumber). We did not include the choice of Phaseolus vulgaris L. (bean), the optimal host plant for our T. urticae population. In past experiments, we never observed a higher performance on other host plants when they were adapting to them compared to bean. Since we wanted to study how choice evolves in concert with local adaptation affecting relative suitability in the choices involved, bean could never evolve to be the less adaptive choice. We used tomato plants of five weeks old and cucumber plants of three weeks old to ensure large enough leaves. Additionally, we used bean plants in further trait tests that were two weeks old.We opted to test the mites to choose 2.2Tetranychus urticae. We maintained this new population on a tomato plant to keep any adaptation to tomato and to keep a consistent developmental environment for any possible induced habitat choice. Following, we submitted this population to ten rounds of artificial selection over ten generations. We did this according to one of three different artificial selection treatments that we replicate four times. We selected for (a) habitat choice for tomato, (b) habitat choice for cucumber and (c) random choice. Note that, although breeding all mites on tomato will influence any induced habitat choice, we still expect this plastic response to evolve to prefer the previously experienced habitat more or less, respectively, in the tomato choice and cucumber choice treatment.We performed artificial selection on habitat choice. First, we sampled 100 individuals from a stock of tomato‐bred One round of artificial selection included testing 100 females in a habitat choice arena for twenty‐four hours and breeding the selected next generation for the next selection round during the following thirteen days. From each replicate population, we transferred 100 random adult females on a piece of parafilm® (1 × 3 cm) on the intersection of a cucumber and tomato leaf both on life plants Figure , we reco2.32.3.1T. urticae. Comparing life‐history traits on tomato and cucumber to that on bean puts these in perspective of the expected optimal fitness.We quantified individual fertility and reproductive success on different host plants to estimate the level of adaptation to a host plant and the adaptive nature of a possible habitat choice. We tested this on tomato and cucumber, both plants involved in the artificial selection, but also on bean. We consider bean to be the optimal host of 2). On this common garden, the female produced the next generation that is controlled for effects of the maternal environment and effects of developmental plasticity. We placed females in their first day of adulthood on an empty patch of the tested host plant . For each experimental population, we started three such common gardens, each from one female, and for every common garden, we tested one female offspring on bean, one female offspring on cucumber and one female offspring on tomato. We recorded the number of eggs laid within the first six days, henceforth fertility, and the number of female deutonymph offspring (i.e. life stage before adulthood) produced the first twelve days by the tested female, here referred to as reproductive success.We transferred a fertilized female from the population we aim to test to a bean leaf  on four touching tomato plants. Except for the occasional watering and replacement of roughly one old plant every week, this stock was not interfered with.2.3.22) and five fertilized females on a cucumber leaf (15.75 cm2) to develop a next generation. From each of these leaves, we placed twelve adults on a habitat choice arena. Such an arena is a petri dish (9 cm) with wet cotton on which a tomato and cucumber‐leaf patch is placed, touching each other (both 1.5 × 2.5cm). Paper towel strips line the two patches at their nontouching edges. The twelve mites were placed on a piece of parafilm® on the intersection of the two patches. After twenty‐four hours, we recorded the distribution of the twelve mites over the two patches and calculated the proportion of mites found on tomato. We performed this test on the stock population of tomato‐bred mites before the experiment, with which we started the artificial selection experiment, with six replicates for each developmental environment. We repeated this after the artificial selection experiment with one test for each of the twelve replicated populations on each developmental habitat.In order to test to what extent habitat choice is induced, we performed an additional induced habitat choice test. We distinguish induced habitat choice mechanisms from that of the other habitat choice mechanisms by varying the developmental environment of the tested populations. For that, we placed five fertilized females on a tomato leaf  with an additional varying intercept and slope of each replica within a selection regime. We alternatively modelled this as only depending on the selection regime with a random intercept for each replica within it. We modelled the log of our reproductive success and fertility data to depend on the tested host plant and with a varying intercept for each replicated population in the life‐history trait tests after the selection rounds. We equally estimated the dependence of the log odds for habitat choice during the habitat imprinting tests on the developmental habitat. Details on the models, their structure and their priors can be found in the supplementary materials  emphasizing estimated model parameters and resulting estimated dependent variables that are (b) expressed as likelihood distributions: the posterior distribution. Overall, we and a growing part of the research community deem Bayesian inference more appropriate because of its analogy with how ecological and evolutionary research is practiced  to the model lacking the effect of time compared to 0.1 (WAIC = 777.8 ± 11.1 SE) to the initial model including the effect of time.We expected a higher tomato preference to evolve in the tomato choice treatment, a lower tomato preference in the cucumber choice treatment and no change in tomato preference in the no choice treatment. However, we recorded no convincing change in the proportion of mites ending up on the tomato plant during artificial selection when given the choice Figure . PosteriWhile we do not record a change in the proportion of mites ending up on tomato during the selection rounds, we do record an overall lower likelihood to end up on tomato than on cucumber over all generations Figure . Consequ3.1.1Reproductive success of the starting population was similar on tomato compared to cucumber at the start of the selection experiment Figure , left. WWe detected no differences in the strength of adaptation in relation to the imposed selection treatments natural systems. Our experiment resembles an agricultural system where our focal species However, there even are evolutionary constraints of adaptive habitat choice to cope with a changing environment. Habitat choice, as evolved behaviour, is expected to promote local adaptation and specialization . This habitat choice involves induced habitat choice mechanisms and likely involves a direct genetic habitat choice mechanism. Our experiment reveals the emergence of an ecological trap. This important but predominantly theoretically approached process can cause habitat choice to be a maladaptation rather than an adaptation and constrain its ability to rescue populations from environmental challenges.In summary, we show the presence of habitat choice in a laboratory population of None declared.Supplementary MaterialClick here for additional data file.