Germany is ethnically diverse and the social climate is more or less welcoming for different immigrant groups. The social climate can be described by stereotypes of members of the receiving society about immigrant groups, which in turn shape receiving-society members’ behavioral tendencies of support or discrimination. We investigated warmth and competence stereotypes about 17 immigrant groups in Germany. Results showed four clusters of immigrant groups in the two-dimensional space of warmth and competence. Groups who immigrated comparatively recently and from regions of conflict  were stereotyped most negatively . Across groups, path analysis investigated the socio-structural relations proposed by the stereotype content model and the BIAS map for immigrant groups in the German context. In a pre-registered model all hypothesized paths were significant but model fit was not good. Therefore, an exploratory model included additional paths as well as intercorrelations between exogenous variables and error terms. The modified model showed good fit and partly replicated the relations proposed by the BIAS map. Threat predicted warmth, whereas status predicted competence. Warmth predicted active behavioral tendencies and competence predicted passive behavioral tendencies. Additional paths from status to warmth, threat to competence, as well as from warmth to passive behavioral tendencies and competence to active behavioral tendencies were also significant. Thus, findings support receiving-society members’ active role in the process of integrating immigrant groups into German society. Based on the results, social-psychological approaches to foster immigrant integration are discussed. Many European countries have ethnically heterogeneous populations and have received large numbers of immigrants during the last decades. Germany, the United Kingdom, France, and Spain are among the top ten countries with the highest proportions of immigrants in the world . FollowiIt is likely that various immigrant groups face different (un)welcoming social climates in Germany. From a social-psychological perspective, these social climates can be operationalized by the attitudes of members of the receiving society towards immigrant groups –9. ReseaWarmth refers to likability and friendliness, and is predicted by perceptions of outgroups’ intentions toward the ingroup. These intentions are reflected by competition or realistic threat  as well as symbolic threat  ”: active facilitation , active harm , passive facilitation , and passive harm  [not at all to 5 = completely. Furthermore, we assessed positive contact (“How often do you have positive contact to immigrants?”) as well as negative contact  or Cronbach’s Alphas (> 2 items). The groups were rated on perceived α > .94) ; perceivs < .05) ; warmth α > .95) , 21. Furs < .01) . AnswersFinally, participants provided demographics including gender, age, and migration background. We also measured majority integration efforts as a more detailed measure of active facilitation behavioral tendencies towards immigrants with 21 items . To reduN = 18, including the 17 immigrant groups as well as the group of Germans) [t-tests   and a hi[t-tests . Cluster[t-tests . With reps < .027). Results of the modified model are shown in To investigate whether threat and status predict stereotypes, which in turn predict behavioral tendencies across the 17 immigrant groups, we restructured the data into long format . The two items measuring symbolic and realistic threat were highly correlated and were thus combined to one scale. We computed a path model in Mplus  accountir = -.38, p < .001). Correlations of residuals were also significant . The model explained the following amounts of variance in the dependent variables: warmth (29%), competence (71%), active harm (7%), active facilitation (21%), passive harm (11%) and passive facilitation (26%). Models excluding participants with non-German nationality (n = 11) or controlling for positive and negative contact with immigrant groups showed similar results.As expected and replicating predictions by the BIAS map, threat negatively predicted warmth and status positively predicted competence. In turn, warmth negatively predicted active harm and positively predicted active facilitation. Competence negatively predicted passive harm and positively predicted passive facilitation. Furthermore, all additional exploratory paths were also significant. Threat negatively predicted competence, whereas status positively predicted warmth. In turn, warmth negatively predicted passive harm and positively predicted passive facilitation. Competence negatively predicted active harm and positively predicted active facilitation. Threat and Status were moderately and negatively correlated (p = .044). This means that active facilitation was predicted by warmth more strongly than by competence. For all other behavioral tendencies model fit of the constrained models was not significantly worse than that of the unconstrained models . Thus, regression weights of warmth and competence for the prediction of active harm, passive facilitation and passive harm did not significantly differ from each other.To test whether the exploratory paths and the pre-registered paths from warmth and competence stereotypes to behavioral tendencies differed in magnitude–and thus to investigate differences in contributions of the newly added paths and the paths included in the traditional formulation of the stereotype content model to the prediction of behavioral tendencies–in a series of constrained models both paths  were restricted to be equal. Model fit of the constrained and unconstrained models was compared using χ² difference tests. For active facilitation the constrained model fit to the data significantly worse than the unconstrained model (Δχ²(1) = 4.04, Germany’s population is highly and increasingly multicultural, and the integration of immigrants remains a societal challenge . ImmigraIn accordance with international studies , 13, 20,In line with the investigation of stereotypes about refugee subgroups by Kotzur et al. (2019), an exploratory investigation showed that factors driving the social perception of different immigrant groups in Germany might be their migration status (labor immigrants vs. refugees) and their religious affiliation (non-Muslim vs. Muslim) . TraditiInterestingly, in the current research groups who predominantly fled from regions of conflict were not stereotyped in the most negative way possible, as their ratings for warmth were higher than for competence. Due to relatively low perceptions of agency and control, it can be expected that refugees are stereotyped as incompetent. In turn, threat perceptions due to immigration by refugees  might lead to refugees being stereotyped as low on warmth as well . HoweverThe current research investigated the relations of stereotypes with the socio-structural variables of status and threat, as well as receiving-society members’ behavioral tendencies towards the immigrant groups in a joint path model. In line with recent methodological critique of traditional analysis strategies to test predictions of the stereotype content model , 28, theThe differential prediction of active/ passive behavioral tendencies by warmth and competence, respectively, was extended by significant paths from warmth to passive behavioral tendencies and from competence to active behavioral tendencies. As expected by the BIAS map, warmth was a stronger predictor of active facilitation than competence . This reThe fact that the path model had to be modified to fit the data well indicates that socio-structural variables, stereotypes, and behavioral tendencies might be more strongly interrelated than originally suggested by the BIAS map–at least in the context of immigration in Germany. Previous research conducted with a broad variety of social groups has mostly supported the differential relationship of socio-structural variables and stereotypes, showing no relationships of status with warmth and competition with competence , 24, 42;The societal integration of immigrants is not only shaped by the immigrant groups themselves, but also by members of the German majority group. The current research showed that socially shared stereotypes are related to receiving-society members’ behavioral tendencies towards immigrants. We investigated stereotypes and behavioral tendencies only across immigrant groups; however, it is an interesting question which immigrant groups are likely to be facilitated and which groups are likely to be harmed in the German context. Becker and Asbrock (2012) showed that when groups receive ambivalent stereotypes, behavioral tendencies are predicted by the more salient stereotype dimension . BecauseBecause portrayals of immigrants in the media interact with receiving-society members’ attitudes to shape their reactions towards immigrants , how speThe current study showed that when an immigrant group is stereotyped low on warmth or competence (or both), active and passive harmful behavioral tendencies are likely to increase. Active harm towards immigrants can be expressed via verbal or physical discrimination based on ethnic group membership. Experiences of discrimination can have adverse consequences for immigrants, for example lower quality of life and poorer health outcomes , 45. In ultimate attribution error [(Negative) stereotypes can also influence majority members’ causal attributions for the poor integration of specific immigrant groups. The attributional model of stereotypes  describeon error , which hon error .stereotype threat [A further education-related consequence of negative competence stereotypes is e threat . In the e threat , 50, 51.e threat . ResultsA prominent way to reduce negative stereotypes and prejudice is intergroup contact . PositivFirst, due to the cross-sectional nature of the current study, causal relations between variables could not be investigated. Longitudinal or experimental studies could be a fruitful agenda for future research. The assessment of socio-structural variables, stereotypes and behavioral tendencies at different time points would allow for testing causal relations. Variables might also be reciprocally related, forming a recursive cycle of stereotypes and integration. Longitudinal investigations could also shed light on how perceptions of immigrant groups develop over time. For example, immigrant groups who have recently migrated to Germany and were stereotyped as moderately warm and competent in the current study might receive more positive stereotypes over time .Second, future studies should include recent developments in research on intergroup stereotypes. There is evidence that the warmth dimension is comprised of sociability  and morality  . MoralitThird, the hypotheses about stereotypes predicting majority integration efforts  as a furIn light of the societal challenges of immigrant integration in Germany, the current study underscores the important role of receiving-society members’ stereotypes for immigrant integration: Whether the social climate towards immigrant groups in Germany is welcoming or unwelcoming predicts stereotypes towards immigrant groups, which translate to behavioral intentions of support–likely leading to higher integration–or harm–likely leading to higher discrimination of immigrant groups. By investigating a broad selection of immigrant groups living in Germany, the current research contributes to the understanding that immigrants of different ethnic origins are not perceived alike, but that members of the (German) receiving society have differentiated social perceptions of immigrant groups.


