Input: Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: (A) Schematic of hyBE4max CBE, consisting of D10A nCas9 with an HNH endonuclease domain (grey), a deaminase domain (-1 [mRNA editing enzyme catalytic polypeptide 1], pink), two uracil glycosylase inhibitor (UGI) monomers (purple), and a RAD51 ssDBD (orange). The editing window is located on the PAM sequence containing strand (yellow) at positions 4–12 (purple). Codons for arginine, tryptophan, or glutamine, can be edited into STOP codons, as shown in the text boxes. While one arginine and two glutamine codons are edited on the coding strand (light blue) into one STOP codon each, the one tryptophan codon on the template strand (dark blue) can be edited into three STOP codons. Suitable single-guide RNAs (sgRNAs) that would convert these four codons into STOP codons were designed for 64 kinetoplastid species. (B) The number of guides per protein-coding gene that yield a STOP codon within the first 50% of the CDS. (C) The percentage of protein-coding genes that could be targeted by at least two guides. We successfully established the hyBE4max CBE in four Leishmania species and have created the online resource to facilitate CBE primer design across 64 different kinetoplastid species. We show that our tool can be used to convert arginine, tryptophan, or glutamine codons into STOP codons with up to 100% editing efficiency in transfected cells that have been selected for CBE and sgRNA expression constructs. We demonstrate that single- and multi-copy genes can be depleted in L. mexicana, L. major, L. donovani, and L. infantum (the causative agents of cutaneous and visceral leishmaniasis) without requiring donor DNA and by expression of just one CBE-bound sgRNA. We then optimised the hyBE4max editor further by replacing the Rad51 ssDBD with a L. major-derived version, giving rise to hyBE4max-LmajDBD.

Output: corroborate


Input: Claim: Bees always live in colonies, with no exceptions. [SEP] Text: Here, we provide empirical evidence that the maturation of the CHC profile of individual honey bee workers is primarily regulated by innate developmental processes associated with age-dependent behavioral tasks and modulated by the social colony environment, and that mature colony-specific recognition cues are primarily associated with the foraging task. Specifically, we find that individual workers exhibit stereotypic quantitative and qualitative changes in their CHC profile as they transition from in-hive tasks to foraging outside, that these changes are associated with innate transcriptional changes in CHC biosynthetic pathway genes, and that only forager honey bees are behaviorally rejected from the entrance of an unrelated hive. Together, our findings suggest that not all members of honey bee colonies display a uniform cuticular chemical profile via the direct acquisition of CHC mixes. Instead, our data indicate that CHC profiles, and likely nestmate recognition cues, in honey bees are more likely a product of a genetically-determined developmental program that is modulated by colony-specific factors. Results CHC profiles of individual honey bee workers exhibit qualitative and quantitative age-dependent changes Given that newly emerged honey bees have lower amounts of total CHCs, and exhibit less chemical diversity compared to older bees (Breed et al. , 2004), we initially sought to determine the age at which the CHC profile of individual honey bee workers matures. To achieve this goal, we analyzed the CHC profiles of individual workers from a single age-cohort that was reintroduced back into its source colony and then collected at different ages. This analysis revealed that the total amount of CHCs increases between one-day post-reintroduction and 14 days post-reintroduction and then remains stable (Figure 1A, Kruskal-Wallis, H = 9. 21, df = 3, p=0. 026, FDR pairwise contrasts: Day 1 vs. Day 7 p=0. 11, Day 1 vs. Day 14 p=0. 036, Day 1 vs. Day 21 p=0. 04, Day 7 vs. Day 14 = 0. 613, Day 7 vs. Day 21 p=0. 691, Day 14 vs. Day 21 p=0. 79; Figure 1—figure supplement 1(3,28) = 6. 40, p=0. 002, FDR pairwise contrasts: Day 1 vs. Day 7 p=0. 036, Day 1 vs. Day 14 p=0.

