Saccharomyces cerevisiae. Previous studies have established that yeast constitutively producing Aβ42 fused to GFP offer a convenient means of analyzing yeast cellular responses to Aβ42. Young cells clear the GFP fusion protein and do not have green fluorescence while the older population of cells retains the fusion protein and exhibits green fluorescence, offering a fast and convenient means of studying factors that affect Aβ42 turnover. In this study the proportion of cells having GFP fused to Aβ after exposure to simvastatin, atorvastatin and lovastatin was analyzed by flow cytometry. Simvastatin effectively reduced levels of the cellular Aβ42 protein in a dose-dependent manner. Simvastatin promoted the greatest reduction as compared to the other two statins. A comparison with fluconazole, which targets that same pathway of ergosterol synthesis, suggests that effects on ergosterol synthesis do not account for the reduced amounts of Aβ42 fused to GFP. The levels of native Aβ42 following treated with simvastatin were also examined using a more laborious approach, quantitative MALDI TOF mass spectrometry. Simvastatin efficiently reduced levels of native Aβ42 from the population. This work indicates a novel action of simvastatin in reducing levels of Aβ42 providing new insights into how simvastatin exerts its neuroprotective role. We hypothesize that this reduction may be due to protein clearance.A large-scale epidemiology study on statins previously showed that simvastatin was unique among statins in reducing the incidence of dementia. Since amyloid beta (Aβ42) is the protein that is most associated with Alzheimer’s disease, this study has focused on how simvastatin influences the turnover of native Aβ42 and Aβ42 fused with green fluorescent protein (GFP), in the simplest eukaryotic model organism,  Statins are highly effective, widely-prescribed drugs that bind to and inhibit HMG-CoA reductase (HMGCR) reducing the liver’s synthesis of cholesterol . In non-fluorescent cells the GFP fused to Aβ42 is absent, having been produced constitutively but then degraded . The GFPThe expression systems to produce GFP fused to Aβ42 are constitutive, so all cells in the population containing the pAS1N.Aβ-GFP and p416GPD.GFP-Aβ plasmids are expected to produce Aβ-GFP or GFP-Aβ. However, as previously reported, only a small fraction of the population is fluorescent and contains GFP fused to Aβ42 ,16. Calcp < 0.01) larger size of 6.7 µm. These are older cells that are a small proportion of the population and can also be distinguished by the numbers of bud scars (not shown). It is this older population that are the focus in this study. This population is a relatively small fraction (~12%). A more quantitative demonstration that these older cells retain GFP fused to Aβ42 is shown in The present study was carried out to assess the effect of lovastatin, simvastatin and atorvastatin, on Aβ42 fused to green fluorescent protein expressed in the yeast. During two hours of statin treatments there was no significant toxicity since propidium iodide stained less than 1.5% of the population, regardless of statin treatment see . In thisIn the untreated population, green fluorescent cells were ~12% of the population. Treatments with 50 μM simvastatin led to an ~70% reduction in green fluorescent cells. Treatments with 100 μM simvastatin led to a greater decrease in fluorescence and 300 μM simvastatin led to an even greater decrease: with 300 μM simvastatin only ~0.58% cells remained fluorescent.Atorvastatin, at levels of 50, 100 and 300 μM, led to an ~60% decrease in green fluorescence, while the effects of lovastatin at these levels caused an ~20% reduction in green fluorescent cells. These studies demonstrate that in a population of exponentially-growing yeast cells statins can aid the clearance of GFP-Aβ from the major proportion of older cells. Simvastatin caused the greatest clearance, followed by atorvastatin and then lovastatin.Statin-induced reduction of GFP-Aβ could be a consequence of lowering ergosterol  where there are reduced opportunities for membrane localisation of the hydrophobic GFP-Aβ. This could make GFP-Aβ more available for proteolytic clearance.In order to examine effects related to ergosterol biosynthesis experiments were performed using fluconazole, an antifungal drug that reduces ergosterol biosynthesis. The IC50 (concentration at which fluconazole caused 50% growth inhibition) was 93 μM: Concentrations above and below this level were tested for their effects on the population see . Fluconazole treatment of BY4743 [pAS1N.Aβ-GFP] resulted in small increases in the population of fluorescent cells. The increases with 25–400 μM fluconazole were statistically significant. Increases were between 5.09% and 7.26% compared to the untreated control. These increases are not due to fluconazole causing cells to fluoresce independent of GFP content, as fluconazole treatment on non-transformed cells did not result in increased fluorescence see . A possiCUP1 locus, which enables copper-inducible expression of foreign genes. Possible complications of copper induction and toxicity were avoided by not using copper as an inducer. Instead our work utilised expression of CUP1 promoter at its basal level (which is around 4% of the copper induced levels). In the absence of leucine there is selection for increased copies of the leu2d gene, which enables a higher number of plasmid copies and higher expression levels of foreign protein . MALDI-BY4743 [pYEX.Aβ] transformants were used to quantitate the levels of native Aβ42 in response to simvastatin treatment. Cellular fractionation showed that the Aβ42 was associated with the membrane fraction, so this was the portion that was analyzed by quantitative MALDI-TOF protein mass spectrometry. The levels of Aβ42 were normalized relative to the amount of cells. The reduction in levels of Aβ42 was found to be statistically significant for the cells treated with varying concentrations of simvastatin. This study shows the utility of examining the levels of native Aβ42 and Aβ42 fused to GFP where it can be observed in living cells. The study showed that young cells of yeast have efficient clearance of the deleterious Aβ42 fused to GFP, but that older yeast cells exhibit low or no turnover of this protein. Furthermore, this is the first report to show that statins aid in reducing levels of Aβ42 fused to GFP and native Aβ42 expressed in yeast, and we favour the idea that statins can affect clearance of Aβ42. Lower levels of Aβ42 fused to GFP, after 2 hours of treatment with statins, could be observed in a portion of the older cells. The effect was greatest with simvastatin, followed by atorvastatin and then lovastatin. The reductions were highly statistically significant and dose-dependent at treatments ranging from 50 to 300 μM. Statins can cause some major effects on yeast. By decreasing ergosterol levels in prolonged cultures statins cause growth inhibition and this also can cause total loss of the mitochondrial genome ,20. For As a comparison, the effects of fluconazole, which also affects the ergosterol biosynthesis pathway, were also investigated. Fluconazole did not reduce levels of Aβ42 fused to GFP: In fact, there was a slight increase. This suggests that the statins are exerting most of their effects by targeting a protein clearance pathway. There are several pathways involved in protein clearance and some of them have previously been shown to be affected by statins. Protein clearance mechanisms affected by simvastatin, lovastatin and atorvastatin include autophagy and the unfolded protein response . The druS. cerevisiae mutants, identified 110 gene deletant mutants with increased green fluorescence  at 60 °C for 60 min. The stock solutions of hydrolyzed statins were stored at −20 °C. Two statins, lovastatin and simvastatin were hydrolyzed to activate them from inactive lactone form to active hydroxyl acid form ,29. Atorg after 2 h. Centrifuged cell pellets were resuspended into 1 mL selective minimal media broth. Statins were added at a final concentration 50–300 μM while fluconazole was added at concentrations up to 400 µM. Incubations were performed at 30 °C and 200 rpm for 2 h. Flow cytometry was performed on strains treated with statins and fluconazole.Yeast cells were analyzed for effect of all three statins and fluconazole on toxic Aβ42 protein clearance. Overnight fresh cultures of yeast cells were obtained by inoculating 3–4 colonies on fresh selective minimal media in a 15 mL centrifuge tube. The tubes were incubated at 30 °C at 200 rpm. Overnight cultures were further grown to exponential phase in order to achieve high levels of fluorescence. The cell culture was obtained after inoculating 1 mL of overnight culture into 4 mL of fresh minimal media in centrifuge tubes. The tubes were incubated at 30 °C and 200 rpm for 2 h. These cells were centrifuged for 2 min at 252× n = 3). Flow cytometry utilised excitation filters with 488 nm (blue laser) as the GFP protein excites in the wavelength ranging from 400–530 nm. Fluorescence emission was measured at 530/30 emission filters. Flow cytometry was done to determine the number of cells with green fluorescence. Flow cytometry was performed using FACScan flow cytometer . The data acquisition was done using BD FACSDiva Software. For each sample, 10,000 cells per sample were analyzed (Kluyveromyces lactis killer toxin) preceding Aβ42 in the construct. The synthetic construct was made by GeneArt and cloned into their vector. A BamHI-EcoRI double digest liberated the desired fragment which was cloned in the BamHI-EcoRI digested target vector, pYEX-BX. The resulting construct, shown in The native Aβ42 expression vector was constructed using the yeast optimized gene construct encoding a secretion signal  and 2 µg plasmids. The solution was vortexed and incubated at 42 °C for 30 min. Following the incubation, the mixture was then transferred into solidified minimal media lacking uracil or leucine for selection of the transformants. The plates were incubated at 30 °C for 2–10 days. A negative control and empty vector transformations were also performed alongside to validate the transformation. Transformed yeast were checked for the correct phenotype.The BY4743 [pYEX-Aβ] transformants expressing native Aβ42 were grown overnight in minimal media containing 20 mg/L histidine at 30 °C. Next day the yeast cells were supplied with fresh media and re-grown for 2 h. The cells were then grown for a further 2 h with simvastatin concentrations of 0, 100 and 300 µM. Cultures were performed in triplicate.Simvastatin-treated cells were harvested from liquid culture by centrifugation at 3000 rpm for 3 min. Cells were washed two times in TBS and resuspended in 500 µL TBS, followed by cell disruption in an MP Fast tissue homogenizer using glass beads (0.5 mm). Six cycles (1 minute each cycle) of homogenization were performed with a resting period of 1 minute in cold ice between each consecutive cycle of disruption. The lysate was transferred into sterile clean Eppendorf tubes and were fractionated using differential centrifugation. Large cell debris were separated by centrifuging the cells at 300 rpm for 3 min and the fraction of insoluble proteins from the supernatant was collected by centrifuging the supernatant at 15,000 rpm for 10 min. The insoluble pellet fraction was then added with 25 µL of TBS buffer and kept on ice until further processing. S. cerevisiae insoluble protein extract as a background. Known amounts of the synthetic Aβ42 were added into the spots to generate a standard curve. For calibration, each of these spots were also added with the same amount of internal calibrant used for the samples. Data from the plate was collected manually using the Flex Control software, which controls the MALDI-TOF mass spectrometer. The data obtained were normalized according to the peak height of the internal calibration standards. Using the standard curve, the relative amount of the Aβ42 in three different samples were calculated and analyzed. The statistical software GraphPad Prism Version 7 has been used to analyze the data collected. The measurement of Aβ42 using MALDI-TOF mass spectrometry in this study was similar to the one described previously  with a fStatins have the potential to reduce the risk of AD, by inducing a protein clearance response in the cells. More extensive research in yeast can be done to decipher the mechanism of actions of statins within the eukaryote and to screen for compounds that might have more significance than simvastatin in clearing Aβ42 fused to GFP or native Aβ42 from the cells. The use of statins for the treatment of other neurodegenerative diseases such as Parkinson’s Disease, Huntington’s Disease and prion diseases should be investigated in the future for their application as therapeutic agents.It is also important to confirm this statin-induced clearance of Aβ42 in studies involving human cells, however, this may require producing Aβ42 independently of APP, to avoid the effects of statins on BACE prenylation.