The chemistry underpinning microbial interactions provides an integrative framework for linking the activities of individual microbes, microbial communities, plants, and their environments. Currently, we know very little about the functions of genes and metabolites within these communities because genome annotations and functions are derived from the minority of microbes that have been propagated in the laboratory. The chemistry underpinning microbial interactions provides an integrative framework for linking the activities of individual microbes, microbial communities, plants, and their environments. Currently, we know very little about the functions of genes and metabolites within these communities because genome annotations and functions are derived from the minority of microbes that have been propagated in the laboratory. Yet the diversity, complexity, inaccessibility, and irreproducibility of native microbial consortia limit our ability to interpret chemical signaling and map metabolic networks. In this perspective, we contend that standardized laboratory ecosystems are needed to dissect the chemistry of soil microbiomes. We argue that dissemination and application of standardized laboratory ecosystems will be transformative for the field, much like how model organisms have played critical roles in advancing biochemistry and molecular and cellular biology. Community consensus on fabricated ecosystems (“EcoFABs”) along with protocols and data standards will integrate efforts and enable rapid improvements in our understanding of the biochemical ecology of microbial communities. There are few studies more fascinating, and at the same time more neglected, than those of the teeming populations that exist in the dark realms of the soil. We know too little of the threads that bind the soil organisms to each other and to their world, and to the world above.Silent Spring, 1962Rachel Carson, Microorganisms are on us and around us, catalyzing reactions on which we critically depend yet poorly understand. Their metabolism has been driving earth’s climate , buildin12 microbial species on earth  of soil microbes that have been cultivated in the laboratory  has reveEscherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae. Even for these well-studied microbes, more than 30% of their gene functions are currently unknown  naturally lends them to laboratory ecologic studies . Howeverecosystem fabrication, producing “EcoFABs” that serve as a much-needed middle ground between model organisms and highly complex communities  printing, sensors, and analytic and imaging technologies has the potential to enable munities . The sucmunities –31. The Native communities will be the inspiration and benchmark for EcoFABs, which in turn would enable perturbation, manipulation, and detailed observation not possible in native environments. Development of EcoFABs will depend on the formation of groups of investigators such as those that have coalesced around the study of single model organisms, such as the fly, mouse, worm, and zebrafish . HoweverArabidopsis or Brachypodium, to leverage extensive knowledge of their biology, powerful genetic tools that have been developed, and advanced imaging technology. For example, the GLO-Roots technology enables visualization of the root system through thin soil sections (We believe that plants and their associated soil microbiota represent particularly attractive targets for ecosystem fabrication and synthetic community design. Such systems would enable investigation of diverse gene and metabolite functions relevant to microbe-microbe and plant-microbe interactions  33). In. In33). sections .Selecting community members for construction of standardized model ecosystems will require careful consideration to make the model ecosystems suitable for diverse lines of investigation. This will require achieving a balance of environmental relevance and experimental tractability . RelevanMicrofabrication technologies that permit control over the “microbial microenvironment” would provide a much-needed experimental platform for deconstructing microbial interactions. While this may sound futuristic, advances in 2D and 3D fabrication of biomaterials could rapidly enable the construction of microbial communities with carefully controlled microenvironments , 38, incUse of advanced technologies in the context of laboratory ecosystems will enable discovery of the relationships underpinning interactions within complex microbial networks. Critically, defined laboratory ecosystems will also help generate a common knowledge base, since all research is performed on a defined system with potentially modified parameters and conditions, allowing for expansion of compatible data and information. However, elucidation of who is communicating with whom requires innovative computational tools. Taking advantage of genomic and metabolomic information and data, genome-scale models have been deployed successfully to unravel complex and intertwined interaction in cocultures and simple communities , 41. TheTo rapidly advance our understanding of the biochemical ecology of microbiomes, we envision the workflow depicted in We see opportunities to integrate a range of innovative technologies within EcoFABs to discover and characterize the connections within communities and chemistry within soils. These techniques include, for example, sequence-based methods , 47, met49–60–65–The chemical signaling and metabolic webs of soil microbial communities are largely unexplored. To investigate the biochemical ecology of these environments, we need new technologies supporting reproducible ecosystem fabrication to enable use of observational and reductionist tools. Community consensus on a few EcoFABs along with protocols and data standards will enable discovery and detailed investigation of the “dark biochemistry” of microbial interactions. Specifically, it will support reproduction of the same microbial ecosystems in labs around the world, enabling researchers to build on each other’s results to advancing microbiome science much as model organisms have advanced our understanding of molecular and cellular biology. However, a critical challenge will be recapitulating sufficient complexity from native microbial communities to provide relevant insights, while at the same time making them experimentally tractable. Striking this delicate balance will require the engagement of scientists with diverse skills to design a series of model ecosystems designed with the appropriate level of complexity need to gaining a predictive understanding of how soil microbial ecosystems function.


Here we constructed a conditional TDP-43 mouse with depletion of TDP-43 in the mouse forebrain and find that the mice exhibit a whole spectrum of age-dependent frontotemporal dementia-like behaviour abnormalities including perturbation of social behaviour, development of dementia-like behaviour, changes of activities of daily living, and memory loss at a later stage of life. These variations are accompanied with inflammation, neurodegeneration, and abnormal synaptic plasticity of the mouse CA1 neurons. Importantly, analysis of the cortical RNA transcripts of the conditional knockout mice at the pre−/post-symptomatic stages and the corresponding wild type mice reveals age-dependent alterations in the expression levels and RNA processing patterns of a set of genes closely associated with inflammation, social behaviour, synaptic plasticity, and neuron survival. This study not only supports the scenario that loss-of-function of TDP-43 in mice may recapitulate key behaviour features of the FTLD diseases, but also provides a list of TDP-43 target genes/transcript isoforms useful for future therapeutic research.TAR DNA-binding protein The online version of this article (10.1186/s40478-019-0674-x) contains supplementary material, which is available to authorized users. Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are both incurable and rapidly progressive neurodegenerative diseases of the central nerves system, and they have overlapping spectra of pathogenic features . While pTardbp gene [TDP-43, or TAR DNA-binding protein-43 , encodeddbp gene  is a RNAdbp gene , 76, 83.dbp gene . Furtherdbp gene .Tardbp gene expression often serve as the biological system for exploring the physiological functions of TDP-43 and its pathogenic roles in neurodegeneration [. Most of the transgenic TDP-43 mouse lines overexpress human TDP-43, wild type or mutants, under the control of pan-neuronal promoter, and the resulting phenotypes appear to be primarily relevant to ALS [Mouse models with transgenic overexpression of TDP-43, knock-out/ knock-down of neration . Most oft to ALS , 63. Stut to ALS , 43, 78.t to ALS . On the t to ALS .Tardbplx mouse line [Moreover, the relative contributions of loss-of-function and gain-of-cytotoxicity to the neurodegeneration in FTLD-TDP or ALS-TDP remain to be better defined , 76, 83.use line  and geneTardbp gene by crossing Tardbp floxP mice with αCaMKII-Cre mice (T29–1 line) [CaMKII-Cre only in neurons of the adult mouse brain [Tardbpflox/flox, Cre+ alleles, referred to as TDP-43 cKO, were born at normal Mendelian ratios and appeared indistinguishable from their wild type littermate controls  at birth. As expected, immunohistochemistry analysis of the TDP-43 cKO mice at 2 months of age confirmed the deletion of Tardbp gene and consequent depletion of TDP-43 expression in the forebrain region, in particular in the CA1 pyramidal cell layer of the hippocampus  , the latse brain . Mice wiTDP-43 has been identified as the major pathological protein in 50% of FTLD patients  and FTLDThe light/dark box test  was usedFTLD patients usually did not display cognitive deficits until later stage of the disease . We usedDefective motor coordination developed in the late stages of a proportion of patients with FTLD . We thusBeside cognitive dysfunction, patients with dementia including FTLD also exhibit decreased activities of daily living (ADL). We analyzed the changes of eating habits in TDP-43 cKO mice and found reduced food intake by the aged TDP-43 cKO mice . Morever, the numbers of neuron with the beading or shorter dendrites [. Immunofluorescense staining showed that the number of neurofilament H marker (SMI-32)-positive neurons in layer III/ V of retrosplenial cortex (RS), but not those in the somatosensory cortex, decreased in TDP-43 cKO mice . Alltogether, these results show that depletion of TDP-43 in the forebrain neurons affects the synaptic functions.TDP-43 has been shown to be a modulator of synaptic plasticity in transgenic mouse models of ALS and FTLD . We inve(see) to examine the gene expression profiles in the neocortex .Transcripts exhibiting alternations of their expression levels or splicing patterns in TDP-43 depleted cells other than cortex have been identified before by RNA-seq analysis , 37, 58.ig. Fig. -4.Fig. 5(Table ). Consistent with the correlation of these up/down regulated cortex genes with the age-dependent pathogenesis of TDP-43 cKO mice, most of the gene expression differences between the TDP-43 cKO cortex and Ctrl cotex became greater magnified as the mice aged  encoded proteins involved in the functions of synapse, e.g. Dlg3, endosome, e.g. Lamp5, and autophagosome, e.g. Tecpr1(Table 1). Furthermore, eight of these 20 down-regulated genes were constitutively repressed in the neocortex of TDP-43 cKO mice at the age of 12 month .Increase of Gfap protein in the cortex and hippocampus of TDP-43 cKO mice at the ages of 3 months and 12 months was confirmed by Western blotting, respectively Fig. . Note thFig.  