Output: contrast


Input: Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: 2016;118(4):535–46. . Estimating the burden of disease attributable to high cholesterol in in 2000. S J. 2007;97(8 Pt 2):708–15. de. A simple method for human peripheral blood monocyte isolation. Oswaldo Cruz. 2000;95(2):221–3. . Calprotectin expression in human monocytes: induction by porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1beta. Journal of periodontology. 2005;76(3):437–42. van Tits LJ, Stienstra R, van. Oxidized LDL enhances pro-inflammatory responses of alternatively activated M2 macrophages: a crucial role for Kruppel-like factor 2. Atherosclerosis. 2011;214(2):345–9. Social and of Index among 15 Years and Older: -1. . 2019;16(20). Different lipid profiles according to ethnicity in the Heart of Soweto study cohort of de novo presentations of heart disease. Afr. 2012;23(7):389–95. van der Merwe L, Chetty N. The relationship between insulin sensitivity and serum adiponectin levels in three population groups. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme. 2005;37(11):695–701. . A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo. Nature immunology. 2000;1(4):298. Synergically increased expression of CD36, CLA-1 and CD68, but not of SR-A and LOX-1, with the progression to foam cells from macrophages. J Atheroscler Thromb. 2002;9(1):57–64. Han J, Nicholson AC. Lipoproteins modulate expression of the macrophage scavenger receptor. Pathol. 1998;152(6):1647. Lusis AJ, Navab M. Lipoprotein oxidation and gene expression in the artery wall: New opportunities for pharmacologic intervention in atherosclerosis. Biochem Pharmacol. 1993;46(12):2119–26.

Output: pass


Input: Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: , 2010]), including 32 Leishmania species (Figure 1B, C). For genes with more than two sgRNAs available, we generated a scoring matrix that ranked guides by their exact edit window (e. g. C to T conversion is more efficient at positions 4–8 than 8–12 [Zhang et al. , 2020]) and based on how many STOP codons each guide would introduce. The primer design for all 64 genomes was then uploaded to our new primer design resource Establishing cytosine base editing in this showed that CBE-mediated genome-wide loss-of-function screens in kinetoplastids would be feasible, we started to develop a strategy to express the hyBE4max CBE in Leishmania. First, we cloned the editor (Zhang et al. , 2020) into pLdCH (Zhang et al. , 2017), enabling episomal expression of the sgRNA and hyBE4max CBE via a ribosomal RNA (rRNA) promoter derived from L. donovani (Figure 2A). Then, we established a reporter assay to test the editor. A L. major clone was isolated, expressing a tdTomato (Shaner et al. , 2004) reporter from the 18S rRNA locus (Figure 2B, C). In the following, two CBE sgRNAs were designed to induce a C to T conversion that results in a neutral substitution (‘control’ sgRNA; codon change but no amino acid change), while four other targeting guides were designed to yield a STOP codon through C to T conversion (‘target’ sgRNA). Three of these six guides were designed to target the coding strand, while the other three would target the template strand. Guide sequences were cloned into pLdCH-hyBE4max (Figure 2A) and then transfectants analysed 11, 21, and 33 days post transfection, while being under constant drug selection to maintain the episome. Strikingly, we observed strong knockdown effects of the tdTomato reporter across all four guides, designed to introduce a STOP codon (‘Target 1’ and ‘Target 2’ on template and coding strand in Figure 2D). For guide 1, targeting the template strand, 98.

Output: corroborate


Input: Claim: Bees always live in colonies, with no exceptions. [SEP] Text: 001, Day 1 vs. Day 18 p=0. 007, Day 7 vs. Day 14 = 0. 412, Day 7 vs. Day 18 p=0. 993, Day 14 vs. Day 18 p=0. 305). Additionally, individual compounds vary in total amount across bees of different ages (Figure 1C,D, Figure 1—figure supplement 1C,D, Table 1, Table 2). Independently of the age-related quantitative changes, we also found that the CHC profiles of workers exhibit age-related qualitative changes in the overall CHC chemical composition (Figure 1(1,31) = 22. 86, R2 = 0. 43, p<0. 001, FDR pairwise contrasts: Day 1 vs. Day 7 p=0. 002, Day 1 vs. Day 14 p=0. 002, Day 1 vs. Day 21 p=0. 002, Day 7 vs. Day 14 = 0. 017, Day 7 vs. Day 21 p=0. 002, Day 14 vs. Day 21 p=0. 31; Figure 1—figure supplement 1(3,28) = 2. 35, R2 = 0. 22, p=0. 038, FDR pairwise contrasts: Day 1 vs. Day 7 p=0. 024, Day 1 vs. Day 14 p=0. 011, Day 1 vs. Day 18 p=0. 018, Day 7 vs. Day 14 = 0. 406, Day 7 vs. Day 18 p=0. 212, Day 14 vs. Day 18 p=0. 524), as well as in the relative amounts of individual CHCs (Figure 1E,F, Figure 1—figure supplement 1E,F, Table 3, Table 4). These data confirm that not all members of a honey bee colony share a common CHC profile (Kather et al. , 2011), and suggest that age-dependent processes might be playing an important role in the regulation of both the quantitative and qualitative dimensions of the cuticular chemical profiles of individual honey bee workers. Figure 1 with 1 supplement see all Download asset Open asset CHC profiles of bees exhibit quantitative and qualitative changes in association with age. (A) Total CHC amounts (μg) extracted from sister bees of different ages. (B) CHC profiles of sister bees of different ages.