Here we show that this novel activity is an editing activity that can add specific ribonucleotides to 3′ ends of RNA or DNA when oligonucleotides, able to form intramolecular or intermolecular hairpin loops with recessed 3′ ends, are added to T7 RNA polymerase in the presence of at least one ribonucleotide triphosphate. Specific ribonucleotides are added to the recessed 3′ ends through Watson-Crick base pairing with the non-base paired nucleotide adjacent to the 3′ end. Optimization of this activity is obtained through alteration of the lengths of the 5′-extension, hairpin loop, and hairpin duplex. These properties define a T7 RNAP activity different from either transcriptional elongation or initiation.Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3′ ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototype of the single subunit polymerases, as a nonspecific addition of nucleotides to the 3′ end of T7 RNAP transcripts  There are two major forms of RNA editing, [A] substitutional, involving nucleotide modification, typically a transamination or deamination, which converts one nucleotide to another, for example, editing of tRNAs in Acanthamoeba castellanni2 or single nucleotide conversions such as A to I3), C to U or U to C4 and [B] insertional in which a nucleotide (or nucleotides) are inserted or deleted to produce a functional RNA7.RNA editing is an alteration of the genetic information in RNA relative to its DNA template. It is an additional step in gene expression, where specific nucleotides at specific positions are substituted for, added to, or deleted from the RNA in order to produce functional RNAs, especially mRNAs with a functional open reading frame. The term “RNA editing” was introduced in 1986 to describe the insertion of four uridine nucleotides into specific sites of mitochondrial cox2 mRNA in Trypanosome mitochondria12. Specificity of the nucleotide insertion or deletion site and the number of nucleotides inserted or deleted at a site is determined by an antisense RNA, termed a guide RNA [gRNA], which has the genetic information to produce functional mRNAs significantly different from the template DNA13.Two different types of insertional editing have been observed, post-transcriptional and co-transcriptional, each exemplified by a mitochondrial DNA system in which that type of insertional RNA editing is used extensively to produce functional mitochondrial RNAs. Post-transcriptional RNA editing, as exemplified by RNA editing in the mitochondria of trypanosomes, requires an endonuclease to break the RNA backbone at a specific site, a terminal transferase to add a nucleotide or nucleotides to the 3′ end of the RNA at the break site, and an RNA ligase to restore the RNA backbone at the site of the insertion or deletionPhysarum polycephalum produce functional mRNAs, rRNAs, and tRNAs17 different from their mtDNA template through non-templated addition of specific nucleotides at specific sites in nascent RNAs by the mitochondrial RNA polymerase (mtRNAP)20. Two major questions in the mechanism of this type of editing are: [A] How is the correct nucleotide inserted without a DNA template? [B] How is the correct site of insertion determined? Miller & Miller20 and Visomirski-Robic & Gott19 have each proposed that an unknown mechanism causes the mitochondrial RNA polymerase to pause during elongation of RNA on the DNA template, and insert a specific, non-templated nucleotide , before continuing DNA-templated elongation. Miller & Miller20 have shown that the Physarum mtRNAP is able to add nucleotides to the 3′ end of RNAs in the absence of DNA in vitro. However, with complex RNA populations without DNA templates in vitro any of the four ribonucleotides can be added.Co-transcriptional insertional RNA editing as exemplified by RNA editing in the mitochondria of myxomycetes such as 21 found in essentially all eukaryotic organisms and have conserved structural and functional similarities that indicate that they evolved from a common ancestor present at or near the advent of the eukaryotic line18. Interestingly, the conservation of these structural and functional similarities extend to other single subunit polymerases, such as the bacteriophage RNAPs24, DNA polymerases (DNAPs) such as the Klenow fragment of DNAP I in E. coli27, and reverse transcriptases (RNA-directed DNA polymerases) such as the HIV-1 reverse transcriptase30, constituting a super-family of single subunit polymerases with structural and functional homology.Both aspects of the specificity of this co-transcriptional RNA editing involve the mtRNAP. Mitochondrial RNAPs are single subunit polymerasesE. coli bacteriophage DNA and it has been extensively studied32. The crystal structure of T7 RNAP has been solved along with the different domains and their functions corresponding to both initiation and elongation processes35. It is a single subunit ~99 kDa protein (883 amino acid)36, found in Bacteriophage T7 and is primarily involved in transcription activity without the need of any other accessory transcription factors. The specificity and robustness of T7 RNA polymerase’s transcription activity comes from its specificity towards its promoter, a 23 nucleotide dsDNA sequence39. This specificity towards the promoter, involves the promoter recognition loop40 as well as the N terminus domain in T7 RNA polymerase42. T7 RNA polymerase can successfully carry out transcription on a synthetic ssDNA oligonucleotide template containing double stranded T7 promoter sequence44.The prototype RNA polymerase for this group of polymerases is the T7 RNAP encoded by the T7 de novo initiation which requires a promoter, single stranded DNA template, and the four ribonucleotide triphosphates; and elongation which requires a duplex with one strand having a recessed 3′ end (primer), the other strand 5′ extended (template), and four ribonucleotide triphosphates; there have been reports of a third activity in which addition of ribonucleotides occurs at the 3′ end of RNAs in the absence of a promoter or DNA template48. This activity was incorrectly attributed to the primer extension activity of elongation. Until recently, this activity of T7 RNAP has not been well characterized and most of the research on it has been focused on ways to prevent the addition of non-templated nucleotides to the 3′ ends of run-off RNAs50. Here we report the characterization of this activity. We show [A] that the T7 RNAP can add nucleotides to the 3′ end of RNAs and DNAs in a process that is neither elongation nor initiation, but is a type of RNA/DNA editing, [B] that the added nucleotide is added specifically as determined by base pairing of either an inter- or intramolecular template with an extended 5′ end relative to the recessed 3′ end, and [C] that the properties of the oligonucleotide that is provided determine the efficiency of nucleotide addition and distinguish this activity from either initiation or elongation activities. We will discuss this activity in terms of a general model of RNA editing by single subunit polymerases that may explain the specificity of nucleotide insertion during RNA editing by the Physarum mtRNAP.In addition to Synthetic single stranded DNA oligonucleotides  were synthesized by Eurofin-Operon. Oligonucleotides were dissolved in water to 200 µM and stored at 4 °C.Each oligonucleotide is named by a five number sequence separated by dashes where the first number is the oligonucleotide length, the second number is the length of the single stranded 5′ extension. The third and fifth number are the length of the double stranded region (stem of hairpin), and the fourth number is the length of the hairpin loop.T7 RNA polymerase (New England Biolabs (NEB) catalog number M0251S) was supplied in: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9), 1 mM EDTA, 20 mM 2-mercaptoethanol, 0.1% Triton X-100 and 50% glycerol and stored in a −20 °C freezer.2, 2 mM spermidine, 10 mM dithiothreitol, pH 7.9 @ 25 °C, 1 mg/ml BSA (Bovine Serum Albumin).NEB 10 × RNA pol reaction buffer was supplied. 1 × RNA Pol Reaction Buffer contains 40 mM Tris-HCl, 6 mM MgClin vitro oligonucleotide labeling protocol was derived from the in vitro transcription protocol recommended by New England Biolabs (NEB) for their T7 RNA polymerase51. For radiolabeling, (α-32P) rGTP, or (α-32P) rATP  were used. In vitro oligonucleotide radiolabeling was assayed in reactions containing 6 µl of 200 µM ssDNA oligonucleotides, 2 µl 10 × RNA polymerase buffer, 2 µl of 5 mM ribonucleoside triphosphates, and 1 µl of α-32P radiolabeled rATP/rGTP to a final volume of 20 µl. The reactions were incubated at 37 °C water bath for 60 minutes. After incubation 2 µl of 6× loading dye (10 mM Tris-HCl (pH 7.6) 0.03% bromophenol blue, 60% glycerol, 60 mM EDTA) was added to individual reaction mixtures and samples were run on 15% TBE (Tris-Borate EDTA) polyacrylamide gels at a constant 200 V and 75 mA, until the bromophenol blue migrated three-quarters of the length of the gel. Experiments were routinely repeated on both denaturing and non-denaturing gels to check their reproducibility.The TM. Detection of incorporated radioactivity was achieved by exposure to a phosphor screen (Molecular Dynamics) and scanned on a Phosphor Imager . Quantification of the phosphor image was done using ImageQuaNT software version 5.2 (Molecular Dynamics). Only the most intense band in each lane was quantitated. Graphical representation of major band intensities below each gel are shown to allow easier interpretation.Nucleic acids in the gels were stained with 1× TBE, Ethidium Bromide for 10 min and then visualized under Bio-Rad Gel Doc52 and that T7 RNAP could add ribonucleotides to the 3′ end of DNA and RNA55. We hypothesized that these observations indicated that these RNAPs had a novel activity similar to the RNA editing observed in mitochondria of Physarum polycephalum7 and other myxomycetes56. To study this activity, we have developed an in vitro assay for labeling the 3′ ends of RNA and DNA oligonucleotides using T7 RNAP and a single radiolabeled ribonucleotide triphosphate. In contrast to using complex mixtures of RNA or DNA, with these simple oligonucleotide