encoded Tecprl gene knockout mice with increased expression of an autophagy substrate, p62 [ and RT-qPCR analysis . Since NMDA receptor (NMDAR)-mediated responses regulated the levels and activities of CaMKII family members [, while the amount of SAP102 was decreased in the cortex and synaptosome of early stage TDP-43 cKO mice. Thus, depletion of TDP-43 in the cortex indeed would down-regulate the expression of a specific set of genes and this could contribute in part to the impairment of synaptic functions , a member of a family of cellular vacuolar protein sorting 10 (VSP10)-domain receptors, was primarily expressed in neurons and a key player in regulating the neuronal viability and function [Sort1 mRNA in mouse striatum and cell lines [Sort1(wt) as the main mRNA isoform encoding sortilin in mouse cortex by RT-PCR and Western blotting  encoding a non-functional progranulin receptor Sortilin 1(e17b) [Sort1 mRNA levels by qRT-PCR in different mouse brain areas confirmed this observation in both 3- and 12-month-old TDP-43 cKO mice  are RNA molecules in which a covalent linkage termed a “backsplice” has formed between a downstream 3′ splice site and an upstream 5′ splice site in a linear pre-messenger RNA . PreviouTDP-43 proteinopathy is assocaited with more than 95% of ALS (ALS-TDP) and more than 50% of FTLD (FTLD-TDP) . A gain-αCaMKII-expressing neurons in the mouse forebrain  often exhibit activation of inflammatory reponse . In TDP-ammation  and in Aammation . SeveralSort1, Adnp2, and Cdh22, the mutations or functional variants of which are associated with aging or neurodegenerative disorders [Dnajc5, Sort1, Pdp1, and Kcnip2, which also occus in TDP-43 knockdown cells of the striatum [Dnajc5 protein in the cortex of TDP-43 cKO and Ctrl mice are similar (data not shown). On the other hand, the truncated sortilin protein accumulates in the cortex of TDP-43 cKO mice at different ages  could act as a decoy receptor [Depletion of TDP-43 in the mouse forebrain also results in aberrant splicing of ~ 50 RNA transcripts in the cortex of 3- and/ or 12-month-old TDP-43 cKO mice receptor  competinCamk1g, Hgsnat, Synj2bp, Adnp2, and Abca8b, in the neocortex of 3- and/or 12-month-old TDP-43 cKO mice  with depletion of TDP-43 in the neurons of cortex and hippocampus. The TDP-43 cKO mice exhibit a spectrum of age-dependent social behaviour change, dementia-like behaviour, impairment of the cognition functions, and decline of ADL. The development of neurodegenerative pathology in TDP-43 cKO mice is closely associated with their behaviour and cognition changes, both of which are well correlated with the age-dependent alterations of the cortex transcriptomes. Notably, the transcriptomopathies of the TDP-43 cKO mouse cortex consist of changes of mRNA levels as well as pre-mRNA splicing patterns of many genes. Related to the latter changes, alternative splicing is known to play an essential role in brain function and mutations in factors involved in splicing regulation cause a range of neurological diseases . TranscrOverall, this study not only supports that loss-of-function of TDP-43 could be a major cause for FTLD-TDP pathogenesis, but also suggests a list of potential TDP-43 target genes that may be useful for future therapeutic development of FTLD-TDP.Tardbp allele was knocked out specifically in the postmitotic pyramidal neurons in the forebrain by crossing mice carrying the Tardbp conditional allele (Tardbplx) with mice carrying a Cre-recombinase transgene driven by the CaMKIIα-promoter. The viability and weight of the mice were monitored regularly. Genotyping of the mice was performed by PCR of genomic DNAs from the tail biopsies.The For RNA-seq, the rRNA-depleted cortical RNAs from three biological replicates of sex-matched TDP-43 cKO as well as littermate Ctrl mice were converted to cDNAs and sequenced them in a strand-specific manner at National Center of Genomic Medicine (NCGM).. The RNAs were extracted from intact mouse cortical tissues and their concentrations determined using NanoDrop 8000 (Thermo Scientific). The RNA integrity was determined by Fragment Analyzer . cDNAs from 5 μg of total RNA was used as an input material for library preparation using TruSeq RNA Sample Preparation Kit v2 (Illumina). Insert sizes of the libraries were confirmed using Fragment Analyzer . The libraries were multiplexed and then sequenced on Illumina HiSeq2000 (Illumina) to generate 50 M of pair end 100 base pair reads per library. Data were processed using the TopHat, Cufflinks  and MISOP value taking in to account the false discovery rate (FDR). We calculated the q value because the expression levels of thousands of genes from a small sample set  were measured. The expression levels of transcripts in the neocortex of TDP-43 cKO mice relative to Ctrl mice were represented by log2 transformed values.The number of mapped fragments per kilobase of exon, per million mapped reads (FPKM) for each annotated protein-coding gene was determined to establish a metric of normalized gene expression. Approximately 80~85% of annotated protein-coding genes in mouse satisfied at least 1 FPKM in either condition. To judge the significance of differences between TDP-43 cKO and Ctrl mice, false discovery rates (q < 0.05) were used as the criteria for different replicates. The q value is an adjusted The ψ  score was defined as the percentage of transcripts containing the alternative splicing events and/ or alternative poly(A) site usage. The mRNAs with increase of splcing events, i.e. conserved exon inclusion/ exclusion, cryptic exon inclusion, and exon extension, as well as change of poly(A) site usage are indicated by Δ ψ > 0. mRNAs with decrease of the processing events are indicated by Δ ψ < 0. Multiple t test was used to calculate the significance from data of 3 independent samples and the Holm-Sidak method was used to correct for multiple t test.For the clustering, transcripts with an estimated expression of a minimum of 0.2 FPKM in both TDP-43 cKO and Ctrl mice were selected for analysis. The list of protein-coding transcripts was compiled based on the RefSeq/Enterz/Vega definitions.https://github.com/TreesLab/NCLscan/TreesLab/NCLscan) [P values were evaluated by the Wald test (DEseq2) and the Fisher’s exact test (edgeR), and then adjusted by the Benjamini-Hochberg procedure. In this study, the significantly differential expression of circRNAs between different stages should satisfy DEseq adjusted P < 0.05 and edgeR adjusted P < 0.05 simultaneously.circRNAs were identified by NCLscan  , which wNCLscan) , 94, on NCLscan)  and edgeNCLscan) , in whicN = 6 for each genotype) using TRIzol reagents (Thermo Fisher Scientific). Synthesis of cDNA followed the manufacturer’s protocol (Invitrogen). qRT-PCR was performed using Roche qPCR FastMix (Roche). Primers were designed using a primer design software (LightCycler Probe Design Software 2.0 from Roche). The expression levels were normalized to gapdh, and data are represented as fold change relative to the Ctrl mRNA levels. Significant differences were determined using un-paired t tests.Total RNAs were extracted from the cortex, hippocampus, and cerebellum of 3-and 12-month-old TDP-43 cKO mice and their Ctrl littermates  in a random corner. After 6, 24, and 48 h, nest building was scored on a scale of 0–5, as previously described . All datThis test has been successfully employed to study social affiliation and interest in social novelty or social discrimination . The test was performed as described previously . BrieflyThe light–dark box was custom made (45 × 20 × 20 cm) and divided into two parts: 1/3 was painted black, covered by the lid, and separated from the white compartment by the wall containing an opening (13 × 5 cm) at the floor level. The light side was illuminated by two 40 W light bulbs 50 cm above the floor. Each mouse was released in the center of the light compartment (facing away from the opening) and allowed to explore the area for 10 min. Video tracking equipment and software  were used for following the animal’s position and movement. In addition, rearings and attempts (stretched attend postures at the opening) to enter either the light or the dark compartment were recorded manually.Mice were trained at the age of 2 months and then subjected to rotarod test monthly. The latencies before falling from the rod was recorded for 3 consecutive days.2O2, blocked in serum with 0.05% Triton X-100 for 30 min, and incubated in rabbit anti-TDP-43 , mouse anti-glial fibrillary acidic protein (GFAP) (1:1000), mouse anti-SMI-32 (1:1000) and mouse anti-NeuN  at 4 °C overnight. After rinsing in PBS, the sections were incubated with biotinnlated goat antibody (Vector) for 60 min, followed by incubation with the avidin–biotin complex (Vector) for 45 min. The reaction products were developed by 3,3′-diaminobenzidine .Mice were anesthetized and perfused with 4% paraformaldehyde. The hemispheres were embedded in paraffin, sliced into 10-μm sections, and mounted on slices. For immunostaining, the sections were washed with 0.1 M PBS buffer, quenched by 1% HTo identify the cellular localization of TDP-43, slices were combined with hematoxylin. The hematoxylin and eosin (H&E) stain was used to visualize the overall morphology of the mouse brain. Nissl staining was applied to characterize the hippocampus size, cortical layer thickness, and neuron number. All data are presented as the mean with standard error, and statistical significance was tested by paired t-test.2 and 5% CO2) at room temperature to allow recovery for at least 90 min before the recording. The field excitatory postsynaptic potential (fEPSP) at Schaffer collateral branches in CA1 region of the hippocampal slices was recorded. Three trains of stimuli at 100 Hz separated by 60 s were applied for LTP induction, and low frequency stimulation (1 Hz for 15 min) were applied for LTD induction. The slopes of the fEPSP were measured and the synaptic responses were normalized to the average of the baseline. All data are presented as the mean with mean standard deviation. Statistical significance was tested by paired t-test.Standard protocol was followed as described previously, with some modifications. In short, the mice were decapitated and their brains rapidly removed and placed in ice-cold artificial CSF (ACSF). 450 μm thick sections of isolated mouse hippocampus were transferred into an interface-type holding chamber in oxygenated ACSF  of TDP-43 cKO mice. Figure S2. Immunohistochemistry staining of brain slices. Figure S3. Persisting reactive astrocytosis in TDP-43 cKO mouse forebrain. Figure S4. Electrophysiology measurements. Figure S5. Mis-regulated genes in TDP-43 cKO mice. Figure S6. Alternations of the processing events of cortical RNAs in TDP-43 cKO mice. Figure S7. The alternative uses of poly(A) sites in the cortical RNAs of TDP-43 cKO mice. Figure S8. qRT-PCR validation of RNA splicing events altered in 3- and 12-month-old TDP-43 cKO mice. Figure S9. Validation of altered splicing events in TDP-43 cKO mice. Figure S10. Calcium signaling and synaptic long term potentiation pathway analysis using Ingenuity Pathway Analysis (IPA). (PDF 2990 kb)Additional file 2:Table S1. List of the chromosome numbers, donor positions, acceptors positions and gene names of the transcripts from which the individual circularRNAs change in the neocortex of 3- and 12-month-old TDP-43 cKO mice, but not Ctrl mice are listed. (PDF 141 kb)Additional file 3:Table S2. Changes of the mRNA levels of social behaviour-related genes in the neocortex of TDP-43 cKO mice relative to Ctrl mice. (PDF 72 kb)


