Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: This also allowed to express sgRNAs under the control of the T7 RNAP promoter either via transcription from short DNA templates or from epsiomes (Figure 3—figure supplement 1B). While the latter strategy increased editing during our tdTomato reporter assay, with all six guides reaching nearly 100% editing rate (Figure 3—figure supplement 1C–E) and controls showing no effect on the reporter expression (Figure 3—figure supplement 1D), doubling times significantly increased once episomal-expressed sgRNAs were transfected into the cell line expressing constitutively hyBE4max and T7 RNAP (Figure 3—figure supplement 1F). We also tested another CBE version (namely AncBE4max [Koblan et al. , 2018]) that, unlike hyBE4max (Zhang et al. , 2020), does not have the Rad51 ssDBD (Figure 3—figure supplement 2A). While we observed also high editing rates of 100% for nearly all sgRNAs when targeting tdTomato (Figure 3—figure supplement 2B–E), we saw strong editing outside of the guide targeting window (Figure 3—figure supplement 2F), depleting the tdTomato reporter signal even in control cells, where only neutral substitutions were intended with no amino acid change (Figure 3—figure supplement 2D). Meanwhile, editing outside the guide target sequence was not observed when using the hyBE4max CBE (Figure 3—figure supplement 2F). We concluded from these initial results that episomal expression of the sgRNA and hyBE4max CBE is a suitable approach for mutant generation. While stable expression systems did not affect growth before expressing the sgRNA and may be attractive too after further optimisation, an episomal expression system seemed to be much more flexible, allowing to transfect wildtype parasites without further modifications. To test the flexibility of our system, we therefore next performed our reporter assay in L. donovani, L. mexicana, and L. infantum, and repeated it in L. major. First, we confirmed the identity of each Leishmania species by sequencing the LPG2 locus (Figure 2—figure supplement 1A, B; Akhoundi et al. , 2017) and generated tdTomato-expressing clones (Figure 2B). [SEP] Justify:

Output: The text corroborates the claim by detailing the effectiveness of sgRNAs in achieving nearly 100% editing rates when targeting specific sequences in a reporter assay. It provides evidence that the episomal expression of sgRNA alongside CRISPR technology (specifically a CBE version) facilitated precise editing, with specific references to editing performance in control and experimental conditions. The passage highlights both the success of targeted editing and the adaptability of the method within different species, reinforcing that CRISPR technology indeed allows for precise genomic editing.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: This suggests that factors associated with the hive environment play a dominant role in specifying the colony-specific chemical signatures used for nestmate recognition. Yet, our data also indicate that CHC profile development in individual workers is a developmentally-regulated process that is closely associated with the age-dependent division of labor among workers. To address this potential conundrum, we next asked whether the effects of task and colony environment on the development of CHC profiles of individual workers are independent by using a reciprocal cross-fostering strategy. To achieve our goal, we introduced cohorts of newly eclosed bees from two different typical colonies back into their source colony, as well as a reciprocal foster colony, and then recollected marked workers from both cohorts in each reciprocal colony at different ages. CHC analyses revealed that through Day 14, the CHC profiles of bees were more similar to the profiles of their same-aged non-nestmate sisters than those of unrelated nestmates of similar age (Figure 3A, Two-way foster colony (environment): F(1,31) = 2. 19, R2 = 0. 06, p=0. 06, source colony (genetics): F(1,31) = 5. 94, R2 = 0. 16, p<0. 001, foster colony*source colony: F(1,31)=0. 46, R2 = 0. 01, p=0. 82; Figure 3B, Two-way foster colony: F(1,31) = 1. 13, R2 = 0. 03, p=0. 33, source colony: F(1,31) = 3. 18, R2 = 0. 09, p=0. 02, foster colony*source colony: F(1,31) = 1. 78, R2 = 0. 05, p=0. 15; sample size assessment depicted in Figure 3—figure supplement 1 indicates sample size is adequate). In contrast, once workers shift to foraging activity, we found that the CHC profiles of fostered bees are different from the profiles of both foraging sisters raised in the source colony and unrelated host foragers of similar age (Figure 3C, Two-way foster colony: F(1,31) = 4. 04, R2 = 0. 10, p=0. 02, source colony: F(1,31) = 7. 65, R2 = 0. [SEP] Justify:

Output: The text contrasts the claim by implying that individual bees may have their chemical profiles influenced by various factors, including their environment and developmental stages, suggesting that bees can exist outside traditional colony structures. The reference to the analysis of CHC profiles and the introduction of bees into different colonies indicates that there are scenarios where bees are not solely determined by colony organization, which challenges the idea that all bees always live exclusively in colonies.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: But unfortunately the difficulty of transit in the large chamber (Sala de las Huellas) led to the destruction of many traces, and only a few footprints have been preserved on large boulders (Figs. 17. 2f and 17. 3a). The cave survey in this sector was finished in 1970, and Almagro invited André Leroi-Gourhan to plan a project to study the footprints, including photogrammetric analysis. Unfortunately, it never materialized. Plaster casts of two footprints were made at that time, and a sediment sample from the first passage was sent to León Pales and Michel-Alain García (Ortega et al. 2013: 178–182; 2014: 43–44). In addition, charred wood remains were collected from in the lower level, at the bottom of a shaft from GH I, and interpreted as thrown or fallen from that gallery. They were dated to 15,600 ± 230 BP (Delibrias et al. 1974: 53). New surveying in 1981 (Grupo Espeleológico Edelweiss 1986) achieved an acoustic connection between GH I and the southwestern passage in San Bernabé Cave (Fig. 17. 1b). This connection motivated the dating of the speleothem plug that separates the two caves. Rainer Grün, who was visiting Spain to sample caves for his PhD, dated the speleothem to about 175 ka (pers. comm. Adolfo Eraso). This age prompted a new study of the footprints and their superpositions, in order to determine the direction of the prints through photographic and photogrammetric analysis (Galaz et al. 2000). These studies showed that the prints followed an entry and exit route within Sala de las Huellas (Galaz et al. 2000; Ortega et al. 2013: 178–182) and that they were, therefore, not connected with the passage in San Bernabé Cave. This new study also identified traces of small carnivores (mustelid-type) especially in the eastern gallery (GH I), in whose initial section bear claw scratches are also preserved on the walls. Some charred wood remains have also been observed on the walls and floors of Galerías de las Huellas, although charcoal remains are more numerous in the lower passages (). [SEP] Justify:

Output: The text is largely focused on the description and study of footprints and the geological context of a specific cave rather than discussing cave paintings or human expression. There is no mention of cave paintings or prehistoric art, making the text irrelevant to the claim.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: mexicana, and L. donovani (Figure 2—figure supplement 2C). CBE targeting of single- and multi-copy genes in the slight toxicity observed in L. donovani and L. infantum, we concluded that our system was efficiently and specifically converting C to T within the target window, thereby introducing STOP codons in all four Leishmania species. This prompted us to generate proof of principle mutants in wildtype cells next. To test our CBE system we decided to target the single-copy genes PF16 (encoding a central pair protein of the axoneme and essential for flagellar motility [Beneke et al. , 2017; Martel et al. , 2017]), IFT88 (encoding a subunit of the IFT-B complex and essential for flagellar assembly [Beneke et al. , 2019]), and the miltefosine transporter (MFT, encoding a P-type ATPase that is linked to miltefosine resistance in Leishmania [Seifert et al. , 2007]), as well as the multi-copy gene PFR2 (encoding a key component of the paraflagellar rod and required for normal flagellar motility [Beneke et al. , 2019; Maga et al. , 1999]). All four genes were targeted with four guides each, which were cloned into pLdCH-hyBE4max plasmids (Figure 2A). While we designed PF16 and IFT88 guides to be universal for multiple Leishmania species, guides for MFT and PFR2 were species specific for L. donovani and L. mexicana, respectively. In addition, guides targeting PFR2 were designed to be universal for all three PFR2 copies (A, B, and C). We then individually transfected plasmids targeting PF16 into L. donovani, L. mexicana, L. infantum, and L. major, while plasmids targeting IFT88 were only transfected into L. infantum and L. major. Meanwhile, plasmids targeting MFT were only transfected into L. donovani and plasmids targeting PFR2 only into L. mexicana. [SEP] Justify:

Output: The text supports the claim by detailing a process where a CRISPR-based system was used to efficiently and specifically convert 'C to T' within targeted genes in various Leishmania species. This process corresponds with the idea of precise editing of genomic sequences, as the authors mention targeting specific genes with designed guides, which is a fundamental aspect of CRISPR technology.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: 19, p=0. 001, foster colony*source colony: F(1,31)=0. 48, R2 = 0. 01, p=0. 67). Together, these data suggest that genetic variations, or other long-term effects associated with the source colony, play an important role in defining the CHC profiles of individuals during the early phases of the age-dependent behavioral development of worker honey bees. However, by the time bees start foraging, the mature CHC profile of individual workers is defined by an interaction between factors associated with both the source and foster colonies (Figure 3C). Figure 3 with 1 supplement see all Download asset Open asset Cross-fostering indicates colony environment drives the signature CHC profiles of foragers. Age-matched cross-fostered bees differ in CHC profile by source colony at Day 7 (A) and Day 14 (B), and by both source colony and foster colony when they are foragers (C). Number to left of arrow in legend represents the bee’s source colony, and the number to the right represents the bee’s foster colony. All statistics using followed by FDR pairwise contrasts, shown as non-metric multidimensional scaling plots depicting Bray-Curtis dissimilarity between samples. Letters in legends denote posthoc statistical significance (p<0. 05). Sample size per group, N = 8. Figure 3—source data 1 Amount (ng) of each compound extracted from each bee sample in Figure 3. Download elife-41855-fig3-data1-v2. xlsx The development of CHC profiles of individual workers is associated with the regulation of CHC biosynthesis genes Homogenization models for the development of colony-specific nestmate recognition cues predict that cue specificity is acquired by individuals via physical contact with other colony members and/or environmental sources of hydrocarbons (Breed et al. , 2015; Crozier and Dix, 1979; Lenoir et al. , 2001; Meskali et al. , 1995; Soroker et al. , 1994; Soroker et al. , 1995b; van Zweden et al. , 2010). [SEP] Justify:

Output: The text contrasts the claim by discussing the developmental processes of worker honey bees and introducing the concept of 'cross-fostering.' This suggests that bees can have interactions and behaviors influenced by multiple colonies, which implies that living arrangements may not be strictly confined to a single colony. Additionally, the mention of genetic and environmental factors affecting the development of individual bees indicates that there can be exceptions to the idea that all bees live exclusively in colonies.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: In 1999, a charcoal sample from the western Galería de (GH II) provided a date of 3820 ± 50 BP (pers. comm. Arsuaga). This date was very different from the Palaeolithic date mentioned above and showed that the passages had been entered again in recent prehistory. Access to a Complicated SectorIt was traditionally thought that the access to Galerías de las Huellas during prehistory was from the blind valley of the Guareña River or the San Bernabé cirque, given its obvious proximity (Ortega and Martín 1986: 342–343). Later, the possibility of access from San Bernabé Cave was discarded, because of the age of the collapse now blocking the cave, which is sealed by a thick speleothem (Ortega et al. 2013, 2014). Finally, direct access from outside to the has also been discarded, given that the current blockage of boulders, a consequence of the receding hillside, seems very stable and was already consolidated in the Neolithic, according to the dates obtained in the framework of this project (Table 17. 3) (Ortega and Martín 2019). In contrast, recent investigations into the archaeological evidence inside Palomera Cave, currently the main entrance to the Ojo Guareña Cave system, indicate that transit inside this large cave was much more frequent and intense than previously thought. This is evidenced by the succession of archaeological remains that extend up to almost a kilometre and a half from the current entrance, towards both the east and the west, belonging to different periods from prehistory to the Middle Ages. On the route from to the surroundings of (Fig. 17. 4), several archaeological remains have been documented in the passages of Museo de Cera, Galería de la Escalada and Galería Macarroni, with confirmed prehistoric dates. The distance from the start of the latter gallery to the Sala del Cacique is barely 100 m; it is very comfortable to walk through and currently included in the tourist route. It is a further 100 m from this point to the first side passage in Galería del Cacique, which leads to Laberinto Otilio. This is the beginning of the sector studied here, where abundant charred wood marks on the walls and pieces of charcoal on the loamy floor and boulders have been identified. Fig. 17. [SEP] Justify:

Output: The text does not address the claim regarding cave paintings and human expression. It discusses archaeological evidence and access to caves without mentioning cave paintings or human expression in prehistory, making it irrelevant to the claim.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: donovani did not result in decreased swimming speed (as expected), we wanted next to assess whether they would instead confer resistance to miltefosine, an orally given antileishmanial drug. To determine the suitability of our method for drug screening applications, such as positive drug resistance screens, we also wanted to test whether miltefosine-resistant parasites could be enriched in the mutant population by treating them with a low dose of miltefosine prior to assessing their actual resistance to the drug itself. To test this, wildtype parasites and parasites expressing MFT targeting guides were treated for 48 hr with 20 µM miltefosine (pre-treated parasites) from 16 days post transfection onwards. Then cells were washed and cultured for another 72 hr. In the following, pre-treated and non-pre-treated parasites were incubated with different doses of miltefosine for 48 hr and subsequently cell viability was measured by using an MTT assay (Figure 6A and Supplementary file 3). Pre-treated with miltefosine or not, wildtype cells were only resistant to small doses of miltefosine (5 and 20 µM). In comparison, parasites transfected with pLdCH-hyBE4max plasmids that allowed to express MFT targeting guides became significantly resistant to high doses of miltefosine (50 and 80 µM) (guide MFT-1 and MFT-3 significantly different in Figure 6C). As expected, when pre-treated with miltefosine the proportion of viable parasites at high miltefosine concentrations then rose further and three out of four guides were able to confer resistance (guide MFT-1, MFT-2, and MFT-3 significantly different in Figure 6B). In addition, viability rates correlated well with high editing rates for all these three guides. For example, guide MFT-3, which conferred the highest level of resistance to miltefosine (Figure 6B, C), resulted also in the highest editing rate of all MFT targeting guides (up to 81. 9% for non-pre-treated cells and 86. 5% for pre-treated cells) (Figure 6D, E). Further analysis of trace plots also highlighted that the introduced mutations resulted in the intended premature STOP codon (Supplementary file 2). [SEP] Justify:

Output: The text corroborates the claim as it discusses the use of a plasmid-based system to express MFT targeting guides, which allows for precise editing of the genomic sequences in parasites. It notes that guide MFT-3 conferred a high level of resistance to miltefosine and that this guide also resulted in a high editing rate, achieving up to 81.9% for non-pre-treated and 86.5% for pre-treated cells. This indicates that the CRISPR technology was effectively used to edit genetic sequences to confer specific traits, thus supporting the precision of CRISPR technology in genomic editing.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: However, because our data indicate that the maturation of the CHC profile of individual honey bees is actually regulated in association with the stereotypic age-dependent division of labor in this species, we next hypothesized that the CHC profiles of worker honey bees develop, at least in part, via an intrinsic age-dependent regulation of the CHC biosynthetic pathways in the pheromone producing oenocytes (Chung and Carroll, 2015; Falcón et al. , 2014; Makki et al. , 2014; Yew and Chung, 2015). Thus, we next examined whether age and/or task are associated with the mRNA expression levels of genes that encode elongases and desaturases, the primary CHC diversity producing classes of enzymes in the CHC biosynthesis pathway (Chung and Carroll, 2015). To identify candidate genes for our analyses, we first used a bioinformatic approach to identify all putative members of both protein families in the honey bee genome (Table 6). Subsequently, we used real-time quantitative RT-PCR to compare mRNA levels of each candidate gene in dissected abdominal cuticles from bees of different ages raised in their source colony (sisters of the bees analyzed for Figure 1- Figure 1—figure supplement 1), as well as foraging sister bees raised in either their source colony or an unrelated foster-colony. Our analyses revealed that the expression levels of at least one elongase and two desaturase genes are associated with either age or colony environment (Figure 4 and Table 7). Thus, our data suggest that individual worker honey bees regulate CHC expression through an innate age-dependent developmental process that is further modulated by other factors such as task and the social environment. Figure 4 Download asset Open asset Age and social environment affect the expression level of CHC biosynthesis genes. (A) Elongase gene. (B–C) Desaturase genes. Only genes with different expression levels between at least two groups are shown (7 for results for all studied genes). Black bars represent bees raised in their own colony. Grey bars represent sister forager bees that were raised in an unrelated colony (‘Fostered’). (D) Heat map of relative expression levels of all genes tested. [SEP] Justify:

Output: The text contrasts the claim by providing information that implies the existence of bees that may not live in their original source colony, as it discusses experiments involving bees raised in unrelated foster-colonies. Specifically, it mentions ‘sister forager bees that were raised in an unrelated colony,’ which suggests that not all bees exclusively live in their original colony, thereby challenging the claim that bees always live in colonies with no exceptions.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: 4Map of the possible access route to the human footprint site. The dark green passages represent the third level of and the dark red, the passages of fourth level. The greenish shadings indicate the sites with rock art and the footprint site. The red marks refer to other archaeological evidences (habitats, burials, transit, etc. ). The blue dashed line represents the route of access from Palomera Cave. (Modified from Grupo Espeleológico Edelweiss 1986; Ortega et al. 2013)Full size imageThere are several points of access to the Sala and Galerías de las Huellas in this area. One of them starts at the northeast end of Sala Negra, which finishes in an impressive boulder blockage (Figs. 17. 2a and 17. 9b), where the presence of charcoal confirms the transit, at least in the (Fig. 17. 9b, number 13 and Table 17. 3). Another access point, which is a little more comfortable, is through a side passage that ascends gently towards until Sala de las Huellas can be reached by climbing between boulders. As already stated, the entire route is full of charred wood marks and pieces of charcoal (Figs. 17. 2b–g and 17. 9). This evidence suggests that, after a 1200-m-long route of easy transit through large underground passages, prehistoric humans explored a sector of that is more complex because of its large size (5652 m2), intricate topography (mazes) and instability (with large embedded boulders that hinder transit). Despite this danger, at least one of the human groups that walked through this sector could have reached the upper level of the Sala and Galerías de las Huellas. Materials and MethodsFootprint DocumentationHuman footprint sites are not very common in the archaeological record because of the special requirements for their formation and preservation, although the number of cases has increased over time (Lockley et al. 2008; Bennett and Morse 2014a). These footprints provide information on human behaviour regarding the environment and on the nature of the sediments, where fine-grained substrates favour the formation and preservation of the prints (Bennett and Morse 2014b). [SEP] Justify:

Output: The text discusses access routes to human footprint sites and does not mention cave paintings or prehistoric human expression. Therefore, it is irrelevant to the claim.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: Since we could not find clones that harboured more than two different guide sequences and since these seemed to be randomly mixed, we concluded that effects of multi-gene targeting in one transfectant should be minimal within the mutant pool. Hence, we proceeded with the viability screen and analysed by Illumina sequencing sgRNA abundances from transfected libraries at 12 and 21 days post transfection (Figure 7A). To compare the sgRNA abundance across the screen, we assumed that each plasmid had an equal chance of being transfected and therefore z-scored the ratio between normalised sgRNA reads at plasmid level (before transfection) and days 12 and 21. Strikingly, we found two of the four CRK3 targeting guides (CRK3-3 and CRK3-4) to be significantly depleted at days 12 and 21 in the Leishmania-optimised CBE library (pLdCH-hyBE4max-LmajDBD), while only one CRK3 guide (CRK3-3) was significant depleted from the non-optimised CBE library (pLdCH-hyBE4max). All other guides, including non-targeting guides, did not significantly change in their representation within the pool over time (Figure 7B and Supplementary file 4). This demonstrates that our method can not only be used to target essential genes but also to pursue loss-of-function screens in Leishmania via delivery of plasmid libraries. and disadvantages of cytosine base editing in Leishmania Here, we report our new toolbox, allowing to knockdown single- and multi-copy genes in Leishmania populations by expressing hyBE4max and just one sgRNA. Our method to convert selected codons into STOP codons by using a base editor bypasses the requirement of DSBs, HR, donor DNA, or isolation of clones in order to obtain functional null mutants. Since our technique resembles a gene silencing approach, it is applicable for targeting essential genes in pooled transfections. Most importantly, this enables genetic screening in wildtype parasites (no engineered cell lines required) through a pooled plasmid library transfection format. While most of these points also apply for RNAi approaches, their application remains restricted to Leishmania species of the Viannia subgenus (Lye et al. [SEP] Justify:

Output: The text corroborates the claim by describing a method that utilizes CRISPR technology for precise genetic manipulation in Leishmania. It highlights the ability to knockdown genes and the technique's functionality in converting codons into STOP codons without requiring double-strand breaks (DSBs) or homology-directed repair (HR). These points emphasize the precision and utility of CRISPR technology in genome editing.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: (2014)LOC100576797Desaturase8E-127 (desat1)ón et al. (2014)LOC552176Desaturase2E-113 (desat1)ón et al. (2014)LOC727166Desaturase4E-56 (desat1)727333Desaturase4E-54 (desat1)ón et al. (2014)eIF3-S8Eukaryotic translation initiation factor7 Genes differ in relative mRNA expression level between bees of different ages (Age), and foraging sister bees raised in two different colonies (Hive). Numbers represent mean relative mRNA expression level ±standard error across four biological replicates. All p-values are from parametric ANOVA or nonparametric (denoted by ‘KW’). Letters denote statistically significant age groups across individual compounds via Tukey’s HSD (ANOVA post-hoc) or Dunn’s Test with FDR adjustment (KW post-hoc) (p<0. 05). GeneDay 1Day 7Day 14ForagerFDR adjusted p-value AgeFosteredFDR adjusted p-value HiveLOC7248671. 22 ± 0. 192. 41 ± 0. 7512. 62 ± 7. 231. 58 ± 0. 880. 328 (KW)1. 22 ± 0. 181 (KW)LOC7245521. 05 ± 0. 261. 28 ± 0. 662. 94 ± 1. 650. 09 ± 0. 020. 177 (KW)0. 03 ± 0. 010. 456LOC5508281. 02 ± 0. 124. 09 ± 1. 996. 17 ± 2. 100. 38 ± 0. 090. 097 (KW)0. 30 ± 0. 060. 53 (KW)LOC4096381. 14 ± 0. 212. 47 ± 0. 414. 36 ± 1. 652. 81 ± 0. 260. 097 (KW)4. 07 ± 0. 670. 530LOC4137891. 03 ± 0. 104. 17 ± 0. 685. 31 ± 2. 071. 98 ± 0. 150. 056 (KW)2. 38 ± 1. 211 (KW)LOC1005788290. 84 ± 0. 082. 30 ± 0. 423. 34 ± 0. 713. [SEP] Justify:

Output: The text contrasts the claim by discussing the differences in mRNA expression levels among bees raised in different colonies, indicating that bees can be situated in distinct environments (e.g., 'foraging sister bees raised in two different colonies'). This suggests that not all bees are always part of a single colony, which undermines the claim that bees always live in colonies without exception.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: The difficulty in balancing the documentation and conservation of these traces has conditioned their study for many years, and it only became possible with the development of non-invasive digital remote sensing technologies applied to archaeological research and heritage management (Bennett et al. 2013; Ashton et al. 2014; D. Webb et al. 2014; Citton et al. 2017). The study of the human footprints in Ojo Guareña started in 2012. It focused on accurate three-dimensional reconstruction of Galería de las and II, using 3D laser scanner technology and GIS methodologies (Benito-Calvo et al. 2013; Ortega et al. 2014). Sala de las Huellas was excluded from this work because of its topographic irregularity and the difficulty of transit in such a chaotic space. The three-dimensional mapping of this site was achieved with a ScanStation C10 Leica laser scanner, with a maximum flight range of 300 m, 4 mm accuracy. Seventeen high-resolution scans were performed (5 mm at 10 m), seven in GH I and ten in GH II. The point clouds obtained were joined by a common reference system with reference targets, obtaining a mean error of 1 mm, with final point clouds that reached resolution means of 3 mm in GH I and 4 mm in GH II. The models of the obtained surfaces were exported to GIS format, generating high-resolution digital elevation models (DEM) that show the topographic relief of the galleries with sufficient resolution to analyse the shape and distribution of the tracks (Benito-Calvo et al. 2013). The terrain roughness index (TRI) model (Riley et al. 1999) allows the irregularities of the floor to be differentiated (white colours) from the softer areas (black colours) by stressing the depressions of the best preserved footprints according to their internal morphology (sole, heel, toes, etc. ). Footprints and TrackwaysThis precise cartography allows the position of the tracks to be observed in relation to the morphology of the passages by identifying not only the human footprints but also the topographic elements (boulders, rocks, speleothems, crusts, channels, etc. ) that form the surface and condition the presence of human footprints. This enables the reconstruction of some of the paths and movements made by these ancient visitors. [SEP] Justify:

Output: The text primarily discusses the technical aspects of documenting and conserving archaeological sites, particularly focusing on the non-invasive technologies used to study human footprints in the Ojo Guareña. It does not mention cave paintings or any specific aspect of prehistoric human expression, making it irrelevant to the claim.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: Cloning strategies to generate sgRNA plasmid libraries typically involve first the amplification of ssDNA oligo libraries and then golden gate assembly (using Type IIS restrictions enzymes) of amplicons into a target vector. Since the vector we here use for base editing is compatible with this cloning strategy, we believe that our base editing method could be scaled up to facilitate genome-wide screens in future studies. The ability of just sharing targeted or potentially even genome-wide plasmid libraries and applying these to different research questions in various Leishmania species in many different laboratories around the globe represents a major improvement to previous methods and overcomes challenges associated with bar-seq screens, such as the LeishGEM project (as outlined in our introduction). Possible improvements to base editing in remains to be improved however, is the long culture time needed for some guides in order to reach high C to T conversion rates because prolonged guide targeting may cause unwanted side effects, such as off-target mutations (Grünewald et al. , 2019; Zhou et al. , 2019). We did not assess possible off-target mutations on a genome-wide scale in this study and this remains to be thoroughly evaluated in the future. However, apart from one guide (PF16-1 in L. donovani; Supplementary file 2), which resulted in editing within 6nt range of the guide sequence, we did not detect any editing outside of the 20nt guide sequence even 33 days post transfection when analysing the entire Sanger sequencing range of all our targeted genes (Figures 3A, B, 5A–C,, 6D, E and Supplementary file 2). Editing time could be reduced by fine-tuning sgRNA and CBE expression, especially through the use of species-specific promoters, synthetic promoters, modified T7 RNAP promoters and stable integration of expression constructs. This could also help to minimise possible toxic effects on growth, as observed in L. donovani and L. infantum (Figure 2—figure supplement 2B). Integrating sgRNA constructs would also ensure that sgRNAs are not lost during in vivo screening and that just one sgRNA per transfectant is expressed, thereby reducing the risk of horizontal plasmid transfer between parasites throughout a screen. Multiple sgRNAs per transfectant could theoretically cause multi-gene knockout effects. [SEP] Justify:

Output: The text corroborates the claim by discussing advanced cloning strategies and base editing methods that facilitate precise genomic editing. It explains how the sgRNA constructs, when integrated correctly, allow for targeted manipulation of genetic sequences, highlighting a method of editing with minimal off-target effects, which aligns with the capabilities of CRISPR technology.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: 104. 96 ± 1. 445. 11 ± 1. 122. 77 ± 0. 910. 0811. 95 ± 0. 090. 53 (KW) Age and task play a role in defining nestmate recognition cues in honey bee colonies Because previous studies have indicated that nestmate recognition in honey bee colonies is likely driven by components of the CHCs profile (van Zweden and D’Ettorre, 2010), and our discovery that the CHC profiles of individual workers seem to mature in association with the well-described age-dependent division of labor in this species (Robinson, 1992; Smith et al. , 2008; Søvik et al. , 2015), we next hypothesized that, in honey bees, nestmate recognition cues themselves mature in association with age-dependent division of labor, and reach maturation during foraging. To test this hypothesis, we investigated the behavioral responses of guard bees to related and unrelated focal bees of different ages (Day 1, Day 7, Day 14, and foragers on Day 21). At each test colony, the behavioral responses of guards to random related and unrelated returning foragers of unknown age were used as the benchmark for the baseline level of nestmate recognition behavior. Behavioral observations revealed that bees are accepted at the entrance of their own colony, regardless of age (Figure 5A, Pearson’s Chi-Squared, Day1: χ2 = 49. 05, df = 2, p<0. 001, Day 7: χ2 = 19. 07, df = 2, p<0. 001, Day 14: χ2 = 44. 89, df = 2, p<0. 001, Day 21: χ2 = 28. 32, df = 2, p<0. 001). In contrast, at the entrance to an unrelated colony, bees were accepted on Days 7 and 14, but rejected as foragers (Day 21) (Figure 5B, Day1: χ2 = 11. 61, df = 2, p=0. 003, Day 7: χ2 = 15. 51, df = 2, p<0. 001, Day 14: χ2 = 11. 91, df = 2, p=0. 002, Day 21: χ2 = 7. 35, df = 2, p=0. 04). [SEP] Justify:

Output: The text contrasts the claim by discussing the behavior of bees in relation to their colonies, specifically noting how guard bees accept related bees regardless of age, and how acceptance or rejection can occur in unrelated colonies. This indicates that there may be instances where bees interact variably with unrelated colonies, suggesting exceptions to the assertion that bees always live in colonies.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: All the measurements and most of the observations of the footprints have been made over the resulting three-dimensional and cartographic restitution with AutoCAD software, as it is not possible to measure them in situ, owing to the nonconsolidation of the sediment recording the traces. Once the footprints were scanned, each of the prints was individualized. The maximum length (FL) was measured parallel to the longitudinal axis of the footprint, while the width of the ball (FW) and the width of the heel (FHW) were measured perpendicular to the FL axis. The longitudinal axis was determined following the technique of D. Webb et al. (2006a). From the FL and FW measurements, the footprint index (FI) was calculated, which consists of the ratio between foot width and foot length: FI = FW/FL × 100. The arch index (AI) is a widely used measurement for the purpose of classifying the foot type according to a high (AI ≤0. 21), normal (AI = 0. 21–0. 26) and flat (AI >0. 26) arch (Cavanagh and Rodgers 1987). AI was measured according to Cavanagh and Rodgers (1987), whereby a perpendicular line to the foot axis was used to divide the toeless area into equal thirds (Fig. 17. 5a): rearfoot (A), midfoot (B) and forefoot (C) regions. Then, AI was calculated as the ratio of the area of the midfoot to entire toeless footprint area: AI = B/(A + B + C). Fig. 17. 5(a) Morphological parameters used in the current study, (1) maximum foot length (FL), width of the ball (FW) and width of the heel (FHW), (2) arch index (AI = B/A + B + C), (3) arch angle; (b) average and standard deviation of all biometric variables used in the current study for each trackFull size imageFinally, arch angle (AA) or Clarke angle is defined as the angle between the medial border line of the footprint and the line connecting the most medial point of the metatarsal region of the footprint and the apex of the concavity of the arch of the footprint (Citton et al. 2017) (Fig. 17. 5a). [SEP] Justify:

Output: The text does not address the claim regarding cave paintings or prehistoric human expression. Instead, it focuses on a technical description of the measurement and analysis of footprints, which is unrelated to the claim about cave paintings.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: LeishBASEedit allows to efficiently transform codons into STOP codons, thereby enabling the functional disruption of single- and multi-copy genes without requiring DSBs, HR, donor DNA, or isolation of clones. We demonstrate that this tool can be used for drug resistance screening, to target essential genes and to pursue loss-of-function screens in a pooled plasmid library transfection format. We therefore believe that this method will enable large-scale loss-of-function screens in Leishmania in many laboratories around the globe, simply by just sharing plasmid libraries. This will potentially even allow for genome-wide screens under different conditions in various clinical isolates and across multiple Leishmania species without limitations due to extreme cases of gene copy numbers and/or aneuploidy or lack of RNAi components. Materials and methods Key resources table Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional informationCell line (Leishmania mexicana)L. mexicana wildtypelaboratoryWHO strain MNYC/BZ/62/M379Used TriTrypDB (release 59, Aslett et al. , 2010) reference annotation: L. mexicana 2001U1103Cell line(Leishmania major)L. major wildtypelaboratory(release 59, Aslett et al. , 2010) reference annotation: L. major FriedlinCell line(Leishmania donovani)L. donovani wildtypelaboratory, Decuypere et al. , 2005Strain BPK190Used TriTrypDB (release 59, Aslett et al. , 2010) reference annotation: L. donovani BPK282A1Cell line(Leishmania infantum)L. infantum wildtypelaboratory, Sulahian et al. , 1997Strain MHOM/FR/91/LEM2259Used TriTrypDB (release 59, Aslett et al. , 2010) reference annotation: L. infantum JPCM5Recombinant DNA reagenthyBE4maxAddgene, Zhang et al. , 2020#157942Recombinant DNA reagentAncBE4maxAddgene, Koblan et al. , 2018#112100Recombinant DNA reagentpTB007Beneke et al. [SEP] Justify:

Output: The text contains information about LeishBASEedit, a tool that enables the functional disruption of genes without the use of double-strand breaks (DSBs) or donor DNA. This aligns with the claim about CRISPR technology since it suggests that the method allows for precise genetic modifications, similar to CRISPR’s capability of precise genomic editing. The mention of targeting essential genes and conducting large-scale loss-of-function screens indicates advancements in gene editing that support the claim regarding CRISPR's precision in genomic editing.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: These data support the hypothesis that nestmate recognition cues in honey bee colonies mature in association with age-dependent division of labor, and suggest that nestmate recognition is specific to behavioral interactions between guards and foragers at the entrance to the hive. Figure 5 Download asset Open asset Nestmate recognition cues are forager-specific. (A) Bees are accepted at a similar rate as Colony one foragers at the entrance to their source colony (Colony 1) at all ages. (B) Bees are rejected at a similar rate as Colony one foragers at an unrelated colony (Colony 2) on Day one and Day 21. However, bees are accepted at a similar rate as Colony two foragers at an unrelated colony (Colony 2) on Day seven and Day 14. All statistics using Pearson’s Chi-Square. Asterisks or letters denote posthoc statistical significance (p<0. 05), ns denotes non-significant comparisons. Sample size per group, N = 18–29. Figure 5—source data 1 Acceptance/Rejection scores for each bee tested in behavioral acceptance assays depicted in Figure 5. Download elife-41855-fig5-data1-v2. xlsx Surprisingly, we also observed that while young Day 1 bees are accepted by related guards, they are often rejected by unrelated guards (Figure 5B). This finding contradicts the broadly accepted ‘blank slate’ hypothesis, which predicts that because day-old bees are devoid of any defining chemical signatures, they should be always accepted by guards independent of relatedness (Breed et al. , 2004). While we do not yet know which specific components of the CHC profile of young bees, if any, might have triggered a rejection by unrelated guards in our colonies, one plausible interpretation of these data is that the observed response of guards to unrelated Day 1 bees is an artifactual experimental outcome of a forced behavioral interaction between two bee groups, which in colonies with a typical demography, do not normally interact in the context of the hive entrance. ability of colonies of social insects to reliably recognize group membership is one of the remarkable adaptations that enabled their immense ecological success. Yet, the molecular and physiological mechanisms that underlie this complex trait remain unknown for most species. [SEP] Justify:

Output: The text contrasts the claim by discussing the behaviors of bees in different social contexts, indicating that young Day 1 bees can be accepted or rejected by guards based on relatedness. This suggests that there can be exceptions to the notion that all bees interact uniformly within colonies, contradicting the claim that bees always live in colonies with no exceptions.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: AA is a conventional measurement that classifies the internal longitudinal arch between tendency to flatness or pronation (AA ≤31°), normality range (AA = 31°–45°) and tendency to cavus foot (AA >45°) (González-Martín et al. 2017). Estimation of Height and WeightMany studies support the use of foot length to obtain an individual’s range of height, while body mass can be estimated by the correlation between foot width and body weight (D. Webb et al. 2006a; Ukoha et al. 2013; Atamtürk and Duyar 2008; Krishan 2008; Robbins 1986). ChronologyAnother objective of this project has been to specify the chronology of the tracks in order to contextualize the activities that took place in this sector of the cave, the deep zone over 1 kilometre from the current entrance of Palomera. To study transit and use in this sector of the system in greater depth, since 2016 we have been conducting a project with the aim of surveying and dating the surroundings of the sector, including Sala and Galerías de las Huellas, on the third level of Ojo Guareña, and Galería del Cacique and its side passages, as well as on the fourth level, i. e. lower than Huellas and the main level within the Ojo Guareña Cave system. Sixteen organic samples were taken for radiocarbon dating. AMS dating of 15 samples was performed at. , between 2017 and 2019. The Gif-1721 sample, dated at the14C Laboratory in 1974, completes Table 17. 3. ResultsFootprints and TrackwaysAlthough the study has not yet been completed, about 700 footprints have been identified and at least 8–9 entry trackways to GH I and about 10–11 exit trackways (Fig. 17. 6). Many superimposed footprints follow an apparently chaotic pattern at the junction with a side passage. This may indicate that some people explored that passage, while the others waited in the main passage. Fig. 17. 6Plan of the Galería de las Huellas I, from the scanner, with the identification of imprints and trackwaysFull size imageAbout 500 human footprints have been identified in GH II, as well as a minimum of 7 entry trackways and 11 exit trackways towards Sala de las Huellas. [SEP] Justify:

Output: The text discusses various scientific measurements and findings related to the study of footprints and trackways found in caves, focusing on the methodologies and chronology of these studies. It does not mention cave paintings or any form of prehistoric human expression, making it irrelevant to the claim.


Input: Task: pass the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: Download as MPEG-4 Download as WebM Download as Ogg Light-induced cortical targeting of NuMA-RFP-Nano and spindle pulling. Light-induced cortical recruitment of NuMA-RFP-Nano (left), and DHC-SNAP (right), and spindle displacement toward NuMA/DHC-enriched cell cortex have been shown in this movie; it is played at five fps. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Light-induced cortical repositioning of NuMA-RFP-Nano and spindle rotation. Light-induced cortical repositioning of NuMA-RFP-Nano (left), and dynamics of SiR-tubulin (right) have been shown in this movie. The spindle rotation was coupled with cortical repositioning of NuMA. This movie is played at five fps. resources table Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional informationChemical compound, drugSiR-tubulinSpirochromeCat# SC00250 nMChemical compound, drugSiR-DNASpirochromeCat# SC00720 nMChemical compound, drugSiR-actinSpirochromeCat# SC00150 nMChemical compound, drugSNAP Cell 647-# S9102S0. 1 μMChemical compound, drug-starBioLabsCat# S9105S0. [SEP] Justify:

Output: The text is irrelevant to the claim as it discusses light-induced cortical targeting and spindle dynamics without mentioning CRISPR technology or genomic editing. It provides detailed descriptions of experimental setups and reagents but fails to address or corroborate the claim regarding the capabilities of CRISPR.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: In the well-studied honey bee, previous studies suggested that the chemical cues that drive nestmate recognition are absent in newly eclosed bees, and subsequently develop primarily through the homogenization and transfer of chemicals between colony members via direct interactions such as allogrooming and trophallaxis, and indirect interactions such as physical contact with wax and other nest materials (Breed et al. , 2015; Breed et al. , 2004). However, the data we present here suggest that the overall development of individual CHC profiles of honey bee workers primarily depends on an innate developmental process that is associated with the stereotypic age-dependent division of labor in this species, and that colony-specific cues are likely only carried by foragers. Therefore, we posit that it is unlikely that CHC profiles in honey bees develop through homogenization and transfer mechanisms between nestmates and hive materials. Furthermore, given the established implicated role of CHCs in nestmate recognition (van Zweden and D’Ettorre, 2010), we additionally posit that CHC homogenization mechanisms are unlikely to play a key role in the production of colony-specific cues in honey bees. A major line of investigation in understanding nestmate recognition of social insects has been to determine how colony-specific cues are determined. Cue specificity has historically been proposed to be determined by mechanisms under genetic control or acquired from the environment (Crozier and Dix, 1979). Although our studies do not directly address the mechanism by which cue specificity is determined in honey bees, data from cross-fostering experiments suggest that cue development and specificity are defined by interactions between factors derived from the colony-of-origin of individual workers and the actual hive environment they develop in. Therefore, our data suggest that CHC profiles of honey bee workers develop via a biphasic process that is governed, at least in part, by the intrinsic physiology of individual workers, the specific behavioral tasks they are engaged in, and the hive environment they age in. In phase one, similar to other social insect species (Soroker et al. , 1995a), the total CHC amount builds up, possibly to increase the resistance of workers to desiccation while still inside the protective hive environment (Chung and Carroll, 2015). [SEP] Justify:

Output: The text contrasts the claim by discussing the specific developmental processes of honey bee workers and their interactions in a colony, suggesting that cues and behaviors might not always be reliant on a strict colony structure. The mention of interactions influenced by the hive environment and individual worker physiology implies that there may be instances where individual bees can exhibit different characteristics or behaviors outside of typical colony interactions, challenging the absolute nature of the claim.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: In addition, the new cartography shows that one of the visitors separated from the group to inspect one of the hidden corners of the gallery (Fig. 17. 7). Fig. 17. 7Plan of the Galería de las Huellas II, from the scanner, with the identification of imprints and trackwaysFull size imageA preliminary study of 39 GH I footprints will be described. They are integrated in seven trackways in Galería de las Huellas I: two in an entry direction (trackways 1 and 2) and the remaining five in the exit direction towards Sala de las Huellas (Fig. 17. 8 and Table 17. 1). All these footprints are located on the sides of the passage and correspond to 22 right feet and 17 left feet. They are in an excellent state of preservation, with the identification of complete footprints with clear anatomical features (toes, balls, heels, etc. ) in the form of a low relief in the soft loam sediment. Fig. 17. 8Identification of the trackways and imprints analysed in this study. Galería de las Huellas I, Ojo Guareña Cave systemFull size imageTable 17. 1 Main measurements of the 39 footprints (grouped in 7 trackways) selected for this study, from Galería de las (mm)Full size tableThe measurements of the maximum lengths and widths of the footprints are given in Table 17. 1. The sample of footprints is characterized by a certain uniformity in foot length, with measurements ranging between 250 and 290 mm, resulting in average estimated heights of between 173 and 188 cm (Tables 17. 1 and 17. 2). This suggests that the footprints correspond to adult individuals, probably males. The foot width range shown in Table 17. 2 varies from narrower traces, about 123 mm wide, to prints about 133, 137 and 144 mm wide (Tables 17. 1 and 17. 2). These values stress the presence of strong individuals. Table 17. 2 Estimated stature and body mass of seven trackways of Galería de las Huellas I, using different height calculation equations (mm)Full size tableDespite the uniformity in foot size among the trackways analysed, the AI and the AA reveal some morphological differences. [SEP] Justify:

Output: The text does not directly support or contradict the claim as it focuses on the analysis of footprints found in a cave rather than discussing cave paintings or human expression. It provides detailed measurements and observations of the footprints but does not connect to the artistic or expressive aspects associated with cave paintings.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: Javascript is disabled in your browser. Please enable Javascript to view PeerJ. Related research -genome editing in human iPSCs with -CAS9 by co-targeting ATP1a1 View 10. 7717/peerj. 9060 Published 2020-05-01 Accepted 2020-04-04 Received 2020-02-17 iPSCs, Genome editing, ATP1A1, Stem cells, Disease models Copyright © 2020 Liu et al. is an open access 2020. Enhanced genome editing in human iPSCs with -CAS9 by co-targeting ATP1a1. PeerJ 8:e9060 The authors have chosen to make the review history of this 1. The introduced mutation confers resistance to cardiac glycosides, which can then be used to select successfully targeted cells. Using this system, we introduced different formats of donor DNA for homology-directed repair (HDR), including single-strand DNAs, double-strand DNAs, and plasmid donors. We achieved a 35-fold increase in HDR when using plasmid donor with a 400 bp repair template. We further co-targeted ATP1A1 and a second locus of interest to determine the enrichment of mutagenesis after cardiac glycoside selection. Through this approach, INDEL rate was increased after cardiac glycoside treatment, while HDR enrichment was only observed at certain loci. Collectively, these results suggest that a plasmid donor with a 400 bp repair template is an optimal donor DNA for targeted substitution and co-targeting ATP1A1 with the second locus enriches for mutagenesis events through cardiac glycoside selection in human iPSCs. genome editing technology provides researchers with a powerful tool to investigate the function of genes in nearly any species. By using programmable nucleases to cut the genome, researchers can mutate or correct the genes of interest for further investigation. Although there are still concerns about safety issues, such technologies may pave the way to develop new therapies for cancer treatment or genetic diseases (Cornu, Mussolino & Cathomen, 2017; Fu et al. , 2013; Hsu et al. , 2013; Ihry et al. , 2018; Pattanayak et al. , 2013). [SEP] Justify:

Output: The text corroborates the claim by discussing genome editing technology, specifically mentioning the use of programmable nucleases to cut the genome, which is a fundamental aspect of CRISPR technology. It highlights that researchers can mutate or correct genes of interest for further investigation, thus supporting the assertion that CRISPR allows for precise editing of genomic sequences.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: In phase two, the total amount of CHCs remains constant but the relative abundances of individual components shift in association with the age-dependent behavioral maturation of workers, at least in part, via the transcriptional regulation of CHC biosynthetic enzymes. Which specific components of the honey bee CHC profile represent the nestmate recognition cue remains unknown. Although it has been shown that CHCs are likely used for nestmate recognition in honey bees (van Zweden and D’Ettorre, 2010), it is unlikely that all components of the CHC profile contribute to this process (Akino et al. , 2004; Dani et al. , 2005; Dani et al. , 2001; Martin et al. , 2008; Ruther et al. , 2002). In fact, it has previously been shown that alkenes seem to play a more prominent role in nestmate recognition in the honey bee than alkanes (Dani et al. , 2005). Our data also indicate that although unrelated foragers raised in the same colony are equally accepted, their overall CHC profiles remain somewhat qualitatively different (Figure 3C). These data provide two important insights. First, guards are not likely using the full CHC profile of individuals to determine group membership. Second, differences in the CHC profiles of co-fostered nestmate foragers of similar age that originated from different source colonies indicate that the chemical profiles of individual workers are not likely to be the product of a stochastic CHC homogenization and transfer between colony members. The observation that the mRNA expression levels of genes that encode CHC-biosynthesis enzymes vary in association with age and/or task further indicate that the primary mechanism for the dynamic regulation of the CHC profile of individual honey bee workers is directly associated with the well-established age-dependent division of labor in the honey bee (Robinson, 1992; Smith et al. , 2008; Søvik et al. , 2015). Although these data do not directly exclude the possibility that some particular CHCs are transferred across colony members, they do indicate that the overall observed qualitative age- and task-dependent changes in the CHC profiles of individual workers are affected by intrinsic molecular dynamics of the CHC synthesis pathway. [SEP] Justify:

Output: The text contrasts the claim by discussing various chemical compounds (CHCs) used for nestmate recognition among honey bees and indicates that differences exist between individuals from different source colonies. This suggests that while honey bees generally live in colonies, the text supports the notion that there may be exceptions regarding the shared chemical profiles and behaviors of foragers that come from different colonies.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: The AA shows a tendency to flatness or pronation in all trackways (AA ≤31°), except in trackway 230, which corresponds to a normal range (Fig. 17. 5b). Furthermore, the AI suggests a normal arch range for all trackways, except for trackway 230, which displays a slightly high arch, and for trackway 2 and trackway 300, which display a flat arch. These flat arches according to the AI are commonly a consequence of weight-bearing activities, so it may suggest that some individuals were carrying an additional weight. The analysed footprints belong to seven trackways, which represent a minimum of five individuals according to the direction of the trackways (two entry trackways and five return trackways) (Fig. 17. 8). The length and width measurements of trackways 2 and 100 are similar, which suggests that these may belong to the round-trip trackway of the same individual, whose estimated height is about 175–179 cm. The weight calculations are more disparate, however. Depending on the chosen equations and markers, the weight could be either about 76–78 kg or 90–99 kg (Table 17. 2). ChronologyThe chronology of the trackways has been determined in order to contextualize the activities that took place in this part of the cave, over 1 kilometre from the current entrance of Palomera Cave. Delibrias et al. (1974: 53) published four radiocarbon dates from Ojo Guareña, and Sample Gif-1720 (OG2), from the footprint site, provided an age of 15,600 ± 230 BP, in the Upper Palaeolithic. This sample, collected in 1970, was taken from the charred remains of a torch at the bottom of a pit that connects (lower level) with Galerías de las Huellas (upper level). In 1999, a new date of charcoal on a footprint in Galería de las Huellas II was more recent, 3820 ± 50 BP (pers. comm. Arsuaga), which suggests that both Palaeolithic hunter-gatherers and Chalcolithic farmers transited this sector of the cave (Ortega et al. 2013: 182). [SEP] Justify:

Output: The text is irrelevant to the claim as it discusses trackway analysis and foot measurements related to Palaeolithic individuals, with no mention of cave paintings or human expression. It does not support or contradict the claim about cave paintings.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: The most widely used technologies to introduce double-strand breaks (DSBs) are zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the RNA-guided cluster regularly interspaced short palindromic repeats-Cas9 (-Cas9) system (Joung & Sander, 2013; Sander & Joung, 2014; Urnov et al. , 2010). ZFNs and TALENs link endonuclease catalytic domains to DNA-binding proteins to generate DSBs. Alternatively, the -Cas9 system uses small guide RNAs that are paired with target DNA to induce DSBs by Cas9. -Cas9 appears to be more flexible, efficient, and easier to design, when compared to the other two systems (Gaj, Gersbach & Barbas 3rd, 2013). Generating DSBs at the target site activates two primary endogenous DNA repair mechanisms in mammalian cells (Valerie & Povirk, 2003). The most frequent repair pathway is non-homologous end joining (NHEJ), which is based on the direct ligation of two DNA ends without the need for a homologous template (Kanaar, Hoeijmakers & Van Gent, 1998). This error-prone mechanism can create an insertion or deletion of bases in the genome (INDEL), which may disrupt gene expression by introducing frameshift mutations. This methodology has been an important tool for deleting genes of interest across many species (Bapteste & Philippe, 2002; Belinky, Cohen & Huchon, 2010; Luan et al. , 2013). Homology-directed repair (HDR) is a more precise mechanism that is based on DNA recombination between genomic DNA and the homologous template (Johnson & Jasin, 2001). HDR allows the genomic sequences to be modified based on the introduced repair template. Utilizing HDR machinery for genome editing provides advantages for genome engineering due to the accuracy and predictable nature of the mutation. Numerous studies have applied this genome editing strategy to therapeutic approaches (Deverman et al. , 2018; Shim et al. , 2017; WareJoncas et al. , 2018). [SEP] Justify:

Output: The text corroborates the claim by detailing how the CRISPR-Cas9 system enables precise genetic modifications through the homology-directed repair (HDR) mechanism. It explains that HDR facilitates genomic sequence modification based on introduced templates, providing 'advantages for genome engineering due to the accuracy and predictable nature of the mutation.' This directly supports the assertion that CRISPR technology allows for precise editing of genomic sequences.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: However, our studies also importantly show that genetically-related bees that age in different colonies exhibit qualitatively different CHC profiles and CHC biosynthesis gene expression levels, which suggests that the CHC synthesis process is also plastic and could be modulated by factors associated with the hive/social environment. We were initially surprised by our observation that Day one bees are accepted at the entrance to their source colony but rejected by guards at the entrance of an unrelated colony since previous studies hypothesized that the lower amounts of total CHCs in young bees represent a ‘blank slate’ in terms of the nestmate recognition cue because these bees are readily ‘accepted’ when introduced into unrelated colonies (Breed et al. , 2004). In fact, this phenomenon was exploited here to introduce cohorts of bees to foster colonies, typically by placing the new bees on the top frames of experimental hives. This apparent conundrum highlights an important, yet often underappreciated, aspect of the nestmate recognition system in honey bees and other social insect species, which is that the ‘rejection’ behavior by guards is highly contextual. Conceptually analogous to other biological systems responsible for the detection of ‘self’ versus ‘non-self’ (e. g. , the acquired immunity system in vertebrates), behaviors associated with nestmate recognition are restricted to interactions between guards and incoming bees at the entrance to the hive (Couvillon et al. , 2013). Therefore, we speculate that because nestmate recognition is spatially restricted to specific behavioral interactions between entering bees and guards at the entrance, the commonly observed ‘acceptance’ of day old bees outside the specific context of the hive entrance actually represents the lack of behavioral ‘rejection’ rather than a true self-recognition-dependent ‘acceptance’. Consequently, the observation that Day one bees are rejected at the entrance of an unrelated colony suggests that nestmate recognition of young bees either depends on components of the CHC profile that are already present in Day one bees, non-CHC chemical cues, or an altogether different sensory modality. Alternatively, because newly eclosed bees usually perform cell cleaning behaviors at the interior of the hive, and therefore do not typically interact with guards at the hive entrance (Robinson, 1992; Smith et al. , 2008; Søvik et al. [SEP] Justify:

Output: The text contrasts the claim by discussing how certain behaviors and recognition of bees can vary based on their social environment and the colony context. It indicates that interactions, such as acceptance or rejection of bees at colony entrances, suggest that the nestmate recognition system can behave differently based on conditions, which implies that not all bees may function strictly within a colony framework.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: The new survey has identified a significant amount of evidence of visits, totalling 60 records: 6 remains in Sala Negra, 23 in and side passages of Galería del Cacique, 22 in Sala de las Huellas and another 9 in Galerías de las and II, different from the footprints (Fig. 17. 9). Fig. 17. 9Map of sector of the Sala and Galería de las Huellas site, separated in the different levels; (a) third level, Sala de las Huellas and Galería de las Huellas I–II; (b) fourth level,. The black points represent the archaeological evidences and the red numbers the dated samples, referred to in Table 17. 3. (Modified from Grupo Espeleológico Edelweiss 1986; Ortega et al. 2014)Full size imageIn all these passages we have documented scattered remains of charred wood as well as small concentrations of charcoal and charred wood marks on the walls and boulders, to mark the rock or rekindle the torches. Several panels of bear claw scratches on the walls and abundant mustelid imprints have been documented at the beginning of Galería de las and also in the initial section of Laberinto Otilio. This survey has also documented the deterioration of the human footprints in Sala de las Huellas, in which only three areas with prints are preserved, all of them in the southern section of the great hall, on the irregular surface of the large inclined blocks covered with loam in which a few human footprints can be observed (Fig. 17. 3a). This study thus presents the results of 15 AMS radiocarbon determinations for samples from different pieces of charcoal and torches in the Otilio-Negra-Huellas sector (Figs. 17. 2b–e, 17. 3b and 17. 9). They were selected from 55 points with charcoal identified in the archaeological surveying. Table 17. 3 reflects all the ages of the samples in the different sectors. The number refers to the identification of the sample on the maps in Fig. 17. 9. [SEP] Justify:

Output: The text does not address the claim regarding cave paintings and prehistoric human expression. Instead, it discusses archaeological documentation of human footprints and charcoal remains in various caves, which, while related to human activity, does not provide insight into artistic expression or cave paintings specifically.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: (2017) used “co-” methods to target exon 4 of ATP1A1 concurrently with a second locus of interest in established human cell lines. By selecting ATP1A1 mutations using ouabain, the percentage of cells harboring an INDEL at the second locus increased (Agudelo et al. , 2017). However, the co-strategy was not applied to human iPSCs and in the cell lines that were tested the HDR rate was not significantly improved. Here, we report the use of a -Cas9 editing system that targets intron 4 of ATP1A1 in human iPSCs. By introducing a repair template to substitute the two N-terminal amino acids Q118 and N129, we specifically created an HDR-directed mutation that confers resistance to cardiac glycosides. We then applied different types of repair templates to investigate the efficiency of HDR. Furthermore, by co-targeting with the second locus of interest, we examined the efficiency of HDR-directed mutation after selection with the cardiac glycoside digoxin. Materials & ofCas9 plasmids and donor templates guide RNAs were designed obeying the sequence rule. The construction of plasmids followed the protocol established by Ran et al. (2013). To generate construct targeting ATP1A1, a guide sequence (5′--3′) was cloned into PX459 pSpCas9(BB)-2A-Puro plasmid following the fast digest protocol (Ran et al. , 2013). The gRNAs targeting the other locus were cloned in to PX459 via the same procedure and the sequences of gRNAs were listed as below: (DGUOK exon1: 5′--3′; DGUOK exon4: 5′--3′; POLG exon10: 5′--3′; GATA6 exon4: 5′--3′; RYR2B exon50: 5′--3′). Single-stranded oligodeoxynucleotides (ssODNs) were obtained as Ultramer® DNA oligonucleotides (). Designed double-stranded DNAs were generated through GeneArt™ synthesis with the addition of restriction enzyme cutting sequences at both ends (). [SEP] Justify:

Output: The text corroborates the claim by detailing the use of a Cas9 editing system to specifically target genomic loci, such as 'exon 4 of ATP1A1', for precise modifications. It discusses the introduction of repair templates and the generation of specific mutations, which showcases how CRISPR technology can facilitate accurate genome editing, supporting the assertion that CRISPR allows for precise editing of genomic sequences.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: , 2015), differences in rejection of Day one bees between these two colonies might represent an experimental artifact resulting from differences in tolerance to the forced behavioral interaction between two bee groups that normally do not interact. Additionally, it has previously been shown that observed levels of guarding behaviors in honey bees are plastic, and could fluctuate in response to various environmental factors such as seasonal weather patterns, overall colony size, food availability, and ‘robbing’ pressures from other colonies or predators (Downs and Ratnieks, 2000). Likewise, more extreme forms of plasticity in nestmate recognition systems have been reported in other social species. For example, some social insects can switch between using visual or chemosensory modalities for nestmate recognition under different circumstances (Baracchi et al. , 2015). Together, it seems that instead of being driven by simple binary decisions, nestmate recognition systems in the honey bee and other social insect species depend on a plastic recognition of ‘friends’ versus ‘foes’ as part of a broader group-level optimization of colony fitness. In conclusion, we propose that nestmate recognition cue production and acquisition in honey bees are not likely to be primarily driven by CHC homogenization and transfer mechanisms as previously described in some ant species (Boulay et al. , 2000; Lenoir et al. , 2001; Meskali et al. , 1995; Soroker et al. , 1994; Soroker et al. , 1995b; van Zweden et al. , 2010). Instead, we propose a new model for the regulation of nestmate recognition in honey bee colonies, which stipulates that unknown factors associated with the hive environment play a direct or indirect role in defining the developmental kinetics and specificity of nestmate recognition cues by modulating the cellular and molecular processes that are responsible for pheromone synthesis. Thus, it is plausible that the colony/social environment drives the intrinsic development of similar pheromone profiles by individual colony members, which in typical honey bee hives, is associated with the physiological processes that drive age-dependent division of labor. If true, this model could resolve previous seemingly contradictory data which suggested that honey bee CHC profiles are defined by genetic (Page et al. , 1991) versus environmental (Downs and Ratnieks, 1999) factors, as well as open the door for comparative mechanistic studies of how complex social traits evolve and function in different social insect clades. [SEP] Justify:

Output: The text contrasts the claim by discussing the dynamics between different bee groups that 'normally do not interact,' suggesting that there can be situations where bees from different colonies may not always behave as if they are in a strict colony structure. The mention of 'plastic' behaviors and environmental factors affecting colony interactions implies variability in social structures, thus challenging the assertion that bees always live in colonies without exception.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: The table has been divided into two groups, the upper one corresponds to the third level, with three dates from Sala de las Huellas, two from Galería de las Huellas II and one from Galería de las Huellas I. Additionally, it should be noted that we have been able to identify the 1999 sample in the field, which corresponds to sample 17. OG17. GH2. C2 (Fig. 17. 9, number 3). Table 17. 3 Summary of the human footprint site14C data from charcoal, all unpublished and AMS except Gif-1721 (Delibrias et al. 1974)Full size tableRegarding the fourth level of the karst, the survey of the northeast end of this sector could indicate a possible access from the San Bernabé blind valley (Figs. 17. 1b, c and 17. 2a), and one sample of the five identified in the blockage in has been dated (Fig. 17. 9b, number 13). Eight samples from have been dated, to add to Sample GIF-1721 OG2 published in Delibrias et al. (1974: 33) (Fig. 17. 9b, numbers 7–12 and 14–16). The distribution map and chronological table of the remains (Fig. 17. 9 and Table 17. 3) reveal the wide chronological range from the to the Chalcolithic in the Laberinto Otilio-sector and the concentration of dates in the third level. sample (GIF-1721 OG2) was collected by members of the Grupo Espeleológico Edelweiss at the base of the pit that separates the two Galerías de las Huellas and connects with (Figs. 17. 2b and 17. 9b, number 7). The new dates from this section of the maze are concentrated in the (Fig. 17. 9b and Table 17. 3). A third period corresponds to the Chalcolithic, with two dates from the upper part of Laberinto Otilio, in areas with difficult access to Sala de las Huellas. The spatial layout of archaeological evidence in Sala de las Huellas displays a clear perimeter distribution towards Galerías de las Huellas II and I. [SEP] Justify:

Output: The text does not address cave paintings or prehistoric human expression. Instead, it discusses archaeological samples and findings, including dating and spatial layouts of evidence in specific sites. Thus, it is irrelevant to the claim about cave paintings.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: In order to generate the amino-acid substitution allele, we designed a 153 single-stranded oligodeoxynucleotides (ssODN) with the sequences that replace glutamine with arginine at position 118 and asparagine with aspartic acid at position 129 (Fig. 1A). After transfecting both sp-Cas9-gRNA (px459-ATP1A1-intron4) and donor template into the cells and treating these cells with a high dose of digoxin, we collected the surviving clones for genotyping (Fig. 1B). Using primers outside the targeted homology arms to perform PCR (out-out PCR) and restriction fragment length polymorphism (RFLP) analysis, we observed that all the cells that were resistant to digoxin or ouabain were successfully repaired through HDR, and both heterozygous and homozygous mutations were identified (Fig. 2A). The genotype was confirmed by genomic sequencing. In order to determine the optimum dosage of digoxin and ouabain, we performed a dose–response assay of both drugs on wild-type and ATP1A1 homozygous mutant iPSCs. We effectively eliminated all wild-type iPSCs at 1 µM after 72 h of exposure. In contrast to the parental iPSCs, homozygous ATP1A1 mutant iPSCs survived in the presence of either drug and were indistinguishable from untreated cells (Figs. 2B–2F). These results demonstrate that cardiac glycosides can be used to effectively identify HDR-driven mutations at the endogenous ATP1A1 locus. Figure 1: Design ofCas9 targeting ATP1A1. (A) The design of ATP1A1 guide RNA and single-stranded oligonucleotide (ssODN) donor. Guide RNA was designed to target intron 4 of the ATP1A1 gene. The expected cut site is 3–4′ nucleotides upstream of the Motif (PAM) sequence and 20 nucleotides downstream of the exon 4 region. The donor template was designed to introduce nucleotide substitutions that result in the replacement of Glutamine with Arginine (Q118R) and Asparagine with Aspartic acid (N129D). The EcoRI cut site was introduced to allow identification of positive clones. [SEP] Justify:

Output: The text corroborates the claim by detailing a process involving CRISPR technology, specifically using sp-Cas9 and a donor template to enable precise genomic editing. It describes the design of guide RNA and the process of homology-directed repair (HDR), which is a key aspect of how CRISPR can be utilized for accurate genomic modifications.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: Materials and methods Animal husbandry and bee collections Request a detailed protocol Honey bee (Apis mellifera) colonies were reared and managed using standard beekeeping techniques across two locations near St. Louis, MO: center (38° 31’N, 90° 33’W) and a residential home. For all experiments that included collections of bees at specific ages, capped brood frames were taken from a colony and placed in a humidified 32°C incubator. Once eclosed, about 1000 bees (<24 hr old) were marked with a spot of paint () on their thorax, and then reintroduced into either their source or a foster colony, depending upon the experiment. For collections of bees at specific ages, marked bees were collected from internal frames of the colony one day post reintroduction (Day 1), seven days post reintroduction (Day 7), 14 days (Day 14) post reintroduction, and as returning foragers, identified by pollen loads on their hind legs or having a distended abdomen due to nectar loads, between 18 and 21 days post reintroduction. Bees used for chemical and molecular analyses were placed in individual 1. 7 mL microtubes and immediately placed on dry ice. All samples were kept at −80°C until further analysis. Single-cohort colonies Request a detailed protocol Single-cohort colonies (SCC) were established as previously reported (Ben-Shahar et al. , 2004; Ben-Shahar et al. , 2002; Greenberg et al. , 2012; Robinson et al. , 1989; Whitfield et al. , 2003). In short, about 1000 newly eclosed bees (<24 hr old) were placed in a small wooden nucleus hive-box with a young, unrelated mated queen, one honey frame from their source colony, an empty comb frame, and three new frames with wax covered plastic foundation. Bees were collected as typical-aged nurses and precocious foragers one week after introduction, and as over-aged nurses and typical-aged foragers at three weeks after introduction. Bee samples were collected and stored as above. Undertaker collection Request a detailed protocol To induce ‘undertaking’ behavior, about 1000 dead bees were placed into the top of two different colonies, and the first 20 bees that were observed removing dead bees from the colony were collected from the entrance. [SEP] Justify:

Output: The text contrasts the claim by indicating that bees can be marked and collected in various experimental setups, suggesting the possibility of keeping bees outside traditional colonies for research purposes. Phrases like 'single-cohort colonies' and the specific handling of bees in non-natural settings (e.g., 'placed in a small wooden nucleus hive-box') imply that under certain conditions, bees do not always live in conventional colonies.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: This southern part of the hall is next to a connection between boulders with the lower Otilio maze, and several charcoal fragments are observed in both levels. The charcoal identified as number 1 from Sala de las Huellas was dated to 4080 ± 30 BP, and the charcoal from the upper part of Laberinto Otilio, number 16, was dated to 3850 ± 30 BP (Fig. 17. 9 and Table 17. 3). In turn, the six samples from the in the third level have ages between those two dates (Fig. 17. 9a and Table 17. 3). This suggests that the Huellas sector was explored in approximately 4300 calBP. In contrast, visits in the immediately lower took place in a wide chronological range from the to the Chalcolithic. DiscussionAn increasing number of sites with prehistoric human footprints are becoming known (Aldhouse-Green et al. 1992; Bell and Neuman 1997, 1999; Ambert et al. 2000; Facorellis et al. 2001; Onac et al. 2005; Bennett et al. 2010; Bennett and Morse 2014b; Citton et al. 2017; Ashton et al. 2014; S. Webb et al. 2006b; D. Webb et al. 2014; Atamtürk et al. 2018; Roach et al. 2016). Recent research approaching this type of site through different disciplines is contributing new knowledge of interaction between humans, the land and the environment in painted caves as special places (Pastoors et al. 2015, 2017). and Galería de las Huellas can be framed within those sites that provide information on the use and transit of the dark zone of caves, where the tracks of old paths are preserved, but whose specific relationship with the concept of the natural, social and cultural environment is unknown (Mlekuž 2012; Moyes 2012). In the case of the tracks in Ojo Guareña, their singularity lies in the fact that they are a long way from the possible entrance point sand without a direct relationship with symbolic spaces (rock art, burials, etc. ), although these exist in other parts of the cave system. The large number of traces is unique, with over 1200 footprints of a minimum of 6 individuals but probably of between 9 and 11, according to the trackways that have been counted. [SEP] Justify:

Output: The text does not address cave paintings or their relation to prehistoric human expression. Instead, it focuses on the dating and significance of charcoal fragments and prehistoric footprints without mentioning artistic expression or cave art specifically.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: (B) The timeline of performing gene-editing on human iPSCs. Human iPSCs were transfected with aCas9 plasmid targeting ATP1A1 intron 4 and a repair template through electroporation. Cells were treated with 10 µM Y27632 for 24 h following 48 h of puromycin exposure to select transfected iPSCs. Cells were maintained for an extra 48 h until colonies were observed and before administering digoxin for 72 h. Surviving cells were isolated and genotype determined by sequence analyses and PCR. Download full-size image DOI: 10. 7717/peerj. 9060/fig-1 Figure 2: Introduction of ATP1A1Q118R/N129D in human iPSCs confers resistance to cardiac glycosides. (A) RFLP analysis of ATP1A1 mutation. Transfected iPSCs were selected through 1 µM of digoxin exposure and genomic DNA from each individual colony was extracted for out-out PCR reaction. PCR amplicons were digested by EcoRI to identify homozygous and heterozygous mutations (for raw data see Fig. S1). (B) Wild-type iPSCs and homozygous ATP1A1 mutants were exposed to 15. 625, 31. 25, 62. 5, 125, 250, 500, and 1000 nM of digoxin and ouabain for 72 h. Cell viability was determined by luminescence assay (B, for raw data see Data S1) and was confirmed by DAPI staining (C–F) (for uncropped images see Figs. S2—S5). The experiment was conducted in triplicate (n = 3) and data are shown as mean ± SD. Statistical differences were determined by ANOVA followed by Tukey’s test (*p <. 01). (Oubain: n = 3, df = 8, F = 5731. 75, p = 4. 85 × 10−29; Digoxin: n = 3, df = 8, F = 37. 33, p = 1. 17 × 10−9). Download full-size image DOI: 10. 7717/peerj. [SEP] Justify:

Output: The text corroborates the claim by describing a gene-editing process that employs aCas9 plasmid to target specific introns within the genomic DNA of human iPSCs. This indicates the use of a technology aligned with CRISPR, which is known for its precision in editing genomic sequences. The description of performing precise sequence analyses and PCR suggests that the editing is conducted with accuracy, supporting the assertion about CRISPR technology.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: Returning foragers and in-hive nurses of unknown ages were also collected from each colony at the same time. Samples were stored and processed as described above. Big-back colony Request a detailed protocol Big-back colonies were established as previously described (Ben-Shahar et al. , 2000; Withers et al. , 1995). In short, bees were introduced in two cohorts to a 5-frame hive box containing three empty comb frames, two brood frames, and a new queen. In the first cohort, 200 day-old bees were collected as described above and marked on the thorax with paint. Half of these bees were marked with a plastic tag attached to the thorax (~3 mm diameter,~1 mm thick; ‘big-back’ bees). Day-old bees in the other cohort were collected and introduced 4 days later as described above to increase the proportion of precocious foragers in the first group. The entrance to the colony was blocked by a piece of Plexiglas with holes in it that prevented ‘big-back’ bees from leaving the hive, but allowed paint marked bees to leave. Bees were collected at 7 days of age: returning foragers were collected as described above, and ‘big-back’ bees were collected as they were attempting to leave the hive via the holes in the plastic. Reversion colony Request a detailed protocol Reversion colonies were made by collecting ~1000 foragers from a single source colony by vacuuming them directly into a sealed 5-frame hive box containing two brood frames, one honey frame, and two empty comb frames. The hive was sealed and moved to a new location ~30 miles away from the source colony, and a new queen was added that night. The hive was sealed for 3 days, and then was opened to allow normal foraging activity to resume. During this time, in the absence of nurses, some foragers reverted back to nursing behaviors (Robinson et al. , 1992). Actively foraging bees were collected at the hive entrance as described above and reverted nurses were collected from internal frames as described above. Cross-fostering experiment Request a detailed protocol 1000 day-old bees from two independent source colonies were collected and marked as above. Half of the bees in each marked cohort were randomly reintroduced to both their own source colony and the reciprocal foster colony. [SEP] Justify:

Output: The text presents a situation where bees are relocated and reintroduced into a new hive, indicating that their living conditions can change based on experimental setups. Specifically, the text describes a 'Reversion colony' where foragers are moved from one colony to another and revert to nursing behaviors in the absence of nurses, demonstrating that bees can exist outside of their original colonies during this process. This suggests that there are exceptions to the claim that bees always live in colonies.


Input: Task: pass the claim using the given text. Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: The superimposition of the footprints, in opposite directions, is indicative of round-trips, which suggests a single inspection of these passages, during which some members approached the recesses of the walls or entered the side passage in GH I. These preliminary observations are visible in both of the Galerías de las Huellas passages. It can therefore be proposed that the human footprints in the Sala and Galerías de las Huellas correspond to a single visit, possibly to explore this deep sector of the cave. However, the human visits or tours that have been documented at different times in the Laberinto Otilio-sector are more difficult to comprehend. This sector is characterized by the absence of archaeological record and the isolation of the sector from places of symbolic activity. Nevertheless, this space was visited on at least four occasions in prehistory: the first about 19,000 calBP, the second moment around 7700 calBP, the third about 6500 calBP and the last time in relation with the exploration of the Huellas sector about 4300 calBP. The results of this project show that the first significant explorations in Laberinto Otilio, a relatively comfortable maze in its initial section, took place in the Mesolithic and Early Neolithic, the times of which the first remains are preserved in Sala Negra. During more intense explorations in the Chalcolithic, the unstable final ramp of large boulders was first accessed in order to reach the upper level where their footprints have been preserved in the Sala and Galerías de las Huellas. The challenges involved in access to this sector, combined with the spaciousness of its passages and the chaotic boulders on the floor, do not facilitate an understanding of the specific activities that took place, although they must be linked with the exploration and knowledge of the underground world. The characteristics of this site mean that it cannot be compared with most human footprint sites, which are generally linked with the zones of habitats, or sacred or rock art sites, both in the open air and in caves (Bennett and Morse 2014b; Ashton et al. 2014; Westaway et al. 2013; Atamtürk et al. 2018). Ciur-Izbuc Cave, Romania (D. Webb et al. 2014); Bàsura Cave, Italy (Citton et al. 2017); Aldène Cave (Ambert et al. [SEP] Justify:

Output: The text does not address the claim directly nor provides information regarding cave paintings or human expressions therein. Instead, it discusses human footprints and the exploration of cave sectors without a connection to artistic expression or cave paintings, making it irrelevant to the claim.


Input: Task: corroborate the claim using the given text. Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: 9060/fig-2 Comparing the HDR efficiency while using different repair templates Although single-stranded oligodeoxynucleotides (ssODNs) have commonly been used as repair donor templates, the efficiency is generally low when using iPSCs (Radecke et al. , 2010; Yoshimi et al. , 2016). Double-strand DNAs with long homologous arms have been shown to improve the efficiency of introducing changes through genome editing in HEK293T cells (Song & Stieger, 2017). We therefore proposed to compare the efficiency of HDR-mediated mutagenesis between ssODNs and dsDNA at the ATP1A1 locus using endogenous selection. We co-transfected human iPSCs with px459-ATP1a1-intron4 and donor template presented in the following formats: ssODNs, dsDNAs, or dsDNAs cloned into a plasmid. HDR-positive clones were then selected using a 72-hour exposure of 1 µM digoxin (Fig. 3A). As shown in Fig. 3B, up to two positive clones were observed when co-transfected with 150 base-pair (bp) template either as ssODN, dsDNA, or linearized plasmid (Fig. 3B). Strikingly, when cells were co-transfected with 400 bp template either as dsDNA or linearized plasmid, the surviving colonies increased 11-fold and 35-fold, respectively when compared to transfection using 150 bp donors. These results indicate that double-stranded repair templates with ≥400 bp length have a higher efficiency of generating HDR-positive cells. Moreover, assembling the dsDNA into a plasmid vector improved the effectiveness by a further ∼3-fold (Fig. 3B). Figure 3: Provision of long HA dsDNA donor template during gene editing increases HDR-mediated mutagenesis. (A) Diagram of approach used to edit ATP1A1 using different donor templates. 1 × 107 of human iPSCs were transfected with PX459 containing guide RNAs which target intron 4 via electroporation. Cells were also transfected with 0. 01 nmole of ssODN, dsDNA, and linearized donor plasmid, respectively. [SEP] Justify:

Output: The text corroborates the claim by detailing experiments using CRISPR technology to edit genomic sequences, specifically referencing homologous directed repair (HDR) efficiency with different donor templates. It highlights the comparative analysis of ssODNs and dsDNAs, illustrating how longer double-stranded templates significantly enhance gene editing efficiency. This directly supports the claim that CRISPR technology can enable precise genomic editing.


Input: Task: contrast the claim using the given text. Claim: Bees always live in colonies, with no exceptions. [SEP] Text: Subsequently, marked bees of defined age were recollected from internal frames of each colony as described above. Nestmate recognition assay View detailed protocol Every day over a three-week period, newly eclosed bees (<24 hr old) from a single source colony were collected as described above, uniquely color-marked, and then reintroduced into their source colony. Subsequently, on each experimental day, bees from the following groups were collected, placed in individual 15 mL plastic tubes (), and chilled on wet ice in an ice cooler up to 10 min before the assay in order to limit heat related stress: bees of the focal age (identified by color of mark), returning nectar foragers (denoted by distended abdomen and lack of pollen) of unknown age from the source colony, and returning nectar foragers of unknown age from an unrelated colony. All foragers, which served as behavioral controls, were painted the same color as the experimental bees just after collection. Tubes were numbered in a randomized order and blinded to the experimenter conducting the behavioral assays. Fifteen bees per group were prepared for each colony each experimental day. Behavioral assays were conducted simultaneously at two colonies (source and unrelated) by two researchers, as well as recorded using digital video cameras. As described previously (D'ettorre et al. , 2006; Downs and Ratnieks, 2000), acceptance at the colony entrance was used as a proxy for nestmate recognition by placing individual bees on a modified entrance platform and recording the behavioral reactions of guard bees for ~5 min. Bees were considered ‘Rejected’ if they were bit, stung and/or dragged by at least one guard bee (Video 1). Bees were considered ‘Accepted’ if they were approached by guards, antennated and/or licked and then left alone (not bit), if they immediately entered the colony and were not removed by other bees, or if they remained on the platform and did not receive aggression (Video 2). After 5 min, focal bees that remained on the platform outside the colony were removed before the next assay. All behaviors were scored in real time, and videos were retained as back-up. All behavioral assays were conducted during a period of 10 days, between 12 and 4pm, with two days focusing on each age of experimental bee (N = 20–30 bees per group). [SEP] Justify:

Output: The text contrasts the claim by detailing a study involving behavioral assays with bees collected from colonies. The mention of 'returning nectar foragers of unknown age from an unrelated colony' indicates that there are instances where bees are associated with colonies but can also interact with non-colony individuals, suggesting exceptions to the claim that bees always live in colonies.