sequences, some oligonucleotides failed to label while others labeled efficiently. We have determined that this labeling specificity depends on the ability of the 3′ end of the oligonucleotide to anneal with an internal complementary sequence which positions the 3′ end next to a nucleotide that is not base paired in the 5′ extension of the oligonucleotide. This can take place within the same oligonucleotide (intramolecular base pairing) or between complementary sequences in two separate oligonucleotides (intermolecular base pairing) as shown in Fig. Several laboratories have reported that single subunit RNAPs could add radiolabeled nucleotide triphosphates to the 3′ end of RNA in the absence of a DNA templateAn example of these experiments is shown in Fig. This experiment demonstrates that when presented with an oligonucleotide with a 3′ end with the potential to base pair as a recessed 3′ end, either inter- or intra- molecularly, the T7 RNAP is able to add the radiolabeled nucleotide to the 3′ end, but only if it can form a Watson-Crick base pair with the nucleotide in the 5′ extension (template) next to the nucleotide base paired with the 3′ end nucleotide (primer).To better understand this self-templated labeling at the 3′ end and to optimize this reaction, the single stranded DNA oligonucleotides have been systematically altered to determine the importance and contribution of [1] the overall length, [2] the length and base composition of the potential duplex, [3] the length of the 5′ extension, and [4] loop length (number of nucleotides separating the complementary sequences) with the goal of optimizing the reaction.To determine the contribution of 5′ extension length, oligonucleotides were designed with increasing 5′ extension lengths (0–16 nucleotides), constant base pairing length (6 pairs), constant loop length (8 nucleotides), and increasing overall length (20–36 nucleotides).Figure To determine the contribution of loop length to labeling efficiency, oligonucleotides were designed with constant 5′ extensions (2 nucleotides), constant double stranded regions (either 9 nucleotides or 6 nucleotides), and variable loop lengths (6–24 nucleotides) and variable overall length (20 nucleotides to 44 nucleotides). Nucleotide labeling efficiencies are shown in Fig. Labeling is seen for oligonucleotides with both intramolecular and intermolecular base pairing. Figure To determine the contribution of the length of the base pairing to 3′ end labeling efficiency, the base pairing length was systematically altered while keeping the overall length (32 nucleotides) and the 5′ extension length (2 nucleotides) constant  and the 5′ extension constant (at either 2 nucleotides or 10 nucleotides) constant. The V-10-V-8-V series is shown in Fig. To determine if this optimum base pairing length for labeling efficiency also pertained to intermolecular base pairing and to eliminate the contribution of loop length to labeling efficiency, a series of oligonucleotides with varying lengths of sequence complementary to a template oligonucleotide were designed. Figure Below the template strands in Fig. Figure Figure On the right side of Fig. While overall oligonucleotide length does not correlate with labeling efficiency, experiments to determine the optimum length of the 5′ extension, the double stranded region (stem), and the loop for the efficiency of labeling the 3′ end with a specific nucleotide give optimum lengths for one of the parameters when the other two parameters are held constant. However, the optimums for a parameter do not appear be independent, since a different optimum for a parameter is observed when the other two parameters are fixed at different lengths. To determine the interdependence of the three parameter lengths, 27 oligonucleotides were created with 3, 6, or 9 nucleotides in the 5′ extension, double stranded region, or loop in all possible combinations Fig. . The relAnalysis of these results allows several generalizations to be made about the interdependence of the parameter lengths. Labeling efficiency generally increases with 5′ extension length for a given stem length/loop length. However, different stem length/loop length combinations give significantly different efficiencies with 3, 6, or 9 nucleotides in the 5′ extension.The labeling efficiencies generally either decrease with the length of the double stranded stem region (3 base pairs >6 base pairs >9 base pairs) for six 5′ extension/loop combinations or peak with 6 base pairs (6 base pairs >3 or 9 base pairs) for the three 5′ extension/loop combinations with loops of 9 nucleotides.Labeling efficiencies vary with loop length in several different ways. For the three oligonucleotides with 6 base pair stems, 9 nucleotides loops have the highest labeling efficiency (9 nucleotides >3 or 6 nucleotides). For the three oligonucleotides with 3 base pair stems, 9 nucleotide loops have the lowest efficiency (3 or 6 nucleotides >9 nucleotides). For the remaining three oligonucleotides with 9 base pair stems, 6 nucleotide loops have the lowest labeling efficiency (3 and 9 nucleotides >6 nucleotides or 3 nucleotides >6 or 9 nucleotides).The highest labeling efficiencies are with oligonucleotides 12-3-3-3-3 and 27-6-6-9-6 followed closely by 30-9-6-9-6, 15-6-3-3-3, and 18-9-3-3-3. Oligonucleotides in the X-X-3-3-3 series have high labeling efficiency, essentially independent of 5′ extension length. Oligonucleotides of the X-X-6-9-6 series have significantly higher labeling efficiencies with 6 or 9 nucleotide (longer) 5′ extensions.Since the formation of a base paired region (stem) is necessary to put a recessed 3′ primer end on the 5′ extension template, it is surprising that longer stem lengths generally give decreased labeling efficiencies. While at least three nucleotides of base pairing complementarity are needed for significant labeling, labeling efficiencies with increasingly longer base pairing potential generally increase up to a peak of about 5 – 7 base pairs and then decline with increasing length, where loop length and 5′ extension length are held constant. This duplex optimum could be caused by at least two possible constraints, [A] a duplex length constraint (static model) in which only duplexes of intermediate length can be bound productively, and [B] a duplex stability constraint (dynamic model) where a duplex is required but where labeling efficiency decreases with duplex stability, indicating that both single stranded and duplex forms are necessary.Efficiency probably does not depend directly on duplex length since different loop lengths and 5′ extension lengths, when held constant, give a different optimum duplex length , constant 5′ extension (10 nucleotides), and duplex regions varying from 3 nucleotides to 7 nucleotides and from 7 to 18 hydrogen bonds Figs  and S1. 3G2T, A3GCT, A2G2T2) and two similar base composition with 15 hydrogen bonds (AG3T and A2G3T) were compared. The four oligonucleotides with base composition A3G2T or A3GCT varied in relative labeling efficiency from 86% to 100%, and the four oligonucleotides with base composition A2G2T2 varied in relative labeling efficiency from 9% to 100%. Likewise, the four oligonucleotides with base composition AG3T varied in relative labeling efficiency from 48–95%. These wide variations in relative labeling efficiency with oligonucleotides with the same base composition in their duplex eliminated base composition as a factor in relative labeling efficiency.In order to determine the cause of the variation in relative labeling efficiency within oligonucleotides with duplexes with the same number of hydrogen bonds, we compared relative labeling efficiencies of oligonucleotides with different base compositions but with the same number of hydrogen bonds in the duplex. There are 15 different base compositions of 6 nucleotides with 14 hydrogen bonds, and there are 16 different base compositions of 6 nucleotides with 15 hydrogen bonds. Three similar six base compositions with 14 hydrogen bonds  than oligonucleotides in which the G nucleotides in the duplex were separated by other nucleotides (9% to 48%).This paper describes and characterizes a novel DNA/RNA editing activity of T7 RNAP with nucleotide specificity. This novel activity is detected when an oligonucleotide is provided that has a duplex with a recessed 3′ end (primer) and a 5′ extension (template) where the first unpaired base specifies the labeling nucleotide through Watson-Crick base pairing. This novel editing activity differs from the two classic transcription activities, initiation and elongation. It differs from initiation in that nucleotide addition occurs in the absence of a promoter, and differs from elongation in that nucleotides are not added processively on the template, but only one or a few nucleotides are added since each nucleotide added to the duplex ultimately decreases the labeling efficiency. Probably the most unique feature of this novel activity is the addition of a DNA templated ribonucleotide to a DNA 3′ end to form a DNA primed, RNA-DNA duplex. Although T7 RNAP will only add ribonucleotides to 3′ ends to create RNA strands, it is able to add ribonucleotides to 3′ ends of either RNA or DNA and will use either RNA or DNA templates to specify ribonucleotide addition. Therefore, either RNA or DNA oligonucleotides able to form intramolecular and intermolecular duplexes with recessed 3′ ends can be specifically labeled with radiolabeled ribonucleotides as well as DNA oligonucleotide templates with RNA primers with recessed 3′ ends or RNA oligonucleotide templates with DNA primers with recessed 3′ ends.57 and other single subunit RNAPs55. These reports did not mention the potential for nucleotide specificity presumably because they were using complex templates that positioned 3′ recessed ends at many positions, and therefore, next to essentially any nucleotide. Rather than studying this activity, most of the focus was on blocking non-templated nucleotide addition which produced in vitro transcripts with unintended sequences. A few studies using less complex templates demonstrated that T7 RNAP could add nucleotides to RNA in the absence of