Bilayer graphene and BNNS were grown separately and transferred subsequently onto the substrates to fabricate 2D device architectures. High-resolution transmission electron microscopy confirmed the bilayer graphene structure and few layer BNNS sheets having a hexagonal B3-N3 lattice. The current vs voltage I(V) data for the G/BNNS/Metal devices show Schottky barrier characteristics with very low forward voltage drop, Fowler-Nordheim behavior, and 10−4 Ω/sq. sheet resistance. This result is ascribed to the combination of fast electron transport within graphene grains and out-of-plane tunneling in BNNS that circumvents grain boundary resistance. A theoretical model based on electron tunneling is used to qualitatively describe the behavior of the 2D G/BNNS/metal devices.Various types of 2D/2D prototype devices based on graphene (G) and boron nitride nanosheets (BNNS) were fabricated to study the charge tunneling phenomenon pertinent to vertical transistors for digital and high frequency electronics. Specifically, G/BNNS/metal, G/SiO Graphene  and otheAlthough free-standing graphene has shown unique and outstanding mechanical and electronic properties, however when it is in contact with a substrate surface its physical properties change due in part to parasitic transport effects. Furthermore, the high carrier mobility of graphene is drastically reduced by grain boundary electron scattering and substrate interactions that disturb the charge distribution, thus limiting its immediate device applications.BNNS is an emerging 2D material with similar crystal structure to that of graphene (G) and a small lattice mismatch of about 1.45% that makes it a strong candidate for developing graphene-based heterostructures . It is aNanothin BN, such as our BNNS, shows in-plane insulating properties and out-of-plane charge tunneling mechanism that contributes in the conductivity of the material underneath . The out2-pulsed laser deposition (CO2-PLD) and hot filament chemical vapor deposition  for BNNS and graphene, respectively. The BNNS synthesis process was carried out by irradiating a pyrolytic h-BN target using CO2-PLD  Ontario, Canada) operating at 10.6 µm with a pulse width of 1-5 µs, repetition rate of 5 Hz, and pulse energy of 5 J. Copper foil (2 × 2 sq. inch), molybdenum disc (1″ diam.), and highly polished SiO2 (1″ diam.) were used as substrates. Substrates were mounted on a holder 3 cm away from the target. A detailed description of the CO2-PLD synthesis has been reported previously [2 using the polymethyl methacrylate (PMMA) technique, frequently used to transfer graphene between different substrates [2, respectively. Finally, the PMMA layer was etched away by dissolving it with acetone leaving behind a G/BNNS heterostructures. The surface morphology and crystal structure of the graphene sheets were analyzed using scanning electron microscope , high-resolution transmission electron microscope , and Raman spectroscopy  equipped with optical microscopy accessories. The quality of the graphene sheets was verified at various stages along the process using Raman spectroscopy: As-grown, on PMMA, and after transfer to SiO2 and BNNS. The devices that ended up with poor quality graphene sheets due to damage/contamination during the transfer process were discarded. For device prototyping and characterization, the sputtering technique was used to deposit metal contacts (Au) on the graphene surface side of the devices. The contacts were 100-nm thick and approximately 1 mm apart from each other. The thickness of BNNS nanolayers was estimated using the XRD method and using an algorithm reported by M. Yasaka [There are detailed reports on the synthesis of bilayer graphene  and BNNSeviously ,20,21,22eviously . To fabrbstrates . First, . Yasaka .2 and we also did not present data in our study. Reason is G/BNNS/SiO2 did not show I(V) characteristics due to the high resistance of the device as two non-conducting surfaces were in contact underneath of the graphene layer. Moreover, at least three devices of each type were fabricated and tested to ensure reproducibility. All experiments were conducted at room temperature.The I(V) data of the 2D/2D devices were obtained by the Van der Paw  method iThe surface morphology and nanoscale structures of as-synthesized BNNS and graphene sheets were carefully evaluated. The BNNS images collected by scanning electron microscope (SEM) are presented in 3–N3 atoms within the sheet as shown in the inset with lattice constant 0.22 Å. 3–N3 structure.To obtain high-resolution images we operated the TEM at 200 kV. Using such high acceleration potential produced the irradiation damages and defects in the BNNS due to a knock-on effect seen in 2D/IG ratio >1 [The identification of graphene layers under the optical microscope is a challenging task due to its highly transparent nature, thickness down to two atomic layers and weak contrast with the substrate. In order to overcome this challenge, we mechanically transferred the graphene sheets onto quartz substrates and collected optical images when illuminated at 600 nm. ratio >1 . The Ram2, G/BNNS/SiO2, and G/BNNS/M heterostructures. 2 heterostructure the sheet resistance was about 103 Ω/sq, which increased three orders of magnitude to about 106 Ω/sq for G/BNNS/SiO2. The high sheet resistance observed for these heterostructures was due to the extensive presence of grain boundaries (50–70 nm grain sizes). Moreover, the lattice mismatch between the hetero-materials as well as overall thickness of the insulating layers contributed together to suppressing any possibility for out-of-plane tunneling effects.We then developed prototype 2D/2D devices by fabricating G/SiO2 and G/BNNS/SiO2, the I(V) characteristics of G/BNNS/M heterostructures show good conductivity. The sheet resistance of this device is seven orders of magnitude lower than that of G/SiO2 down to 10-4 Ω/sq. The low sheet resistance of this particular configuration may be understood as arising from BNNS charge tunneling mechanism in the vertical direction  when it is sandwiched between two conductive materials [In contrast to the G/SiOaterials ,25,26. Waterials . If thisThe I(V) characteristic measurements conducted for G/BNNS/M having 10 nm (Curve 1) and 100 nm (Curve 2) thickness are shown in 2, G/BNNS/SiO2 showed no I(V) characteristics due to the high resistance of the devices. However, the measurements conducted for G/BNNS/M showed I(V) characteristics of a typical Schottky diode [−5 V. The Dc power source used for measurement offered uncertainty better than 0.1 × 10−5 V, which was sufficient to get correct measurement in both cases. It is worth noticing that in our previous study on BNNS/M device [The performance of each individual device was analyzed by measuring electrical current vs. voltage I(V) characteristics between two individual metal contacts and by estimating the corresponding sheet resistance of G layer using Van der Pauw method . The carky diode  with lowM device  we obserM device .0 is the potential barrier height and ϕM is the work function of metal, respectively. aBN parameter represents the barrier width and A is the layer critical thickness. We assume that the effect of applied V was to round off the corner of the potential barrier U0 and to narrow the average barrier width, as shown in The observed decrease in current with an increase of BNNS film thickness b was anan0 is the number density of the conductive electrons in the metal, |e| is the absolute value of the electron charge, *m is the effective electron mass, h is the Plank constant, α(E) is the parameter dependent of an applied electric potential difference and increases with an increase of potential difference (dimension is (eV/nm)), U0 is the potential barrier amplitude, e,metE is the energy of the conductive electrons in the metal, Bk is Boltzmann constant, and T is temperature. To carry out the numerical analysis of interest, we assumed that α(E) = aE, where E is the electric field strength developed in the sample of interest. In our case, such field was defined as V/s, where V is the applied potential difference and s is the thin film thickness. The U0 value can be assigned with good accuracy to the BN bandgap  for data shown in a and U0 parameters to be 2.71 ± 0.48 (eV/nm) × (cm/V) and 5.7 ± 0.6 eV, respectively. This value is the result of averaging of a parameter values obtained by fitting procedure for both curves shown in U0 variation around the ionization threshold of BN sheets, the tunneling current dependence on thin film thickness was simulated, where analysis was carried out for the average interval of the applied potential difference used in the current study. Results for the tunneling current through the G/BNNS heterostructure analysis described by Equation (1) are presented in Numerical calculations of the resulting tunneling current were carried out for the general system shown in p (~6 eV ). Here, , thermo emitted charges passing above barrier) that can be approximated by the relation:0 ~6 eV  and the applied potential difference in the mV range, it is justified to assume that the thermionic emission is small and can be neglected in the case studied. Therefore, we assumed that the main mechanism of electron transport through the BN layer in the G/BNNS/M heterostructures was only tunneling. We could thus state that the I(V) experimental data of G/BNNS/M were properly accounted for by the tunneling charge transport mechanism. When we substituted the metal substrate for an insulating substrate the results changed dramatically. 2  data for the G/SiO2 and G/BNNS/SiO2 devices show very high sheet resistances, while the G/BNNS/Metal devices show 10−4 Ω/sq sheet resistance and Fowler-Nordheim behavior. This result is explained as the combination of fast electron transport within graphene grains and out-of-plane tunneling in BNNS that circumvents grain boundary resistance. A theoretical model based on electron tunneling was used to qualitatively describe the behavior of the 2D G/BNNS/Metal devices. Taken altogether, the results hereby presented indicate that G/BNNS/M heterostructures might help to circumvent grain boundary resistance in graphene layers by drastically reducing the effective sheet resistance. This finding may be useful for improving the performance of 2D device architectures.Bilayer graphene and BNNS were grown separately and transferred to fabricate 2D device architectures: G/BNNS/Metal, G/SiO