Output: contrast


Input: Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: AbstractIn 1969, members of Grupo Espeleológico Edelweiss discovered the Sala and Galerías de las Huellas in Ojo Guareña Cave system (Burgos, Spain). These contained hundreds of ancient human footprints, preserved in the soft sediment on the floor. These footprints represent the tracks of a small group of people who walked barefoot through these complex passages in the cave. Owing to the difficult compatibility of the documentation and preservation of these prints, it was not possible to study them before the development of new non-invasive remote sensing techniques. However, since 2012 optical laser scanning and digital photogrammetry have been used in Galerías de las Huellas, in combination with GIS techniques, to obtain a model of the cave floor, where the footprints and their internal morphology can be observed in detail. We have identified over 1000 prehistoric human footprints and at least 18 distinct trackways through the passages, which could have been left by around 8–10 individuals. Since 2016, an archaeological field study has been conducted in this sector, in order to determine and explore its surrounding area and find other archaeological evidence that may be directly associated with these tracks. Numerous remains of torches are preserved on the walls and floor in the immediate surroundings of the footprint sites. Some of them have been dated, which has revealed the intensive use of this underground landscape from the to the Mesolithic-Neolithic. However, the remains in Sala and Galerías de las Huellas date solely to the Chalcolithic, around 4300 calBP. You have full access to this open access 17. 1a). This cave system developed in the northern flank of the Mesa-Pereda syncline (del Olmo et al. 1978), in the Coniacian (Late Cretaceous) limestone and dolomite unit. Ojo Guareña is an extensive multilevel cave system, formed by the connection of 14 caves and over 110 km of passages, distributed on 6 main interlinked levels, from a relative height of +70 m to the current level of the River Guareña (Grupo Espeleológico Edelweiss 1986; Ortega et al. 2013: 45–53). Fig. 17.

Output: pass


Input: Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: 27:1,028,462–1,030,089; LmjF. 27:1,041,477–1,043,104; LmjF. 27:1,052,607–1,054,234; LmxM. 27:982,640–984,268; LinJ. 27:1,068,199–1,069,826; LinJ. 27:1,078,759–1,080,386; LinJ. 27:1,093,368–1,094,995; Ld27_v01s1:1,021,892–1,020,265). (C) FACS (fluorescence-activated cell sorting) plot showing tdTomato-expressing and wildtype L. major parasites. (D) FACS plot of tdTomato-expressing L. major parasites, transfected with pLdCH-hyBE4max-tdTomato targeting plasmids (see description main text). Cells were analysed 11, 21, and 33 days post transfection. Percentages in (C) and (D) represent the remaining proportion of tdTomato-expressing cells. (E) Doubling times for analysed parasites in (C) and (D). Error bars show standard deviations of triplicates. PAR: tdTomato-expressing cells; WT: wildtype cells. Asterisks indicate Student’s t-test: *p > 0. 05. To validate the observed changes of the tdTomato reporter, we then Sanger sequenced the reporter sequence before and after the editing (Figure 3A). Sanger sequencing reads were analysed by using ICE (inference of edits; Sythego), allowing to quantify the discordance (editing rate) between the normalised edited and non-edited read for the entire sequenced region (from 2. 7% to 25% of the tdTomato CDS) (Figure 3B). For all six guides, editing was only observed within the guide target sequence and not elsewhere in sequenced regions (Figure 3A, B). While selected C to T conversions within the editing window (positions 4–12) were up to 100% effective (100% editing rate) for three of the six guide target sequences, not all possible C to T conversion targets were effectively edited.