DNA, presumably through inter- and intramolecular formation of RNA-RNA duplexes48. Konarska and Sharp44 have reported ribonucleotide addition to RNA templated, RNA primed oligonucleotides but did not explore the mechanisms of this activity and did not report activity on DNA templated or DNA primed oligonucleotides. Krupp45 observed promoter independent RNA synthesis on DNA templates able to form intermolecular or intramolecular hairpin loops that could be used as DNA primers for RNA synthesis forming RNA-DNA duplexes. However, none of these reports discussed the actual mechanism and criteria for T7 RNAP to add these apparently non DNA-templated nucleotides to the 3′ end. These reactions were all done with four ribonucleotide triphosphates present and often produced complex gel patterns that were complicated to interpret. Here we used DNA oligonucleotides with a single radiolabeled ribonucleotide triphosphate present to simplify interpretation.There have been a few reports of non-templated nucleotide addition to 3′ ends of nucleic acids with T7 RNAPTo determine their contribution to labeling efficiency, we have systematically varied the three parts of the oligonucleotide . In general, labeling efficiency increases with 5′ extension length up to about 10 nucleotides. The loop length can affect labeling efficiency depending on duplex length, but is essentially optional since intermolecular base pairing between two separate oligonucleotides can also produce 3′ end labeling. Small loops when present seem to destabilize a duplex and longer than optimal duplexes can be activated by a small loop while optimal length duplexes may be inactivated by a short loop. On the other hand, longer loops may be detrimental since they increase the distance between the component strands of the duplex decreasing the probability of duplex formation. The most critical portion of these oligonucleotides is the duplex which positions the 3′ end recessed relative to the 5′ end. The duplex length for optimal labeling is about six base pairs for duplexes 33% GC rich (14 hydrogen bonds), about 9 nucleotides at 0% GC (18 hydrogen bonds), or about three base pairs at 66% GC (8 hydrogen bonds).The observation that duplex stability is a major factor in labeling efficiency implies a dynamic, two-step mechanism for labeling. Although the duplex must transiently form in order to prime for labeling, duplex instability implies that an unfolded single strand intermediate is required for polymerase binding. If the polymerase binds the unfolded single strand oligonucleotide in a productive way, so that the duplex can form with the 3′ end base paired and at the active site of the T7 RNAP, a nucleotide complementary to the template base binds, and a phosphodiester bond is formed incorporating the base into the duplex. In this model, phosphodiester bond formation is coupled to the release of the T7 RNAP. This non-processive mechanism is in contrast to elongation which uses a processive, primer extension on template mechanism. Although duplex stability appears to be a major factor in labeling efficiency, additional sequence motifs may enhance RNA polymerase binding and therefore contribute to labeling efficiency. One motif that seems to enhance 3′ end labeling in oligonucleotides is consecutive G nucleotides (GG motif).de novo by binding to a promoter and then transitioning to elongation through promoter clearance. Early RNAPs probably lacked promoter recognition domains and initiated through primer extension like their closely-related DNA polymerases or through some other non-promoter mechanism. Several contemporary examples of such RNA polymerases exist52. It is likely that the RNA/DNA editing activity of T7 RNAP derives from such a primal activity. Mutations in the gene for T7 RNAP produce altered enzymes which support this idea. For example, some mutated enzymes have been found to be catalytically active although they bind promoter weakly, and in some instances mutated enzymes which are transcriptionally inactive have strong binding to the promoter58. Also, mutated T7 RNA polymerase59 with two amino acid substitutions (Y639F and S641A) has altered specificity towards promoter, but gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA60. This Y639F mutant retains the ability to use RNA or DNA templates and can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA-directed RNA polymerase or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction55.One of the major differences between RNA polymerases and DNA polymerases is the ability of RNAP to initiate synthesis 61. One of the major questions about Myxomycete RNA editing is how the identity of the inserted nucleotide is determined. Based on the observed in vitro mechanism of editing in T7 RNAP, we propose a model of nucleotide specificity in RNA editing for the related Physarum mtRNAP. At an RNA editing site, the Physarum mtRNAP pauses and then may either stay in place, release, or back up on the template to allow the 3′ end of the RNA to loop back on a complementary sequence either in the RNA or in the non-template strand of DNA, and add a nucleotide at the 3′ end. This positioning of the 3′ end allows specific addition to the 3′ end using the vacant position next to the 3′ end to specify the added nucleotide through Watson-Crick base pairing. The transient base pairing of the RNA is then released resulting in the insertion of a nucleotide at the 3′ end not templated by the template strand of DNA. Elongation is then resumed as a phosphodiester bond is formed between the unpaired 3′ nucleotide and the next templated nucleotide. In vitro presentation of the Physarum mitochondrial RNAP (mtRNAP) with oligonucleotides, similar to those described above, would be a test of this hypothesis. Confirmation of this novel activity in Physarum mtRNAP would be a major step toward understanding the co-transcriptional mechanism of RNA editing by the mtRNAP in mitochondria of myxomycetes.This novel DNA/RNA editing activity in T7 RNAP is probably present in all single subunit polymerases and may be the basis of nucleotide specificity during RNA editing by the mtRNAP in mitochondria of myxomycetesSupplementary Information


Upon recognition of peptide-MHC complexes by T cell receptors (TCR), the cognate T cells expand and differentiate into effector T cells to generate protective immunity. Despite the fact that any immune response generates a diverse set of TCR clones against a particular epitope, only a few clones are highly expanded in any immune response. Previous studies observed that the highest frequency clones usually control viral infections better than subdominant clones, but the reasons for this dominance among T cell clones are still unclear. Here, we used publicly available TCR amino acid sequences to study which factors determine whether a response becomes immunodominance (ID) per donor; we classified the largest T cell clone as the epitope-specific dominant clone and all the other clones as subdominant responses (SD). We observed a distinctively hydrophobic CDR3 in ID responses against a dominant epitope from influenza A virus, compared to the SD responses. The common V-J combinations were shared between ID and SD responses, suggesting that the biased V-J recombination events are restricted by epitope specificity; thus, the immunodominance is not directly determined by a bias combination of V and J genetic segments. Our findings reveal a close similarity of global sequence properties between dominant and subdominant clones of epitope-specific responses but detectable distinctive amino acid enrichments in ID. Taken together, we believe this first comparative study of immunodominant and subdominant TCR sequences can guide further studies to resolve factors determining the immunodominance of antiviral as well as tumor-specific T cell responses.The online version of this article (10.1007/s00251-019-01134-9) contains supplementary material, which is available to authorized users. T cells play a crucial role in human adaptive immunity. The recognition of an antigenic peptide bound to the MHC molecule (pMHC) by T cell receptor (TCR) leads to proliferation of the antigen-specific T cell clones  and J genetic segments. Within this domain, the antigen recognition site is formed by three complementarity-determining regions     and selecting both TCR genes (“TRA” and “TRB”). Further filtering of the data was done using an in-house pipeline . First, naive T cell sequences (CD45RA+CCR7+) were removed, as well as sequences with missing frequency because their dominance could not be determined. Because very few CD4+ sequences were present, only CD8+ sequences were used for further analysis. Next, MHC allele groups  as “only paired records.” The downloaded data was processed as previously described and the epitope-specific α/β pairs were matched using the “complex.id” column.The paired To extract epitope-specific responses for each individual, we parsed a subject identification and CDR3 clonotype frequency which were provided in the JSON string format. The subject ID was stored in the “meta” column while the frequency can be extracted from the “method” column. Since the subject ID was often missing or insufficient to define an individual response, we combined the original subject.id, replica.id, epitope, reference, and denominator of frequency and used this combination as the subject ID, with “*” as both separator and replacement for missing data. The total clonotype frequency was recalculated if the sum of frequencies in each subject ID was higher than 100%. This was often due to a repetitive measurement of the same CDR3, so this filtering ensured that each clonotype was counted only once in each individual. After correcting for the frequency and sequence redundancy per individual and epitope, the highest CDR3 clone frequency was defined as the ID response while the rest was classified as an SD response. This resulted in a highly imbalanced dataset where for each pMHC combination in an individual, a single ID and several SD were identified. Since it is possible that an ID response of one individual can be an SD response in another individual, it is