Marophrys sp. SRT127 belonging to the Centroheliozoa, as the first report on this lineage. The circular-mapped mitochondrial genome, which is 113,062 bp in length, encodes 69 genes typically found in mitochondrial genomes. In addition, the Marophrys mitochondrial genome contains 19 group I introns. Of these, 11 introns have genes for homing endonuclease (HE) and phylogenetic analyses of HEs have shown that at least five Marophrys HEs are related to those in green algal plastid genomes, suggesting intron transfer between the Marophrys mitochondrion and green algal plastids. We also discovered a putative mitochondrial plasmid in linear form. Two genes encoded in the circular-mapped mitochondrial genome were found to share significant similarities to those in the linear plasmid, suggesting that the plasmid was integrated into the mitochondrial genome. These findings expand our knowledge on the diversity and evolution of the mobile genetic elements in mitochondrial genomes.Mitochondrial genomes exhibit diverse features among eukaryotes in the aspect of gene content, genome structure, and the mobile genetic elements such as introns and plasmids. Although the number of published mitochondrial genomes is increasing at tremendous speed, those of several lineages remain unexplored. Here, we sequenced the complete mitochondrial genome of a unicellular heterotrophic eukaryote,  Monocercomonoides sp. PA2031 (now classified as Monocercomonoides exilis2). Although most of the genes in the α-proteobacterium that gave rise to the ancestral mitochondrion have been lost or transferred to the host nucleus during organellogenesis, typical mitochondria, which can carry out aerobic respiration, still retain their genomes3 . The gene repertories in mtDNAs have differently reduced in individual lineages in eukaryotes from the ancestral mtDNA that encoded at least about 100 genes for oxidative phosphorylation, translation, transcription, protein transport, protein maturation and RNA processing as found in mtDNA of jakobids5. In contrast, apicomplexan parasites and their relatives have only three to five genes in their mtDNAs6. mtDNAs also vary with regard to genome architecture, from a simple monocircular molecule  to a complex network comprising thousands of chromosomes . Monolinear mtDNAs were reported from separate branches of eukaryotes as well as mtDNAs consisting of multiple linear chromosomes11. Likewise, the size range of mtDNA is broad among eukaryotes. Apicomplexan parasites are known to have the smallest mtDNAs, approximately 6 Kb in size8. On the other hand, land plants tend to contain large-sized mtDNAs, up to 11 Mb in Silene conica12.Mitochondria have emerged by the endosymbiosis between the last common ancestor of eukaryotes and an α-proteobacterium. All extent eukaryotes have mitochondria or mitochondrial remnant organelles, with only one exception of 13. Group I and group II introns are also known to be able to catalyze self-splicing by forming distinct structures, which enable us to distinguish two types of introns from DNA sequences: Group I introns form the ribozyme structure consisting of 10 stem-loops14, while group II introns require a characteristic secondary structure, such as a central wheel with six stems, for the self-splicing reaction15. Both of group I and group II introns show patchy distributions, which are considered to be formed by intron invasion from an intron-containing locus to a homologous, but intron-less locus in the same species and/or a distantly related organisms17. Group I intron transfers are facilitated by homing endonucleases (HEs) encoded by introns themselves (Note that HEs can act as maturases that facilitate intron splicing as well13). The HEs introduce a double-strand break in the recipient (intron-less) allele that leads to the homologous recombination between intron-containing and intron-less alleles. Individual HEs possess distinct specificities for the nucleotide sequences to recognize and digest. Therefore, a particular HE can introduce the corresponding intron only into a certain position in a genome18. Conversely, the group I introns (and the corresponding HEs) found in the homologous position in different genomes are expected to share their evolutionary origin21. The mechanism of group II intron transfer is different from that of group I introns, but intron-encoded proteins and their sequence recognition specificities play a central role in invasion for group II intron, as in group I intron. Thus, the evolutionary history of group II introns can be retraced by analyzing intron positions and intron-encoded proteins23.mtDNAs of extant species vary markedly in terms of their gene repertories, genome structure, and genome size, as described above. On top of that, mobile genetic elements confer additional layers of mtDNA diversity. Group I introns and group II introns are the examples of the mobile genetic elements found in bacterial, mtDNA and plastid genomes24. Typical linear plasmids in mitochondria contain terminal inverted repeats and one or some open reading frames (ORFs) usually encoding virus-type DNA and/or RNA polymerases, designated as dpo and rpo, respectively, suggesting their independent replication and transcription25. Linear plasmids are extrachromosomal elements in mitochondria, but can occasionally integrate into mtDNA through recombination26. To date, linear plasmids in mitochondria or plasmid-derived dpo/rpo sequences in mtDNA have been reported from fungi27, a slime mold28, plants24 and a ciliate29. Consistent with the genetic mobility proposed for the linear plasmids, the phylogenies of dpo/rpo were found to be inconsistent with the organismal phylogeny30.Another type of mobile genetic elements in mitochondria is the linear plasmids31. At present, Heliozoa comprises two classes, namely Endohelea and Centrohelea33. The phylogenetic position of Heliozoa in the eukaryotes has been controversial for a long time, but recent phylogenomic study suggests that Heliozoa and haptophytes form a monophyletic clade, Haptista, as a sister to the supergroup consisting of stramenopiles, alveolates, and Rhizaria36. Although data on the nuclear-encoded genes have been accumulated for heliozoans, as far as we know, no complete mtDNA sequence has been obtained from any member belonging to this group.Heliozoa was traditionally defined as a taxonomic group of the axopodium-bearing, heterotrophic unicellular eukaryotes mainly living in freshwater environmentsMarophrys sp. strain SRT127. The circular-mapped mtDNA of Marophrys contains 69 typical mtDNA-encoded genes. Up to 20 group I introns were found in six out of the 69 genes, and phylogenetic analyses of the corresponding HEs suggested that at least five introns share their origins with those in green algal plastid genomes. We also identified a linear plasmid carrying genes encoding dpo and rpo. The plasmid is likely to localize in mitochondrion because both mtDNA and plasmid share a deviant genetic code in which UGA codon assigns tryptophan. We further provide the evidence for the linear plasmid being integrated into the mtDNA. These findings expand our knowledge of the diversity of mobile genetic elements in mtDNAs.Here, we first present the complete mtDNA of a member of the Centrohelea, Marophrys sp. SRT127 is 113,062 bp in length and mapped as circular  and the subunit of cytochrome c oxidase assembly (cox11), which are notable as most of the mtDNAs do not retain these genes except those in a limited number of eukaryotes42. In addition, the Marophrys mtDNA includes genetically mobile genes, namely those of dpo, rpo and 11 intronic HEs (see below). The Marophrys mtDNA also contains 12 functionally unidentified open reading frames (ORFs) longer than 100 amino acid residues. Large and small subunits of rRNA genes plus 5S rRNA gene were detected. We identified twenty kinds of genes for tRNAs that could translate 50 codons that cover 17 amino acids in total. During our BLAST analyses of the Marophrys mtDNA sequence against NCBI nr database, we observed UGA at the conserved tryptophan positions in the putative amino acid sequences. Thus, we concluded that UGA in the Marophrys mtDNA is used as tryptophan codon, instead of the termination signal for translation. Genes of the tRNAs that bind the codons UUA for leucine, UGA for tryptophan, ACN for threonine, AAR for lysine and CGN for arginine  were not found , apocytochrome b (cob), ATP synthetase subunit 1 (atp1), and large and small subunits of rRNA (rnl and rns) appeared to be split by a single or up to nine introns. The sequence motif shared among the canonical group I introns was detected from all of the introns in the Marophrys mtDNA (Table Marophrys mtDNA and those of the HEs harbored in the introns as follows. Introns are designated by adding ‘_i’ (intron) to their host gene names with the ascending numbers from the 5′ terminus . The HEs are distinct from each other by adding the corresponding superscripted host intron names .In the NA Table . Hencefonad5) appeared to be split into two distant loci in the mtDNA, which were designated as nad5_a and nad5_b encoding the N- and C-terminal halves of the protein, respectively -PCR using cDNA as template and two primers—one specific to the nad5_a nucleotide sequence and the other to the nad5_b nucleotide sequence  introns in the nucleotide sequences flanking the Marophrys mtDNA harbor intronic ORFs encoding LAGLIDADG motif-containing HEs, which have been typically been found in group I introns16. All of the HEs in the Marophrys mtDNA belong to either LAGLIDADG_1 (pfam00961) or LAGLIDADG_2 (pfam031611). Here, we explored the evolutionary origins of the 11 group I introns harboring HEs by combining phylogenetic affinities of the HEs and the insertion positions of the introns. We are aware of the cases in which the evolution of a group I intron and that of the corresponding intron-encoded protein disagreed to one another47. Unfortunately, we could not exam whether introns and their intron-encoded proteins coevolved, as the intron (nucleotide) sequences dealt in this study are too diverged for phylogenetic analyses. Thus, we assumed the coevolution of each pair of a group I introns and its HE in the following sections. Table Marophyrs mtDNA and their putative origins.Overall, 11 of the 19 introns identified in the Marophrys atp1 intron (atp1_i1) harbors an HE (HEatp1_i1). The HE phylogeny grouped HEatp1_i1 with those harbored in atpA introns, which were found in the plastid genomes (plDNAs) of green algae belonging to core Chlorophyta, with an ML bootstrap value (MLBP) of 97%  in green algae. The HE harbored in Marophrys rnl_i3 (HErnl_i3) grouped together with the HEs in two mitochondrial and one plastid rnl genes in green algae with an MLBP of 97% .We also detected four Marophrys rnl_i4 (HErnl_i4) branched at the base of the clade of nucleus-encoded HEs in land plants with an MLBP of 87% , and Marophrys rnl_i4 and eight plastid rnl introns in green algae appeared to be inserted between T2151 and C2152 (nucleotide numbering is based on the