Output: corroborate


Input: Claim: Bees always live in colonies, with no exceptions. [SEP] Text: , 2008; Søvik et al. , 2015). Consequently, under natural colony settings, it is impossible to separate the possible independent impacts of ‘age’ and ‘task’ on the expression of forager-specific CHC profiles. Therefore, we next analyzed the CHC profiles of individual nurse and forager bees from single-cohort-colonies (SCC), a well-established experimental approach to uncouple behavioral maturation from chronological age (Ben-Shahar et al. , 2004; Ben-Shahar et al. , 2002; Greenberg et al. , 2012; Robinson et al. , 1989; Whitfield et al. , 2003). Because these artificial colonies are initially comprised of a single age-cohort of day-old bees, a small proportion of these young workers will accelerate their behavioral maturation to become precocious foragers that are the same age as typical nurses (~7 days old) (Ben-Shahar et al. , 2002; Greenberg et al. , 2012; Huang and Robinson, 1992). The comparison of the CHC profiles of typical young nurses and precocious foragers of identical age revealed a significant effect of task on the CHC profile of individual workers (Figure 2(1,15) = 13. 79, R2 = 0. 50, p<0. 001). Similarly, we observed a significant effect of task on the CHC profiles of individual ‘over-aged’ nurses and typical-aged foragers at three weeks of age (Figure 2(1,15) = 45. 41, R2 = 0. 76, p<0. 001). In contrast, task and age had no effect on total CHC amount (Figure 2C, Two-way ANOVA, age: F(1,28) = 0. 55, p=0. 46, task: F(1,28) = 0. 37, p=0. 55, age*task: F(1,28) = 5. 37, p=0. 03). Together, these data suggest that processes associated with the behavioral maturation of honey bee workers, not chronological age, are primarily responsible for the observed forager versus nurse CHC profiles of individual honey bee workers. Figure 2 with 1 supplement see all Download asset Open asset Effect of task on the CHC profile of bees is independent of age.

Output: contrast


Input: Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: It covers an area of 5652 m2 and connects with in the fourth level. This third level is perched at 40 m above the Guareña River level and comes very close to and Bernabé Cave, whose passages belong to the second karst level (Fig. 17. 1c). Their western passages descend until they link topographically with the end of Galería de las (GH I), although without a physical connection because of the collapse of the cave ceiling and the growth of a large speleothem (Grupo Espeleológico Edelweiss 1986, Annex mapping 153–3; Martín 1986: 141–147; Ortega and Martín 1986: 342–343). Additionally, sediment on the San Bernabé hillsides has completely silted up the old entrances in this sector, so current access is from Palomera Cave, 1250 m to the west. Sala de las Huellas is an enormous chamber 80 m long, 50 m wide and more than 5 m high, with large boulders that have collapsed from the ceiling and obstruct transit (Figs. 17. 1 and 17. 2f). Galerías de las and II (GH I and GH II) are located on the south side of this chamber (Figs. 17. 1b, 17. 2c, g and 17. 9a). The first gallery (GH I) consists of a main passage, 60-m-long and a 25-m-long side passage (Figs. 17. 2g and 17. 3d). The main axis of the second gallery (GH II) is approximately 100 m long. Both galleries are about 5 m wide and 4–5 m high (Fig. 17. 3f, g). Fig. 17.

Output: pass


Input: Claim: CRISPR technology allows for precise editing of genomic sequences. [SEP] Text: For example, three C to T conversions were 100% effective for ‘Target 1’ on the template strand at positions 6–8, yielding a STOP codon at the intended position. However, the fourth conversion target of that guide at position 12 was not edited (Figure 3A), confirming previously observed differences in editing efficiencies across the editing window (efficiency higher at positions 4–8 than 8–12 [Zhang et al. , 2020]). Strikingly, both control guides, which did not show any changes in the reporter expression (Figure 2D) as they were designed to only change the DNA sequence but not the amino acid sequence, showed also high editing rates (100% for Ctrl(+), 79. 2% and 45. 9% for Ctr(−)), confirming that the hyBE4max CBE was highly specific even though expressed for 33 days (Figure 3A, B). Figure 3 with 2 supplements see all Download asset Open asset Sanger sequencing following cytosine base editing in L. major. (A) Alignment of reference sequences (Reference) with Sanger sequencing trace plots before transfection of pLdCH-hyBE4max-tdTomato targeting plasmids (D0) and 33 days after transfection (D33). Six guide loci are shown as described in the main text. Blue shading: 20nt guide target sequence. Red dotted lines: 4–12 editing window. Red marked nucleotides: expected cytosine to thymine conversion. Red boxed nucleotides: edited nucleotides. (B) Discordance between D0 and D33 trace plots from (A) was measured and plotted as editing rate versus Sanger sequencing coordinates. Each data point represents one C to T conversion. The position and orientation of each guide are indicated above, with the first guide sequence starting after 2. 7% of the CDS and the last guide sequence ending after 25% of the CDS. Since editing rates did not reach 100% for all guides after 33 days, we thought that stable expression of the hyBE4max CBE would increase editing rates and inserted hyBE4max into the β-tubulin locus using the pTB007 plasmid as a vehicle (Beneke et al. , 2017; Figure 3—figure supplement 1A).