not trivial to make non-overlapping ID and SD sequence sets. As the number of ID sequences was much smaller than the number of SD sequences (due to our definition of ID and SD), we decided to remove all SD sequences if these sequences were ever found as an ID response in at least one individual in our dataset. In addition, we only selected the CDR3 sequences with a frequency over 0.01% to minimize the bias from very rare clonotypes found in a few publications.The naive T-cell dataset  was derived from two healthy donors from an independent study . The unique CDR3 clones with an absolute naive count > 1 and not observed among non-naive sequence reads were included. The CDR3 nucleotide sequences were available for these datasets and we implemented a Python (version 3.5.2) script to calculate the non-VJ peptide sequences containing unidentified D segment and random nucleotide insertions.P value was not displayed if the P > 0.05 except in the supplementary figures in which “ns” was presented as “non-significant.” The two-sided Mann–Whitney statistics (two-sample Wilcoxon test in R) was used for quantitative comparisons such as length and hydropathy. The same criteria for P value was used as previously mentioned. An alternative method other than the two statistics was indicated if performed.The frequency distributions were tested for similarity using the Kolmogorov–Smirnov (KS) test. The The conserved V and J regions of the CDR3 sequences were removed at the terminal ends. The remaining sequence was defined as “non-VJ” region comprising of encoded D segment and randomly inserted nucleotides. The 20 naturally occurring amino acids were clustered into 3 subgroups based on hydropathic properties including hydrophobic , neutral , and hydrophilic . These three groups were made following the Kyte–Doolittle hydropathy scale :In order to compare CDR3 sequences varying in length, we mapped the CDR3 sequences to the 2D peptide representative of TCR structure Lefranc . The CDRi ∈  indicates the set of sequences, while k is a set of 20 amino acids and gap (“-”) and Xij is the amino acid at position j, sequence i of the alignment. We then calculated a log ratio of amino acid probability of ID (PMMID) to SD PMM (PMMSD), which we called the “positional log enrichment score” (pLES). A positive ratio indicated the enrichment of amino acids at a certain position in ID compared to SD. Occasional ∞ and − ∞ values appeared in the pLES matrix due to missing amino acids in SD and/or ID. These values were replaced with 5 and – 5, respectively, since the maximum |pLES| were observed around 4. The matrix was displayed as a heatmap colored based on pLES. The positive pLES was subsequently converted into a sequence logo for improved visualization.The parameter β detected both as ID and SD responses. This suggests that an ID response from one individual only in very rare cases (< 2%) is an SD response in another individual. In other words, immunodominance of a clone is more universal than originally thought. The redundant CDR3 sequences were removed to obtain a unique CDR3 dataset for ID and SD responses. After this processing, 5811 CDR3 sequences remained which were described as responses against nine virus species, namely HIV-1, CMV, influenza A virus (IAV), EBV, yellow fever virus (YFV), HCV, and four serotypes of dengue virus (DENV1–4)  were restricted to HLA-A*02 molecules  from IAV, NLVPMVATV  from CMV, and GLCTLVAML  from EBV.We obtained human CDR3 sequences from the VDJdb . The length distributions of both chains were normally distributed  as has been observed previously ; thus, we expected the longer non-VJ region in the CDR3β chain. Interestingly, we observed significantly longer J regions in the CDR3α chains while the V and non-VJ segments were longer in CDR3β chains  and SD (n = 5305) sequences. As shown in Fig. It has been suggested that shorter CDR3 sequences are more likely to be generated, as they closely resemble the encoded peptide and need very deletion events , and therefore, it might be expected that they share a similar distribution of CDR3 lengths. To examine the difference between non-naive and naive T cells in their CDR3 length distribution, we compared the sequences in our dataset (ID and SD combined) with CDR3 lengths of the naive CD8+ T cell β chain see the “” sectionr = − 0.14, P < 0.001) between epitope length and CDR3 length -J recombinations, and repertoire diversity . Previous studies on the GIL-MHC complex discovered a plain interacting site with fewer protruding side chains of the CDR3 sequence


This work demonstrates the use of polymer thin films and graphene to support and maintain the hydration of protein microcrystals on fixed targets for serial femtosecond crystallography at X-ray free-electron lasers. Rapid encystment protein (REP24) provides a benchmark for this encapsulation approach. For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering. Tape drives mitigate challenges related to sample consumption by synchronizing drop arrival to the X-ray pulse timing  at a rate that matches the pulse repetition rate of the XFEL (120 Hz in the case of LCLS), (2) in vacuum to minimize background scattering from air, especially in the case of small or weakly diffracting objects, and (3) in such a way that sample hydration is maintained to prevent degradation. Various types of continuous sample delivery systems have been developed  and serial synchrotron crystallography including microgrids based on silicon ] to provide a benchmark for polymer/graphene sandwich performance. By performing a comparative study of REP24 diffraction encapsulated in-vacuum and without encapsulation in humidified helium, we determine that the device is robust against evaporative losses.Therefore, our strategy towards background minimization for weakly diffracting samples was to explore the use of large-area few-layer graphene (FLG) in conjunction with polymer thin films, which would impart mechanical robustness, flexibility and allow for easy handling while minimally adding to the X-ray background scatter. We used these hybrid films as enclosing layers to maintain sample hydration for room-temperature studies in the Coherent X-ray Imaging (CXI) end station  CXI in vacuum with the crystals encapsulated in polymer/graphene hybrid films and at (2) MFX in a humidified environment on a bare Si chip (without encapsulation or sandwiching) to benchmark the performance of the sandwich towards preventing dehydration. The following descriptions of the protein crystallization, the chip design and the performance of the SFX experiments pertain to experiments in both conditions unless otherwise noted.2.1.a)]. The wafers were rinsed with a 2 vol.% Hellmanex III and MilliQ water (18.2 MΩ) thoroughly, and dried with high purity nitrogen prior to use, to remove any silicon particles generated during cutting and handling.Chemical vapor deposited FLG on nickel grown on a 4 inch silicon wafer with a 300 nm silicon oxide layer was purchased from Graphene Supermarket  and cut to desired dimensions as required. The deposition process on nickel results in a continuous surface consisting of 3–10 µm sized patches of 1–7 monolayer thickness , a putative virulence factor protein from the intracellular pathogen Francisella tularensis, was expressed recombinantly and purified as described previously  with a precipitation solution containing 54%(v/v) PEG-MME 750 and 100 mM Na-acetate pH 4.5 in a 1:1 ratio for final conditions of 7.2 mg ml−1 REP24, 27%(v/v) PEG-MME 750, 50 mM Na-acetate pH 4.5 in 25 mM NaCl, and 5 mM HEPES pH 7.5. The crystals of REP24 used in the experiment were between 15 and 20 µm in length and had the appearance of two square-based pyramids connected at the peak, with a maximum thickness of 10 µm. Crystal concentrations were estimated at 2.2 × 106 crystals ml−1 based on counts done via optical microscopy of a fixed volume.REP24 .The resulting PMMA-FLG coated chips (composing the bottom layer of a sample sandwich) were mounted film side up to custom-fabricated aluminium frames bearing magnetic mounts for loading onto the goniometer of the fast-scanning Roadrunner systems used for the experiments at the LCLS . For experiments in humidified helium, the chips were immediately transferred to the Roadrunner III sample chamber flushed with humidified helium (99 to 100% humidity) after loading. Humidity at the sample was monitored throughout the course of the experiment to ensure >99% humidity.et al., 2013−1 because of (1) the saturation of detector pixels within Bragg spots at higher flux and (2) concerns regarding damage to the chip resulting from the lower-intensity wings of the X-ray beam around the central focus spot . All chips were assessed for damage immediately following measurement with optical microscopy. The nominal pulse duration was 40 fs for both experiments. During each sample scan, diffraction images were recorded on a shot-by-shot basis at the full 120 Hz repetition rate of the LCLS by the Cornell-SLAC Pixel Array Detector  and 3 × 3 µm FWHM for experiments at CXI  of REP24 microcrystals on the bare chip, of which 5500 (69%) were successfully indexed. The indexed unit-cell dimensions were 1° Fig. 2. The max0° Fig. 2.4p5p; Segelke et al., unpublished work), with unit-cell parameters of a = 44.4 Å, b = 44.4 Å, c = 183.5 Å, α = 90.0°, β = 90.0° and γ = 120.0°. Previous room-temperature measurements of REP24 using fixed-target SFX also yielded a comparable unit cell as synchrotron single-crystal measurements: a = b = 44.4 Å, c = 183.5 Å, α = β = 90°, and γ = 120°, indicating that cryogenic cooling does not significantly impact the unit cell. They utilized similar micro-crystallization conditions but adopted a very different approach to preventing dehydration in which the microcrystals were suspended in Paratone-N and then spread onto a silicon nitride support, which is not applicable to weakly diffracting samples  can be explained by the difference in crystal deposition methods for each experiment. The sample was unsupported in humidified helium and excess liquid was removed after deposition by wicking on the opposite side of the chip, eliciting a focusing