Marophrys rnl gene). Taking these findings together, we propose that Marophrys rnl_i4 and the eight rnl introns in green algal plDNA are derived from a single rnl intron.The HE harbored in eny Fig. . HoweverMarophrys rnl_i6 (HErnl_i6) and those found in four plastid and four mitochondrial rnl introns in green algae formed a clade with an MLBP of 91% . We concluded that Marophrys rnl_i6 and the rnl introns found in green algal organellar genomes share the common ancestor.The HE harbored in Marophrys rnl_i8 (HErnl_i8) and that in the rnl intron in the Acanthamoeba castellanii mtDNA grouped together with an MLBP of 94%, and the two introns appeared to be inserted between G2482 and A2483  found in the Marophrys mtDNA share origins with those in organellar genomes in green algae as well as those of atp1. The atp1 and rnl introns described above were likely transmitted from green algae to Marophrys by considering the predator–prey relationship between centrohelids and green algae in the wild (see above). We presume that, once mobile genetic elements resided in a centrohelid mtDNA, these elements may have persisted in the descendant genomes, as centrohelids are unicellular and asexual eukaryotic lineage53. Unfortunately, the data obtained in this study failed to (i) pinpoint the green algal species that donated the five introns or (ii) clarify whether mtDNA or plDNA was the origin of each of the four rnl introns. It is also important to re-examine the putative green algal origins of the five introns (and their HEs) in future studies incorporating organellar genome data sampled from much broader eukaryotes .By combining the phylogenetic affinities of HEs and intron positions, we propose that the four Marophyrs mtDNA, as the intron phylogenies were not examined due to their extremely divergent natures. Nevertheless, the HE phylogenies  and those in two green algal and three fungal cox1 genes with an MLBP of 78%  were found to be totally different from those surrounding dpo and rpo in the mtDNA  triggered the duplication of a ~7.6 Kb region containing nad9, cob, cox1_a, two ORF and three tRNA genes observed in the current Marophrys mtDNA  was established using micropipetting method from seawater sample that had been collected from Tokyo Bay, Tokyo, Japan , on July 30, 2011. Based on the morphological characteristics, strain SRT127 was identified as a member of the genus yrs Fig. .Marophrys sp. strain SRT127 was maintained in MNK medium (http://mcc.nies.go.jp/02medium.html#mnk) with a green alga Pyramimonas sp. as prey at 20 °C under a 14-h light / 10-h dark cycle until it died in March 2016.Genome annotation section).The centrohelid cells were collected by centrifugation once they had reached confluence and the algal cells were hardly observed in the culture medium under a light microscope. Genomic DNA was extracted with 25:24:1 of phenol:chloroform:isoamyl alcohol and purified by ethanol precipitation. Total RNA was extracted using TRIzol (Thermo Fisher Scientific), following manufacturer’s instructions. The extracted RNA was used to synthesize random hexamer-primed cDNA with SuperScript II reverse transcriptase (Thermo Fisher Scientific). The cDNA was used for the PCR experiments to confirm intron splicing . A total of 125,733,479 paired-end reads of 100 bp were generated  and assembled by SPAdes59 search against the assembly data using the dpo and rpo genes identified in the circular-mapped mtDNA as queries. This 5,877 bp-contig appeared to bear inverted repeats of 102 bp in length at both 5′ and 3′ ends, and to carry only dpo and rpo. To determine whether the contig is a linear or circular molecule, we mapped the paired-end reads to the contig using Bowtie260. We also conducted a PCR experiment to connect both ends of the contig as done to the circular mtDNA (see above). As neither of the aforementioned experiments positively supported the circular structure of the contig (data not shown), we concluded that the 5,877-bp contig represents a linear DNA molecule.A contig of 5,877 bp in length was found by a TBLASTNMarophrys mtDNA was annotated by Prokka61, followed by manual curation. NCBI translational Table https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi#SG4) was applied during the aforementioned annotation, because UGA codons are most likely assigned as tryptophan, not as the termination signal for translation in the Marophrys mtDNA. When a gene appeared to contain one or more introns, the corresponding cDNA was amplified by PCR with the primers listed in Table http://megasun.bch.umontreal.ca/cgi-bin/RNAweasel/RNAweaselInterface.pl). The secondary structural motif conserved among group I introns was also searched by Infernal62 with covariance models built by Nawrocki et al.63.The Marophrys mtDNA were used as queries for a BLASTP59 search against the NCBI nr database  to generate phylogenetic datasets of HEs. The HE sequences retrieved from the NCBI nr database, which shared >90% identity at the amino acid sequence level, were clustered by CD-HIT65. To reduce the redundancy, the representative sequences from each cluster were selected to build the phylogenetic datasets analyzed in this study. The HE sequences harbored in the third and sixth introns of cox1 gene appeared to share the same evolutionary origin, and we prepared a single dataset including both HEs and their related sequences. For a similar reason, we prepared a single dataset containing HEs harbored in three of the introns found in rnl gene, a single intron in rns gene and one of the introns found in cob gene in the Marophrys mtDNA. Individual datasets were subjected to MAFFT66 with the LINSI option for calculating the alignments. The ambiguously aligned sites in the alignments were discarded manually. Maximum likelihood (ML) analyses using IQ-TREE67 were conducted on each alignment with 1,000 ultrafast bootstrap replicates68. The substitution models were selected by ModelFinder69 implemented in IQ-TREE.The conceptual amino acid sequences of the HEs identified in the Supplementary Figures


An energy density of 68.1 W h kg−1 and a high maximum power density of 558.5 W kg−1 in 6 M KOH electrolytes were recorded for the optimum sample. The NG samples showed an appropriate cyclic stability with capacitance retention of 87.7% after 5000 cycles at 4 A g−1 with high charge/discharge duration. Thus, the prepared NG herein is considered to be promising, cheap material used in energy storage applications and the method used is cost-effective and environmentally friendly method for mass production of NG in addition to opening up opportunities to process waste materials for a wide range of applications.3D sponge nitrogen doped graphene (NG) was prepared economically from waste polyethylene-terephthalate (PET) bottles mixed with urea at different temperatures using green approach via a novel one-step method. The effect of temperature and the amount of urea on the formation of NG was investigated. Cyclic voltammetry and impedance spectroscopy measurements, revealed that nitrogen fixation, which affects the structure and morphology of prepared materials improve the charge propagation and ion diffusion. The prepared materials show outstanding performance as a supercapacitor electrode material, with the specific capacitance going up to 405 F g Electrochemical capacitors (ECs) are considered as one of the most promising energy storage technologies responding to the increasing demand of the clean energy related technologies and applications such as portable/mobile electronics, electrical vehicles, or other storage systems based on sources like windmills and solar cells.1.The graphene-based materials are promising for applications in supercapacitors and other energy storage devices and there are many researches on the refinement of their structures and development of effective low cost synthesis techniques as well as possible clarification of their electrochemical performance4. Among a variety of nitrogen sources, urea has a high nitrogen content (46 wt %), and can react with oxygen-containing groups. Hence, urea is a typical precursor that has been used widely as a nitrogen source in experiments on graphene7.Chemical doping has been exceedingly used to enhance capacitor property of graphene. Doping of graphene with nitrogen is of much interest, since it improves the overall conductivity and supercapacitive properties because introducing N atoms intrinsically improve the electronic properties of graphene in particular the performance as supercapacitor electrodes8. However, the extremely low yield and high cost of these methods limit their application only to fundamental studies. Later, it was found out that GO can be used as a platform for N incorporation via thermal annealing with NH3 or nitrogen plasma treatment but these methods involve either toxic precursors or need sophisticated equipment5. Many researchers aim to develop one-step simple preparation way for inquiring nitrogen doping into graphene and most of their efforts have used aqueous mixture of graphene oxide (GO) and nitrogen source as starting the materials. For instance, Hao et al. prepared novel cross-linking three-dimensional (3D) graphene layers utilizing gas-foaming technology by using colloidal solution of exfoliated GO and NH4Cl solution; the obtained material showed a capacitance of 231.2 F g−1 at a current density of 1 A g−1 in 1 M H2SO49. Chen et al. produced 3D graphitic carbon nitride functionalized graphene networks by the hydrothermal reduction of GO sheets and exfoliated carbonized melamine in their mixed aqueous dispersions at 180 °C for 12 h. The obtained material displayed 264 F g−1 at a current density of 0.4 A g−1 in 0.1 M LiClO410. Xiao et al. inserted nitrogen into graphene using nitric acid through thermal treatment at 500 °C, which showed a capacitance value of 370 F g−1 at 1 A g−1 in 6 M NaOH11. Zhang et al. produced nitrogen doped graphene using nitric acid and a high temperature co-processing method at 500 °C for 4 h in a quartz tube furnace with a high specific surface area (436.7 m2 g−1) and a high specific capacitance 12.There are numerous methods to synthesize nitrogen doped graphene, which can be categorized into two kinds of methods: direct synthesis such as chemical vapor deposition (CVD), arc-discharge, segregation growth, solvothermal methods, and post treatment such as hydrazine hydrate, thermal and plasma treatments+ ions induce the hydrogenation of dangling bonds on the formed graphene layer14, and hence, forming a strong C–H bond. Bonding configuration changes from sp2 to sp3 and less oxygen functional groups are present in grapheme, that in turn enhances the capacitive behavior because the oxygen functional groups would impede the migration of electrolyte ions into micropores and as a result reduced the effective pore volume of the graphenic materials. Both raw materials are low cost and are manufactured in large quantities. The great challenge is the conversion of waste PET bottles using a one step thermal decomposition method into supercapacitor electrode material through simple and cost effective method. and that was demonstrated by using only urea. In this method, graphene gets