Output: corroborate


Input: Claim: Bees always live in colonies, with no exceptions. [SEP] Text: Single cohort colony bees differ in CHC profile by behavioral task at one week of age (typical nurse age, (A)) and three weeks of age (typical forager age, (B)). (C) SCC bees do not differ in total CHC amount due to age and/or task. (D) Undertakers and nurses differ from foragers in CHC profile. (E) ‘Big-back’ bees differ from same-aged actively foraging sisters in CHC profile. Total CHC statistics (C) using ANOVA followed by Tukey’s HSD with FDR correction. CHC profile statistics (A, B, D, E) using followed by FDR pairwise contrasts, shown as non-metric multidimensional scaling plots depicting Bray-Curtis dissimilarity between samples. Letters in graphs and legends denote posthoc statistical significance (p<0. 05). Sample size per group, N = 8. Figure 2—source data 1 Amount (ng) of each compound extracted from each bee sample in Figure 2 and Figure 2—figure supplement 1. Download elife-41855-fig2-data1-v2. xlsx Previous studies in Harvester ants suggested that exposure to the environment outside the nest is sufficient to induce stereotypical changes in the CHC profiles of individual social insects (Wagner et al. , 2001). Therefore, we next asked whether spending time outside the hive is sufficient to induce the observed forager-specific CHC profile by comparing the CHC profiles between ‘undertakers’, nurses, and foragers from typical colonies. ‘Undertakers’ are a small group of highly specialized older pre-foraging workers (2–3 weeks of age), which are responsible for removing dead bees by carrying them outside and away from the colony (Robinson, 1992; Smith et al. , 2008; Søvik et al. , 2015; Trumbo et al. , 1997). Therefore, because undertakers and foragers perform their respective tasks outside the hive, while nurses and other younger, pre-foraging bees rarely do, we reasoned that if outdoor exposure defines the distinct forager-specific CHC profile then the CHC profiles of undertakers should be more similar to foragers than to nurses.

Output: contrast


Input: Claim: Cave paintings offer a glimpse into prehistoric human expression. [SEP] Text: 2(a) General overview of the Sala Negra; (b) charcoal samples (No 11 and 12) located in the connection shaft between Galería de las (c) charcoals in the block (No 10) of Laberinto Otilio; (d) block decorated with torch remains and charcoals in the ground (No 14, Laberinto Otilio); (e) torch remains on a block, with charcoals in the floor (No 4), of the Sala de las Huellas; (f) general view of Sala de las Huellas; (g) cross section view of the beginning of Galería de las Huellas II. (Photos (a) P. Carazo-Grupo Espeleológico Edelweiss; (b–f) M. A. Martín -Grupo Espeleológico Edelweiss)Full size imageFig. 17. 3(a) Human footprints on a large block of the Sala de las Huellas; (b) remains of a torch under a block of Sala de las Huellas (No 1); (c) detail of a footprint of Galería de las Huellas II; (d) view of Galería de las Huellas I; (e-g) several views of the traces of the Galería de las Huellas II. (Photos M. A. Martín-Grupo Espeleológico Edelweiss)Full size imageThe floor of these galleries is characterized by interior cave sediments, with a loamy texture and composition mainly of calcite, quartz, feldspar and to a lesser extent phyllosilicates (Benito-Calvo et al. 2013: 220). Small channels have incised the floors of both galleries, particularly in the final section of GH II. The entry sections have calcite crusts, which do not register any footprints. On the contrary, the deposition of fine calcite crystals makes it difficult to recognize many of the impressions, especially in the GH I gallery. The human traces located in these passages are very well preserved in both galleries, thanks to the protection measures that were taken at the time of the discovery, limiting access to the sites and waymarking alternative routes (Fig. 17. 3).

Output: pass


Input: 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).

Output: corroborate


Input: 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.

Output: contrast


Input: 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 ().

Output: pass


Input: 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.

Output: corroborate


Input: 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).

Output: contrast


Input: 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.

Output: pass


Input: 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).

Output: corroborate


Input: 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.

Output: contrast


Input: 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).

Output: pass


Input: 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.

Output: corroborate


Input: 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.

Output: contrast


Input: 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.

Output: pass


Input: 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.

Output: corroborate


Input: 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).

Output: contrast


Input: 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).

Output: pass


Input: 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.

Output: corroborate


Input: 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.

Output: contrast


Input: 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.

Output: pass


Input: 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.

Output: pass


Input: 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).

Output: contrast


Input: 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.

Output: pass


Input: 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).

Output: corroborate


Input: 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.

Output: contrast


Input: 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).

Output: pass


Input: 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).

Output: corroborate


Input: 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.

Output: contrast


Input: 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.

Output: pass


Input: 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 ().

Output: corroborate


Input: 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.

Output: contrast


Input: 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.

Output: pass


Input: 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.

Output: corroborate


Input: 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.

Output: contrast


Input: 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.

Output: pass


Input: 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.

Output: corroborate


Input: 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.

Output: contrast