effect where crystals are drawn into pores by the wicking process. The sandwiching method employed in vacuum relies on capillary action to spread the solution applied to the first PMMA–FLG film when the enclosing PMMA–FLG layer is applied. While the hit rate reported for the in-vacuum experiment was relatively low compared with both the MFX experiment and the ideal hit rate , higher hit rates can easily be achieved by concentrating the microcrystal slurry by centrifugation before application to the chip to be sandwiched. The crystal density across the chip is not uniform because of this process of spreading and this was observed as an unevenness in the hit rate as the chips were scanned in these studies [Fig. 3c)].The relatively low hit rate for the in-vacuum experiment compared with the humidified helium experiment  after measurement, suggesting that hydration is locally maintained despite physical damage to the chip and the membrane substrates. Currently, damage to both the PMMA–FLG films and the Si membrane itself as a result of beam exposure makes the assembly a one-time-use under these measurement conditions. While in the case of REP24 permissible X-ray flux on the chip was limited to avoid detector saturation within Bragg spots, the Si chip itself imposes a limit to incident flux. While the nominal beam sizes (FWHM) for both experiments are well within the pore sizes used, there are spatially broad wings that are still sufficiently intense to cause significant damage to the chips themselves. This becomes problematic if this damage either causes melting of the Si substrate and thus significant amorphous Si scatter signal, or if this damage causes cracks that propagate down a row of pores resulting in physical deformation of the chip. While strong single-crystal Si Bragg reflections are at higher angles than typically observed for macromolecular crystals, rotation of the Si membrane due to this physical damage may result in these potentially damaging reflections appearing on the detector. This bolsters the case for development of amorphous polymer chip materials both to prevent unintended exposure of the detector to strong reflections resulting from damage and to reduce manufacturing costs.et al., 2018et al., 2015et al., 2017et al., 2016et al., 2015et al., 2019et al., 2018et al., 2018The unit cell of REP24 found by analysis of diffraction data derived from in-vacuum and humidified atmosphere experiments was in good agreement and indicates that the presented method of sample enclosure with graphene and polymer films is robust against evaporative losses. To our knowledge, other methods utilizing polymer films and/or graphene as supporting or enclosing substrates for XFEL or synchrotron studies either used much thicker polymer films (micrometres thick) alone (Doak 3.3.c)–3(e)].In an effort to understand the contributions to the background of the enclosed sample, device and sample environment, median scattering intensities were calculated for REP24 within the PMMA-FLG sandwich, a PMMA-FLG sandwich containing a non-crystalline thin film (∼5–10 nm) sample consisting of protein and lipid and deposited onto the chip without water, and REP24 on the bare Si chip in humidified He at MFX. These median intensities were arranged to spatially represent variations in background across the chip. Radial averages of each detector frame for both MFX and CXI experiments were calculated, normalized according to the incident pulse energy and degree of beam attenuation, and converted to photons per pixel by identifying the detector response corresponding to the single photon peak in a histogram of single-pixel detector response for the whole detector. The median photons per pixel was calculated per shot for each scattering profile to exclude sharp peaks resulting from Bragg reflections. This intensity was sorted by time stamp and the chip-row number recorded by the Roadrunner translation software . The majority of the high-intensity shots correspond to the location of the Kapton frame used to support the second enclosing layer of PMMA–FLG film. The shots of moderate intensity are not uniformly distributed across the rest of the chip but are clustered in regions. Representative radial scattering profiles for shots distributed through these regions [Fig. 4a)] indicate that the dominant component of this varying signal is a broad peak, which corresponds to liquid water ]. The localization of hits around pockets of thicker water layer is probably caused by the manual method of deposition that relies on the capillary force of the buffer to spread the sample. To optimize sample density and hit rate while minimizing water-film thickness, methods could be imagined that deposit many more concentrated drops over the sample area before encapsulation, redesign the chip to allow controlled wicking for water removal, or employ surface functionalization to fix crystals to the film surface before water removal.In general, the chip with sandwiched REP24 contains three populations: very high intensity shots above 40 photons pixel−1 mJ−1 [Fig. 3e)]. These shots are similar to the low-intensity population of shots for the sandwiched REP24 chip, both in their median intensities and in their radial scattering profiles [Fig. 4a)]. The median intensities of the chip measured at MFX have a higher floor, and the areas of highest intensity are also clustered in certain regions of the chip that mostly correspond to higher hit rates [Fig. 3d)]. Scattering profiles of shots both at relatively low intensity and high intensity have a broad sloping background that is probably caused by scattering from the humidified He environment. High-intensity shots also have a broad peak that is probably caused by residual water [Fig. 4b)]The PPMA-FLG sandwich containing the thin-film sample at CXI is extremely uniform and the vast majority of shots have median intensities of <1 photon pixelNscat) onto the CSPAD for each component of the enclosure, PMMA, FLG and water, was modeled based on the following formula,N is the number of incident photons, Ab is the area of the beam, M is the number of atoms or molecules in the beam, and xraylib library; Schoonjans et al., 20115 and 1.8 × 106 photons, respectively, per 1 mJ of incident pulse energy at 7.5 keV. A 1 µm film of water scatters 7.8 × 106 photons and we anticipate the water thickness surrounding the crystals to be the same dimension as the crystal size, ∼20 µm, thus the total contribution from water is about 1.6 × 108 photons. This is consistent with our observation that the water signal dominates the background for our encapsulated sample. While more water is removed from the chip at MFX and water scatters less at 9.5 keV (6.6 × 106 photons for 1 µm) the contribution from water vapor for He at 100% humidity at room temperature is likely to be significant (2.6 × 108 photons cm−1). By contrast, Mylar films used to sandwich microcrystals in ], the enclosed sample in vacuum shows most images have low background below 10 photons pixel−1 mJ−1 with no effective lower limit, whereas the MFX sample in humidified He shows most images contain above 10 photons pixel−1 mJ−1, with a lower limit of 5 photons pixel−1 mJ−1. These results demonstrate that the background scattering of the enclosed chip in vacuum can be significantly less compared with the background scattering of the humidified He environment.The very low background intensities across the chip for the thin-film sample and the nature of the varying background for the enclosed sample indicate that the vast majority of the enclosed sample background originates from an encapsulated water layer of varying thickness. The polymer, graphene and Si components of the device together contribute on average <1 photon pixel4.et al., 2019et al., 2016et al., 2017et al., 2019New fast-scanning systems have made fixed-target data acquisition at the 120 Hz repetition rate of the LCLS possible, drastically reducing measurement time for fixed-target SFX experiments and making studies of low-order samples feasible in a relatively short time frame. Recently, even higher scanning rates up to 1 kHz have been demonstrated using a Roadrunner system together with the JUNGFRAU 1M detector at the European Synchrotron Radiation Facility where precise alignment with pores is not a concern  and provide a modular, well controlled sample environment will be better adapted to take advantage of the faster rate of data acquisition. To this end, we are currently adapting this sample-support-enclosure approach to include low-cost polymer frames that can maintain sample hydration over long periods of time, and substrate functionalization and patterning to control sample deposition and location. This could include patterning to control wetting during deposition or on-chip 10.1107/S2052252519014003/ec5015sup1.pdfAdditional characterization of the graphene/polymer films and time invariance of the REP24 unit cell after vacuum exposure. DOI:


These coals exhibit a three-stage evolution in atmospheric δ13C from the Artinskian to the Kungurian. Early Kungurian coals effectively record the presence of the short-lived Kungurian Carbon Isotopic Excursion (KCIE), associated with the proposed rapid release of methane clathrates during deglaciation at the terminus of the Late Palaeozoic Ice Age (LPIA), with no observed disruption to peat-forming and terrestrial plant communities. δ13Corg variations in coals from the Moatize Basin are cyclic in nature on the order of 103–105 years and reflect changes in δ13Corg of ~±1‰ during periods of stable peat accumulation, supporting observations from Palaeozoic coals elsewhere. These cyclic variations express palaeoenvironmental factors constraining peat growth and deposition, associated with changes in base level. This study also demonstrates the effectiveness of vitrain in coal as a geochemical tool for recording global atmospheric change during the Late Palaeozoic.The end of the Late Palaeozoic Ice Age (LPIA) ushered in a period of significant change in Earth’s carbon cycle, demonstrated by the widespread occurrence of coals worldwide. In this study, we present stratigraphically constrained organic stable carbon isotope (δ This global event is critical to understanding changes in the carbon cycle associated with the highest rates of global organic carbon burial (up to 6.5 × 1018 mol/Myr) in the past half billion years4, and coal formation across the terrestrial lithosphere.The end of the Late Palaeozoic Ice Age (LPIA) represents one of the most extreme climate transformations in geological history, transitioning from icehouse to greenhouse conditions6. The multiple ice centres of the LPIA across Gondwana are evidenced by widespread glacial deposits in Palaeozoic-aged basins of Australia, Antarctica, South America, Arabia, India and Africa7. Recently, growing evidence suggests that the LPIA may have been triggered, and subsequently terminated, by uplift and erosion of the Hercynian Mountains8.The terminal deglaciation of the LPIA was an irregularly distributed, asynchronous event occurring from the Late Palaeozoic, where polar ice melted due to continental scale warming at high-latitudes over Gondwana13C data has been increasingly utilised to understand both palaeoclimatic and palaeoenvironmental changes associated with the LPIA11. Recently, a negative isotopic shift in δ13C was observed in both inorganic and organic carbon sources, in sediments from high and low latitude Palaeozoic basins11. This δ13C excursion has been proposed as the Kungurian Carbon Isotopic Excursion (KCIE), and hypothesised to record the release of extremely depleted (ca. −35‰) methane clathrates during glacial melting11.δ13Corg records of coals deposited during the LPIA have not yet been used to demonstrate changes in the global carbon cycle. The slow accumulation of peat, ~0.9 mm/yr in modern high-latitude settings12, allows subsequently formed coals to continuously record palaeoclimate and palaeoenvironmental change at high-temporal resolutions.Despite coal occurrence recording the high rates of carbon burial during this period, high-resolution δ13Corg of Early Permian coals of the Moatize Basin, Mozambique. These coals were selected for this investigation as they are associated with the final occurrence of widespread glacial deposits, and interbedded with deglacial lacustrine sediments, noted throughout other stratigraphically equivalent basins in Southern Africa16.This study investigates if the proposed KCIE is evidenced in δ15. The glacial sediments and the lacustrine sediments above the first correlatable coal seam, Sousa Pinto seam, to the base of the Chipanga seam, aided in the correlations between drill cores across the basin15, in the absence of better chronostratigraphic markers .Samples from core recovered by Vale Moçambique were utilised from available core across eight (8) locations in the Moatize Basin, Tete Province, central Mozambique Fig. . These c13Corg for the coals of the Moatize Formation falls within the typical range of C3 plant organic matter17, ranging from an absolute maximum of −20.0‰, to an absolute minimum of −26.9‰. The data were collected across all locations domained by each respective ply within the Bananeiras, Chipanga, and Sousa Pinto seams , exhibiting a shift to more positive δ13Corg with time.Stage 1 coals, encapsulate the Sousa Pinto seam . The average δ13Corg, suggesting some variation in palaeoenvironmental factors controlling low-magnitude (~±1‰) δ13C cycling. A weak (~1.5‰) positive shift in δ13Corg of Stage 1 coals suggests a more long-lived change in atmospheric CO2 concentrations and δ13C. These changes are concurrent with the development of widespread peat deposits, resulting in increased rates of carbon burial coincident with the Artinskian8.Stage 1, Artinskian coals of the Sousa Pinto seam exhibit variable ranges of δ13Corg value for this stage is −24.7‰, with a high standard deviation . Stage 2 coals have the most negative δ13Corg of all the data domains. A striking, high-magnitude (~3.5‰) negative excursion is observed δ13Corg in Stage 2, coincident with the base of the Chipanga seam in the early Kungurian. This negative excursion is relatively short-lived compared to smaller-scale δ13Corg cycles (~±1‰) in Stage 1 coals of the Artinskian, and Stage 3 coals of the Kungurian. The compiled δ13Corg record from the Moatize Formation coals is time equivalent to other, continuous δ13Corg records from sediments in both low and high-latitude sediments . The mean δ13Corg value for this stage is −22.6‰ , and remains stable throughout each ply domain, regardless of seam and age distribution. When the total sample set  is normalised for each ply domain, the cyclic variation of δ13Corg within these coals can be observed . The average δved Fig. . Mechani13C from the Artinskian to the Kungurian. In this study, δ13Corg cycles  trends inferred from Palaeozoic palaeosol development27, and in palaeofloral communities28. These short-term changes observed in low-latitude sediments, attributed to pluvials, result in changes in base level. These base-level changes, coincident with δ13Corg cycling, are also observed in coals from Eastern Australia20, and the 103–105 year time-frame is coincident with Milankovitch-scale orbital frequencies28, also observed in Mesozoic and Cenozoic coals30.From these calculations, it is likely that δ3–105 year cycle. The short-lived nature of this isotopic excursion suggests the rapid injection of 13C-depleted carbon into the atmosphere, rather than any relatively long-lived changes in CO2 concentration. It is possible that this negative carbon isotopic shift is due to the release of methane clathrates (CH4) into the atmosphere during terminal deglaciation. Furthermore, the contribution of deep soil organic carbon (SOC) loss and CH4 from terrestrial permafrost may also have contributed to widespread δ13C perturbation31.The observed KCIE is equivalent to the duration of a 104 release is equivalent to the development of euxinic lake deposits across Southern Africa as a result of deglaciation marking the end of the LPIA15. The stratigraphic equivalent of these euxinic lacustrine deposits is represented by organic rich black shale separating the Sousa Pinto and Chipanga seams, at variable thickness at each sample location . Great care was taken to only sample bright (vitrain) bands from coals, as to minimise δ13Corg variation with coal lithotype or biochemical composition35. Vitrains were hand-picked at a millimetre scale to avoid any potential carbonate contamination from mineralised cleats. The typical low taphonomic diversity of peat-forming ecosystems28, minimises the likelihood of δ13Corg variation dependent on taxa9.Samples were taken from plys of the Bananeiras , Chipanga  and Sousa Pinto  coal seams  at the University of Queensland using a stable isotope ratio mass spectrometer (Isoprime), coupled in continuous flow mode with an elemental analyser (Elementar Cube) (EA-CF-IRMS). Calibration was performed by use of two standards, USGS24 (−16.1‰ δ13CPDB) and NAT76H (−29.26‰ δ13CPDB), interspersed throughout analytical runs. Each sample was analysed in duplicate, using 50–200 μg of concentrate combusted at 1020 °C in 3.5 mm × 5 mm tin capsules. Any sample with a beam size outside the working range of 1 × 10−9 to 9 × 10−9 Å, or with a δ13Corg result variation between duplicates of >0.4‰, was re-analysed, in accordance with laboratory quality control practices. Final data values were normalised and are reported in ‰ VPDB.The δ


Infographics represent a potential means of improving public knowledge about cancer. However, there is little experimental evidence of their efficacy. This preliminary study investigates whether infographics are superior to text for the communication of information about cancer risk in old age via a three armed randomized controlled trial. Trial involved allocation concealment and block randomization of 30 male participants aged over 50 to receive text information (control) or one of two infographics (interventions). Participants who viewed an infographic were more likely to know the correct association between cancer risk and old age compared with those viewing text information . Participants had limited understanding of the phrases “cancer incidence” and “cancer prevalence” but good understanding of the phrases “cancer risk factor” and “cancer stage.” Possession of good numerical skills appears to be a key determinant of ability to extract meaning from statistical information provided; regardless of format. Initial results suggest icon array infographics may be more effective communication mediums than text but further study with more participants and an updated infographic is necessary to confirm this finding. Trial registration number: ISRCTN33951209. Public awareness of the link between cancer risk and getting older is low. In the United Kingdom, less than 15% of people surveyed in 2011 knew that cancer was most common in those over the age of 70 compared to those younger. This lack of awareness may be linked to poorer cancer survival due to delay in presentation to a doctor and delay in diagnosis. This study aims to address low awareness of cancer by developing and testing picture-based information resources called “infographics” that are easier to understand by the public to help make information about cancer more accessible and allow people to make more informed decisions about cancer. In this small study of 30 Northern Irish men over the age of 50 we show that infographics may be better than text for communication of information about cancer risk but further study with more people is necessary to confirm this finding.1.Infographics are a colorful and innovative means of communicating with the general public  is superior to text alone for improving awareness of the association between cancer risk and old age. Second, to assess the feasibility of quantitatively measuring efficacy of icon array infographics using randomized-controlled-trial (RCT) design with a view to making any required methodological or infographic design changes prior to a second trial phase.2.2.1.This study employed a mixed-methods approach whereby results of the RCT were quantitatively analyzed by way of odds ratios (OR) and risk ratios (RR) reporting while quotes from participants gathered during the