simultaneously doped with nitrogen.Therefore, developing a low-cost, scalable, and eco-friendly method is still of great interest. Herein, the nitrogen (N) doping was used to improve the capacitance performance of graphene through a novel synthesis using PET waste bottles and urea. Urea has two amine groups with nitrogen content about 46 wt% joined by a carbonyl (C=O) functional group. Therefore, it is a good candidate to be used as nitrogen precursor. Moreover, the presence of H6. Urea totally decomposes into volatile products with a total weight loss close to 600 °C. While, for PET, the degradation started at 360 °C and achieve maximum degradation rate at 480 °C. The curve exhibits one major mass loss which is attributed to thermal decomposition and the residue at 800 °C is 0.9 wt%. This is obviously associated with the segment corresponding to the carboxylic acid and the benzene ring appearing in the chemical unit of PET15. Compared to PET and urea curves, the curve of PET/urea is coalesce of their two curves. A mass loss of 60% took place in the first step at the temperature range of 150–240 °C, which is in good agreement with urea decomposition to biuret. This process was followed by two steps which ended at 480 °C similar to PET curve. During the thermal decomposition process, the out-gassing as CO, CO2, NO2, and H2O molecules could introduce force to expand the graphene layers and help the formation of porous frameworks16. That is confirmed by SEM and TEM images. Meanwhile, the embedded nitrogen atoms in the carbon lattice cause the formation of NG frameworks. In order to prove the presence of nitrogen doped in graphene structure, an elemental analysis was conducted on the dried product with XPS and FTIR. The mass fractions of carbon, oxygen, and nitrogen for all samples are presented in Supplementary Table Thermogravimetric analysis (TGA) is used to investigate the behaviors of PET and urea in the thermal decomposition process. Supplementary Fig. −1 corresponding to the stretching vibration mode of -OH and a characteristic peaks appearing at 1532 cm−1 correspond to the skeletal vibration of C=C (sp2 hybridization)10. It is noticeable from the spectrum of undoped graphene samples (5 G and 6 G) that the intensities of characteristic peaks decreased by rising the temperature of PET thermal decomposition and this indicates that the quality of produced graphene increases with increasing the treatment temperature due to decreasing amount of oxygenous groups. However, for NG samples, the spectra show marked changes in the 1000–1700 cm−1 region, particularly in the C–O stretching bands, in addition to the C=N stretching vibration modes which appeared at 1631 cm−1 and that confirm the success introducing of N atoms into graphene lattice. Complementary information on nitrogen functional groups were obtained by X-ray photoelectron spectroscopy (XPS) analysis as shown in Fig. 16. The XPS spectra of the graphene sample derived from PET bottles waste at 800 °C (sample 6 G) confirmed that carbon and oxygen were the only present atoms and nitrogen signal could be clearly observed in the XPS spectra of NG samples. For NG samples  was observed and this demonstrate that C atoms were arranged in a honeycomb lattice and the fitted peaks centered at 285.55, 286.7, 287.85, and 288.95 eV are assigned to C=N, C-N, C=O, and O=C-O, respectively. It is worth noting that a small amount of oxygen containing functional groups was in samples because the presence of oxygen functional groups on the surface of graphenic materials reduced its surface conductivity and prevented ions from entering into the pore17. But they have a great influence on the capacitive performance18 because it improves the hydrophilicity of the surface which enhancing the ions diffusion of the aqueous electrolyte and reduced the mass transfer resistance on the surface of electrode; subsequently make it easier for ions to form electrochemical double layer and as a result the capacitance will be increased18.As shown in Fig. 1. The second way for N-doping is that carbon nitride, produced by the decomposition of the N precursors, acts as an intermediate for the formation of N dopant in the NG. This way had a low handout here because the yield of carbon nitride from urea was very low and FTIR spectroscopy had confirmed the absence of the signature peak for carbon nitride at ~810 cm−1 indicating that it is unlikely that carbon nitride is involved in this process. Moreover, it was notable from XPS high-resolution N1s spectra for sample 1NG and 4NG  to 2.514 at.% (sample 4NG).However, the high-resolution N1s spectra of the N-doped graphene in Fig. As shown in Fig. 13 with distinct hysteresis loops at the range of relative pressures (P/P0) from 0.45 to 1.0 which can result from the coexistence of micropores and mesopores. BET surface areas of all samples are illustrated in Supplementary Table 21. The pore size distributions of N doped samples are shown in Supplementary Information Fig. 13.Nitrogen adsorption–desorption isotherm curves for NG samples as shown in Fig. 22.XRD patterns, illustrated in Supplementary Fig. −1 correspond to the D, G, and 2D bands of graphene-based materials, respectively23. The former D band originates from the defects in sp2 lattice structure (amorphous nature), the latter G band corresponds to the first order scattering of sp2 (graphitic nature) domains while 2D bands originate from two phonon double resonance processes and are related to the band structure of graphene layers24. Raman spectrum of NG shows a shoulder to G peak, known as D’ which generally appears when there is lattice defect or doping of another atom and that confirm N-doping20. The 2D band for NG samples is hardly detectable in the spectra due to significant structural defects induced by thermal synthesis and intercalation of N atoms in addition to further increase in layers leads to a significant decrease of the relative intensity of the lower frequency 2D peaks20. As shown in Fig. D/IG ratio of graphene blank samples is slightly lower than NG samples, demonstrating that N-doping increases the defects of graphene lattice and structural distortion. In addition from Supplementary Table However, Fig. 3 hybridization caused by structural distortions within the graphene network which induced by the thermal synthesis and embedded N atoms. Moreover, selected area electron diffraction pattern  were displayed in Fig. 27.The unique 3D structure combines high surface area in addition to nitrogen doping content leading to the prominent capacitive and cycling performance of these supercapacitor electrode materials using two similar electrode cell. In Fig. −1 in 6 M KOH, and no distinct IR drops appear in any curves, indicating excellent electrochemical reversibility, good rate capability and outstanding carrier transport within the electrodes for the electrochemical double layer capacitors (EDLCs). Furthermore, the capacitance of the 4NG electrode is four times higher than that of graphene electrode. The detailed GCD curves of the 4NG sample at various current densities are shown in Fig. −1 in 6 M KOH at current densities of 1, 2, 4, and 6 A g−1, respectively. However, the unchanged curves with linear and symmetrical shapes at various current densities are observed from this test. As a result it can be revealed that, the NG possesses considerable capacitive performance in 6 M KOH and that ascribed to the combination of microporous in the NG sheet and the mesoporous between the NG sheet in 3D structure with high active area which facilitates ion transport, thus contributing synergistically to the high rate performance.To assess the capacitive property, a two-electrode (2E) cell was used to conduct the galvanostatic charge–discharge (GCD) tests. As shown in Fig. −1. The 4NG electrode with the point power density of 558.5 Wkg−1 displays maximum energy density of 68.1 Wh kg−1 at a current density of 1 A g−1, which is over three times higher than that of 6 G (29.6 Wh Kg−1). The results illustrated in Supplementary Table The energy density (E) and power density (P) are calculated according to Equations  and 3)  3) from 4 than other prepared samples which exhibit straight lines at the low frequency region and a small depressed semicircle in the high-frequency region. Moreover, more vertical line was observed for N doped electrodes than undoped electrodes, which suggested high electrochemical activity of N doped graphene towards supercapacitors. However, the obtained semi-circle represents the charge transfer impedance at the interface of the current collector/electrode material while, the vertical line represents ion diffusion in the structure of electrode materials and the slope of this line is related to the speed of EDL formation28.The supercapacitive properties of all prepared samples as electrodes in 6 M KOH were studied through Electrochemical Impedance Spectroscopy (EIS). Nyquist plots as shown in Fig. s corresponds to the resistance of the electrolyte is 0.003 Ω, Rct is the charge transfer resistance (0.005 Ω), Cdl is the double layer capacitance (4-E6 Fg−1) and W represents the diffusion Warburg impedance (0.9) which is very low comparable to other prepared electrodes as illustrated in Supplementary Information Table 29 and the relaxation time for the 4NG electrode was found to be (335 ms). Therefore, it is confident to say that, 4NG has got faster frequency response capability, with shorter ionic transport paths that in turn reduced internal resistance and increase the electrical conductivity30.As shown in the inset of Fig. −1 using the galvano static charge/discharge technique. The capacitance retentions of 4NG were 87.7% of their initial capacitances, retained after 5000 charge/discharge cycles . The closed stainless steel reactor was placed inside the center of an electric furnace and the experiments were repeated at two different temperatures 600 °C and 800 °C with a rate of 10 °C min−1 under nitrogen gas flow. The composition of the prepared materials was studied using Fourier transform infrared (FTIR)  and X-ray photoelectron spectroscopy (XPS) Phi 5300 ESCA system  with the Mg (Kα) radiation (X-ray energy 1253.6 eV). The XPS samples were prepared by dropping 100 μL of NG aqueous dispersions (1.0 mg/mL) onto silicon substrates followed by air drying. Elemental analysis of the NG samples was measured using vario-Micro CHN Elemental analyzer (Germany).The thermal properties of raw PET, urea and a mixture of PET and urea with 1:1 wt. ratio were investigated by TGA using a thermogravimetric analyzer (Shimadzu TGA-50) from 20 °C to 800 °C at a rate of 10 °C min2) adsorption-desorption isotherms were recorded with Shimadzu, Micromeritics ASAP 2010 Instrument) cooled by liquid N2. Prior to recording sorption isotherm, the sample was degassed at 120 °C under vacuum for 12 h.Nitrogen (NThe morphology of the prepared samples was explored using scanning electron microscopy (SEM), JEOL  instrument with an operating voltage of 15 kV, and transmission electron microscopy (TEM), JEOL  under an accelerating voltage of 200 kV.−1 were employed to analyze the crystal structure of prepared materials.Raman spectroscopy  with a 514.5 nm excitation wavelength and X-ray diffraction  and dried at 60 °C overnight. The mass of active materials in the active surface was around 1–1.2 mg cm−2.To prepare the working electrode, the prepared active materials (90 wt %), carbon black (5 wt %), polyvinylidinefloride (5 wt %), and 1-methyl 2-pyrrolidinone as a solvent were mixed forming slurry. The slurry was then coated onto nickel sheet (1 cm−1 to 100 mV S−1. The charge–discharge measurements were performed at different current densities (from 0.5 to 6 Ag−1). Cyclic stability tests were conducted at a current density of 4 A g−1. The specific gravimetric capacitance of the supercapacitor was determined from the galvanostatic cycles by means of the formula:31.The electrochemical measurements were performed in a two-similar electrode cell using 6 M potassium hydroxide (KOH) electrolyte solutions, nickel sheet as current collector and Whatman fiber glass filter paper (GF/D) as a separator. The electrochemical characterization was performed at room temperature using a computer-controlled Potentiostat  involving cyclic voltammetry (CV), galvanostatic charge/discharge (GCD), and electrochemical impedance spectroscopy (EIS). The CV studies were performed between − 0.2 to 1 V at increasing sweep rates from 5 mV S32. Electrochemical Impedance Spectroscopy (EIS) tests were recorded in a frequency range from 0.001 to 100 kHz with a sinusoidal signal of 5 mV.Energy density (E) and power density (P) were calculated using the formulas,Supplementary Information


To describe and analyze a new protocol for the extraction of platelet-rich plasma (PRP) for use in clinical practice and compare this technique with methods that have been previously described in the medical literature.We extracted PRP from 20 volunteers using four different protocols . In another group of 12 individuals, we extracted PRP with our new technique (named ‘turn down-turn up') consisting of a double spin (200 ×g and 1600 ×g) closed system using standard laboratory equipment (including an ordinary benchtop centrifuge), where the blood remained in the same tube during all processes, reducing the risk of contamination. Platelet counts adjusted to baseline values were compared using analysis of covariance (ANCOVA).p=0.063 compared with the double spin at 600 and 900 ×g method). The total cost of the disposable materials used in the extraction process was less than US$10.00 per individual.Using the four previously described protocols (mentioned above), we obtained concentrations of platelets that were 1.15-, 2.07-, 2.18-, and 3.19-fold greater than the baseline concentration, respectively. With the turn down-turn up technique, we obtained a platelet count that was 4.17-fold : 3.09 to 5.25) greater than the baseline platelet count (In the present study, we described a simple and safe method for obtaining PRP using low-cost devices. The incidence of articular degenerative diseases is increasing PRP is defined as an autologous preparation obtained from the centrifuged peripheral blood of patients with a concentration that is 3-5 times greater than the basal concentration The first step to carry out a clinical study on PRP is the standardization of the process for collecting and preparing the concentrate. Several previous studies have reported different methods of obtaining PRP using different centrifugation speeds and numbers of spins In the present study, we aimed to describe and evaluate a simple, inexpensive, and reproducible protocol for obtaining PRP for use in clinical practice.The first part of the study involved the reproduction of four different protocols based on a literature review. Healthy blood donors were selected from a hospital blood bank. After explaining the research objectives to patients and obtaining signed informed consent forms from the patients, 42.5 ml of peripheral venous blood was obtained from each patient. For coagulation analysis, blood was collected using blood collection tubes (BD Vacutainer ACD Solution A Ref. 364606) containing 3.2% sodium citrate at a volumetric ratio of 1.5 ml of anticoagulant to 8.5 ml of blood. A Sorologic Kasvi centrifuge (model K14-0815A) with a fixed angle rotor was used.The blood samples were analyzed on a Sysmex XE5000 hematology counter prior to the centrifugation procedures to establish the baseline values of platelets, red blood cells, and leukocytes in each patient. After obtaining the concentrate, a new count was obtained to enable the evaluation of the platelet concentration factor . The extraction of PRP was performed following four different protocols. Protocol 1 was used to obtain samples based on commercial kits mentioned in previous studies In the second part of the study, we investigated our protocol (‘turn down-turn up') for obtaining PRP. The procedures are described in Box 1. The estimated time for preparation of PRP using this protocol was approximately 35 minutes.1. Collect the desired volume (8.5 ml) of blood through peripheral venous access directly into a vacuum tube with acid citrate dextrose (ACD) (1.5 ml).2. Equalize the remaining vacuum in the tube.3. Centrifuge the tube at 200 ×g for 15 minutes with the tube cap facing down .4. Carefully remove the tube from the centrifuge and maintain the tube in the downward position without turning the tube.5. Under aseptic conditions, aspirate 3.5 ml of the hematic layer through the rubber cap .6. Turn the tube to an upright position (cap facing up).7. Centrifuge the tube at 1600 ×g for 10 minutes with the lid facing up.8. Under aseptic conditions, aspirate 3.5 ml of the superior portion of the material .9. Aspirate the desired amount of PRP (1-2 ml) from the lower portion of the tube .t-test and analysis of covariance (adjusted for baseline platelet counts) using IBM SPSS Statistics for Windows, Version 20.0 . The 95% confidence intervals (CIs) were estimated for the ratio between the PRP and baseline concentrations.The platelet counts were compared with Student's The present study was approved by the Institutional Review Board of our institution  and registered at Plataforma Brasil (the Brazilian government's registry of scientific studies) under CAAE  number 53166416.1.0000.5530.For the first part of the study (the investigation of previously described protocols), 20 male donors aged 26 to 54 years : 9.9 years) were selected. The baseline counts of the blood components were within the normal range of values .The PRP extraction results are described in 3 platelets/μL . Analysis of the PPP revealed negligible values  of red and white blood cells. The number of platelets and erythrocytes lost at the end of the first centrifugation was also evaluated, and the median count was 48.75 x 103 platelets/μL (26.4% of the baseline platelet count), which varied from 29 to 66 x 103 platelets/μL. Therefore, an average of 73.6% of the total number of platelets was recovered by our PRP preparation protocol.In the second part of the study (the investigation of the turn down-turn up PRP protocol developed by our team), another group of donors  was selected . Our protocol achieved a mean platelet concentration that was 4.07 times greater than the basal concentration with a mean platelet count of 749x10elets/μL . This plwww.mibo-statement.org. Some authors proposed methods of PRP classification. A more comprehensive and comparative investigative strategy has been proposed by Lana et al. The use of PRP in various medical fields has resulted in the exponential growth of the number of studies using PRP therapy. In orthopedics, PRP serves as an intermediate between conservative and surgical treatments In addition to the lack of standardization of the methods to obtain PRP, the frequency and number of PRP injections remain controversial. In previous studies, the protocols for injections are highly variable, with up to 6 weekly applications in the lumbar muscles for the treatment of back pain In the present study, we performed PRP extraction using four methods adapted from previously reported protocols. We obtained a low volume of platelet concentrate using single spin protocols in accordance with previous reports. Cavallo et al. 3 platelets/uL, which was 4.3 times greater than the basal concentration of platelets, with a nonrefrigerated centrifuge. Using this method, we obtained a platelet concentration that was 3.09 times greater than the basal concentration. This difference may be explained by several factors, which we briefly discuss here. The results may have been influenced by the design of the centrifuge. Rotors with a fixed/nonfixed angle and the distance to the center of the rotor may result in different forces, leading to different concentration values, and the size of the blood tube may also influence the results Among the previously established protocols investigated in this study, the highest concentrations of platelets were obtained using the method reported by Jo et al. There is still no consensus or scientific basis for the concentration of platelets that is necessary to induce tissue repair. However, there is evidence that even relatively low platelet concentrations may be effective. Sanchez et al. found that, compared with hyaluronic acid injection in the knee, using the single spin technique, which produced a concentration of platelets that was two-fold greater than the baseline concentration, showed a superior effect After reviewing the current literature and investigating four different techniques of PRP extraction, we developed a reproducible and low-cost closed protocol, which we named the turn down-turn up technique. This two-spin method follows the principles of low-speed centrifugation to preserve the maximum volume of platelets and growth factors In the present study, we described a simple and safe method of obtaining PRP using low-cost devices. Our results were consistent with current standards of quality for PRP preparation for clinical trials suggested by the American Academy of Orthopedic Surgeons working group, who presented the MIBO statement Machado ES provided substantial contribution to the concept and design of the study, data acquisition, analysis and interpretation, manuscript drafting, critical revision for important intellectual content and final approval. Leite R, Santos CC, Artuso GL provided substantial contribution to the concept and design of the study, data acquisition, manuscript critical revision for important intellectual content and final approval. Caldas JM provided substantial contribution to the concept and design of the study, manuscript critical revision for important intellectual content and final approval. Gluszczak F provided substantial contribution to data acquisition, analysis and interpretation, manuscript critical revision for important intellectual content and final approval. Jesus LG provided substantial contribution to the data analysis and interpretation, manuscript drafting and final approval. Bredemeier M provided substantial contribution to the data analysis and interpretation, manuscript drafting, critical revision for important intellectual content and final approval. All of the authors take public responsibility for appropriate portions of the content.