pre-trial questionnaire phase were qualitatively analyzed via content analysis to gain an insight into baseline knowledge of some of the words and phrases used in cancer statistics reporting.2.2.Data from the N. Ireland Cancer Registry. were used2.3.This was a three-armed non-blinded RCT involving allocation concealment and computer-generated block randomization. Eligible participants  were invited to take part during separate 1-day visits to four Men Shed’s in Northern Ireland   of 0.80, and four cancer definition questions  to be answered within 15 min . Participant demographics were summarized and tabulated. Differences in participant demographics between RCT wings were compared using one-way analysis of variance (ANOVA) for continuous variables and chi-squared test for categorical variables. Knowledge of the association between cancer risk and old age was dichotomized (correct or incorrect) and was collected for each participant before and after viewing assigned intervention.correct association between cancer risk and old age post-intervention. RR and 95% confidence intervals were compared between control and intervention groups. OR and 95% confidence intervals were calculated to determine the effect of numeracy score, education status, and pre-intervention cancer knowledge on post-intervention knowledge of the association between cancer risk and old age. A value of P < 0.05 indicates significance.Unadjusted risk ratios (RR) were calculated to identify the probability of identifying the 2.6.Free text written responses to the four phrases participants were asked to define were analyzed using qualitative content analysis. This is a widely used method of analyzing information such as text to derive themes in psychological and sociological studies . Trial is registered with the International Standard Randomized Control Trial Number (ISRCTN) registry (ISRCTN33951209).3.3.1.A total of 31 men agreed to participate across four study sites in Northern Ireland during July 2017. One participant was excluded from analysis for being aged less than 50 . This increased to 46.7% post interventions (N = 14). Baseline knowledge among those assigned to view intervention A or B (N = 20) was 10% (N = 2). This increased to 60% post interventions (N = 12). When compared with text control, viewing either intervention A or B increased the probability of having knowledge of the correct association between cancer risk and old age, however this was not statistically significant (RR = 3.0 95% CI = 0.82–10.90). Viewing infographic intervention A increased probability of correctly answering the post-intervention question threefold (RR = 3.2 95% CI = 0.85–11.88) with similar effect seen in those viewing infographic B (RR = 2.8 95% CI = 0.71–10.94) (see Table Baseline knowledge of the correct association between cancer risk and old age before interventions were viewed was 13.3% (or B N = 0 was 10%3.3.P < 0.001). An above average numeracy score doubled the odds of correct knowledge of cancer risk and old age post-intervention (OR = 2.1 95% CI = 1.21–3.50) (P = 0.008). Having at least one formal educational qualification also increased the odds of getting the post-intervention question correct (OR = 7.3 95% CI = 1.27–42.29) (P = 0.026) (see Table P = 0.886) . The mean score of participants who incorrectly answered the post-intervention question about cancer risk and old age was 2.94 (SD = 1.77). This compares to a mean score of 5.57 (SD = 2.10) in those who answered correctly post-intervention. The difference in scores between these two groups was highly significant  seemingly confused “cancer incidence” with a diagnostic phrase such as “detection” and “diagnosis.” Similarly, the majority of participants chose to define the phrase “cancer prevalence” using either mathematical terminology or confused it with a phrase that meant increased cancer risk. While no participants gave an exact definition for either of these two phrases, those who used mathematical terms such as “percentage” appeared to have greatest insight into the meaning of these phrases.The majority of participants who attempted to define “cancer incidence” either did so using a combination of mathematical terms like “odds,” “chances,” or “numbers” or by using negative emotive phrases like “bad news,” “scare from the doctor,” or “hurt and pain.” A minority of participants (N = 15) chose to define “cancer risk factor” using an example of what they considered to increase the risk of cancer. These included in order of respondent frequency, “smoking,” “alcohol,” “food,” “pollution,” and “sun exposure.” A number of participants responded with more general definitions that mainly fell under the umbrella of lifestyle related risk. A small number (N = 3) of participants attempted to define “cancer risk factor” using probability terminology including “increased likelihood” and “increased chances.”Half of all participants (N = 17) was the concept of growth or development as a means of defining “cancer stage.” Some participants used words and phrases associated with the concept of time such as “phase,” ‘1-2-3ʹ, and “beginning-middle-end.” Others used words or phrases describing spatial awareness such as “position,” “level,” or “localized or metastasized.” Six participants chose to further develop this theme by linking it with the concept of diagnosis or treatment by using phrases such as “early detection,” “catching cancer early,” “degree of invasion and treatability,” and “a measurement of how treatable a cancer is.” The remaining participants had little or no understanding of the term “cancer stage” and either rephrased the question or used negative emotive phrases such as “death” or “concern.”Three themes emerged when analyzing responses of those attempting to define “cancer stage.” The first and most numerous . This contrasts with those with no formal educational qualifications who actually saw a 15% decline in their knowledge. This would seem to indicate that additional information confused or misled the group with no formal educational qualifications. Confusing individuals with visual information is something Arcia et al.  identifipercentage of population affected by cancer.” This implies that while exact knowledge of these terms is lacking, there is an understanding that they are statistical terms. The use of named examples  while defining “cancer risk factor” shows that there is awareness among the study population of exposures that have a multiplicative effect on cancer risk. One participant in particular gave an extremely cohesive and complete explanation for what a cancer risk factor was—“lifestyle habits such as smoking or drinking, or certain jobs where there are pollutants such as smoke or smoke producing fumes all contribute to the risk of cancer.” Another participant chose to outline less commonly known risk factors for cancer—“exposure to sun without sf cream, unhealthy eating, and lack of exercise.” No participants chose to mention familial risk factors such as genetic mutations. This is likely a consequence of lower educational attainment of participants. Finally, in defining the phrase “cancer stage,” participants used a variety of words and phrases that tell us that there is an appreciation that cancer stage is related to progress and development of cancer. A sizeable minority were also able to further develop this concept to create a semantic link to diagnosis and treatment. For example, one participant wrote that “the early stages where it is localized and operable or later when it has metastasized.” In our opinion, this implies good understanding of what the phrase “cancer stage” means.Qualitative analysis of participants attempts to define the four phrases used in cancer statistics yielded some interesting results. Overall, participants had limited knowledge and understanding of the phrases “cancer incidence” and “cancer prevalence” while understanding of the phrases “cancer risk factor” and “cancer stage” was generally good. Those who were closest to accurately defining the phrases “cancer incidence” and “cancer prevalence” did so using vague numerical terminology. For example, one participant chose to define cancer incidence as “the percentage of population who are diagnosed with cancer” while another wrote that cancer incidence was “the number of people within a certain sample who get cancer.” Multiple participants described cancer prevalence as a “4.2.The strengths of this study include its novelty, use of an RCT design, and inclusion of participants who are more socially isolated and traditionally more difficult to recruit. The mixed-methods design of this study allows for integration of quantitative and qualitative findings, thus augmenting our understanding of both intervention effects and trial methodology. To our knowledge, this is one of the first trials involving public health infographics and will therefore be of interest to public health decision makers, bioinformatics researchers, and healthcare professionals. Hill et al.  and ZikmLimitations of this study include small sample size and limited generalizability of results. It was our original intention to recruit as many participants as possible to this study but it proved difficult to recruit to this non-incentivized RCT. Recruitment to research studies where there is no financial, material, or therapeutic incentive for participants is known to be challenging (Adams, Caffrey, & McKevitt, 4.3.Cancer survival is lower in the UK compared to other socioeconomically similar countries (Coleman et al., 5.Infographics can potentially make statistical information more accessible to the general public. We have identified a knowledge gap that will likely benefit from infographics and have piloted a methodology to test this theory. Based on our experience with this initial trial phase, some changes to methodology and recruitment will be beneficial for a future larger scale trial. Potential changes include recruitment of males and females, simplifying to a two-armed RCT design, and creation of a new more colorful icon array intervention with actionable public health message based on Public Health England guidelines (Public Health England,